Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2014-08-31.


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Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2014-08-31.
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Skurupey, Leigh A
University of Florida
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Gainesville, Fla.
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Master's ( M.S.)
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University of Florida
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Animal Sciences
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Warren, Lori
Committee Members:
Johnson, Sally
Pratt-Phillips, Shannon


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Animal Sciences -- Dissertations, Academic -- UF
Animal Sciences thesis, M.S.
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by Leigh A Skurupey.
Thesis (M.S.)--University of Florida, 2012.
Adviser: Warren, Lori.
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lcc - LD1780 2012
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2 2012 Leigh Ann Skurupey


3 To my parents m other La nette and s tepfather John Collins and father Brian Skurupey whose support and encouragement is instrumental to my courage, my success and my future. My parents and two brothers are the most important people in my life and any success I achieve is testament to their boundless support. It is my brothe r James Skurupey who is my hero and has leaded me to believe anything is possible if I want it bad enough. I am also truly thankful to all my amazing friends who support and encourage me in all my endeavors.


4 ACKNOWLEDGMENTS I would not have reached this mile stone without the help of Dr. David Denniston, my remarkable Horse Judging Team C oach from Colorado State University and Dr. Saundra TenBroe ck for both believing in me as a Horse Judging Team. These two incredible individual s, along with Joel McQuagge and Justin Callaham at the University of Florida have provided me with so many opportunities for which I am immensely grateful. I am greatly appreciative of my advisor, Dr. Lori Warren, for giving me the opportunity to work for her, as it is her encouragement knowledge, patience, and guidance which has helped me through this thesis. I especially thank Dr. Sally Johnson and Dr. Shannon Pratt Phillips for serving on my committee and reviewing my thesis. I would like to thank Mrs Jan Kivipelto for her endless guidance and technical assistance in the lab with procedures and assays. I am also grateful for the helping hand of Dr. Joel Yelich for working tirelessly with me on my insulin assay and statistical codes I also significant ly thank Dr. William W. Thatcher Toa Sha and Eduardo de Souze Riberio for their constant and genuinely needed help with my statistical questions. Further, I would like to thank the consistent guidance of Dale Kelley with the amino acid analyzer, because without his initial guinea pig role with the machine; I would not have been successful in a timely manner given the complex equipment I am grateful for my mother and Aunts Karen Amsbau gh and Charell Shillo spoiling me rotten with horses, which ha s led me on a quest to involve my passion of the horse. Lastly, I acknowledge those horses involved in my study without them this study would not have existed; they too helped make this milestone possible.


5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 10 ABSTRACT ................................ ................................ ................................ ................... 13 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 15 2 LITE RATURE REVIEW ................................ ................................ .......................... 17 Insulin Resistance ................................ ................................ ................................ ... 17 Insulin Sensitivity versus Insulin Effectiveness ................................ ................. 18 Prevalence of Insulin Resistance in the current horse population .................... 18 Insulin mediated Glucose Transport ................................ ................................ 19 Non Insulin Mediated Glucose Uptake ................................ ............................. 22 Insulin Resistance: Obesity and Fatty Acids ................................ ..................... 23 Mechanisms of Insulin Resistance ................................ ................................ ... 27 Assessment of Insulin Resistance ................................ ................................ .... 31 Oral glucose tolerance test ................................ ................................ ........ 31 Intravenous glucose tolerance test ................................ ............................ 32 Insulin tolerance test ................................ ................................ .................. 33 Frequently sampled intravenous glucose tolerance test ............................ 34 Hyperglycemic and hyperinsulinemic clamps ................................ ............. 36 Basal glucose and insulin measurement ................................ .................... 38 Proxies and reference quintiles for basal glucose and insulin .................... 38 Equine Metabolic Syndrome ................................ ................................ ................... 40 Equine Metabolic Syndrome and Obesity ................................ ......................... 41 Equine Metabolic Syndrome and Laminitis ................................ ....................... 42 Hyperinsulinemia and Vascular Dynamics ................................ ....................... 44 Management of Equine Metabolic Syndrome and Insulin Resistance .............. 45 Diet ................................ ................................ ................................ ............ 45 Exercise ................................ ................................ ................................ ..... 49 Pharmacologic intervention ................................ ................................ ........ 51 Dietary Supplements ................................ ................................ .................. 51 Arginine ................................ ................................ ................................ ................... 52 Dietary Arginine Supply ................................ ................................ .................... 53 Role of Arginine in the Body ................................ ................................ ............. 54 Arginine Metabolism ................................ ................................ ......................... 55


6 Endogenous Synthesis of Arginine ................................ ................................ ... 55 Arginine and the Urea Cycle ................................ ................................ ............. 59 Arginine Degradation ................................ ................................ ........................ 60 Regulation of Arginine Metabolism ................................ ................................ ... 61 Citrulline ................................ ................................ ................................ .................. 62 Role of Citrulline in the Body ................................ ................................ ............ 63 Citrulline Biosynthesis ................................ ................................ ...................... 64 Citrulline Malate ................................ ................................ ............................... 66 Arginine and Citrulline Supplementation and Insulin Resistance ............................ 67 Arginine and Metabolic Disease ................................ ................................ ....... 67 Arginine Mechanisms that Improve Insulin Sensitivity ................................ ...... 72 Advantage of Citrulline over Arginine Supplementation ................................ ... 74 Citrulline .......................... 75 3 MATERIALS AND METHODS ................................ ................................ ................ 80 Hors es ................................ ................................ ................................ .................... 80 Dietary Treatments ................................ ................................ ................................ 80 Insulinemic Response to a Meal ................................ ................................ ............. 81 Sample Analyses ................................ ................................ ................................ .... 82 Plasma Glucose ................................ ................................ ............................... 82 Serum Insulin ................................ ................................ ................................ ... 83 Plasma Amino Acids, Urea and Ammonia ................................ ........................ 84 Statistical Analyses ................................ ................................ ................................ 85 4 RESULTS ................................ ................................ ................................ ............... 88 5 DISCUSSION ................................ ................................ ................................ ......... 98 APPENDIX A PLASM A GLUCOSE ASSAY ................................ ................................ ................ 108 Materials ................................ ................................ ................................ ............... 108 Preparation of the Internal Control ................................ ................................ ........ 109 Preparation of the Glucose Standards ................................ ................................ .. 109 Assay Part I Pipetting Standards and Samples ................................ ................. 109 Assay Part II Enzyme Mixture Preparation ................................ ........................ 110 Assay Part III Incubation and Plating ................................ ................................ 110 B SERUM INSULIN ASSAY ................................ ................................ ..................... 111 Preparation of Radioactive Tracer and Insulin Standards ................................ ..... 111 Tube Labeling ................................ ................................ ................................ ....... 111 Radioimmunoassay Procedure ................................ ................................ ............. 112 Decant Samples ................................ ................................ ................................ ... 115 Packard Cobra Auto Gamma Counter ................................ ................................ .. 115 ................................ ................................ ................................ ... 116


7 C AMINO ACID, UREA, AND AMMONIA ANALYSIS ................................ .............. 118 Materials Needed ................................ ................................ ................................ .. 118 Reagents f or Physiologic Fluid Analysis of Amino Acids ................................ ...... 118 Recipes for SSA, HCL, AEC ................................ ................................ ................. 119 Calculating Percent Recovery of AEC ................................ ................................ .. 11 9 Preparation of Plasma Samples for Amino Acid Analysis ................................ ..... 120 LIST OF REFERENCES ................................ ................................ ............................. 122 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 143


8 LIST OF TABLES Table page 3 1 Nutrient composition of feeds included in the basal ration ................................ .. 87 4 1 Measures of glucose and insu lin response to a grain meal containing citrulline malate (CIT) or an isonitrogenous amount of urea (CON). ................... 92 A 1 Preparation of glucose standards. ................................ ................................ .... 109 B 1 Preparation of standards and samples for the serum insulin radioimmunoassay. ................................ ................................ .......................... 114 C 1 Composition of buffers used in Hitachi L 8900 Amino Acid Analyzer ............... 121


9 LIST OF FIGURES Figure page 2 1 Arginine synthesis. ................................ ................................ ............................. 78 2 2 Metabolism of citrulline and arginine in the hepatic urea cycle and related pathways.. ................................ ................................ ................................ .......... 79 4 1 Plasma glucose and serum insulin responses before (0:00 h) and after horses consumed a grain meal containing citrulline malate (CIT) or an isonitrogenous amount of urea (CON). ................................ ............................... 93 4 2 Plasma citrulline and arginine concentrations before (0:00 h) and after horses consumed a grain meal containing citrulline malate (CIT) or an isonitrogenous amount of urea (CON).. ................................ .............................. 94 4 3 Plasma ornithine, proline, and glutamate concentrations before ( 0:00 h) and after horses consumed a grain meal containing citrulline malate (CIT) or an isonitrogenous amount of urea (CON). ................................ ............................... 95 4 4 Plasma lysine, methionine, and threonine concentrations before (0:00 h) and after horses consumed a grain meal containing citrulline malate (CIT) or an isonitrogenous amount of urea (CON). ................................ ............................... 96 4 5 Plasma urea and NH 3 concentrations before (0:00 h) and after horses consumed a grain meal containing citrulline malate (CIT) or an isonitrogenous amount o f urea (CON). ................................ ............................... 97 B 1 Packard Cobra Auto Gamma Counter ................................ .............................. 115 B 2 Swipe protocol. ................................ ................................ ................................ 117


10 LIST OF ABBREVIATION S ADP Adenosine Diphosphate AEC Aminoethyl Cysteine Hydrochloride AGC1 Aspartate Glutamate Carrier 1 AHC American Horse Council apoB Apolipoprotein B ASS Argininosuccinate S ynthase ASL Argininosuccinate L yase ATP Adenosine Triphosphate BCS Body Condition Score BMI Body Mass Index BW Body weight CALS College of Agriculture and Life Sciences cGMP Cyclic Guanosine Monophosphate CIT Citrulline Malate treatment group CON Control Urea CSP I Carbamoyl P hosphat e S ynthase I DM Dry M atter EDRF Endothelium Derived Relaxing Factor EDTA Ethylenediaminetetraacetic acid, anticoagulant EMS Equine Metabolic Syndrome ESC Equine Sciences Center FFA Free Fatty Acid FFAs Free Fatty Acids FSIGT Frequently Sampled Intravenous Glucose Tolerance Test


11 GLUT Glucose Transporter GSIS Glucose Stimulated Insulin Secretion HCl Hydro chloride HOMA Homeostasis Model Assessment IFAS Institute of Food and Agricultural Sciences IRS 1 Insulin Receptor Substrate 1 ITT Insulin Tolerance Test IVGTT Intravenous Glucose Tolerance Test MIRG Modified Insulin Response to Glucose mRNA Messenger Ribonucleic Acid NAGS N Acetylglutamate Synthase NAHMS National Animal Health Monitoring System NH 4 + Ammonium NO Nitric O xide NOS Nitric Oxide S ynthase NSC Non Structural Carbohydrate OAT Ornithine A minotransferase O CT Ornithine C arbamoyltransferase OGTT Oral Glucose Tolerance Test P5C S P yrroline 5 C arboxylate Synthase PPID Pituitary Pars Intermedia Dysfunction QH Quarter H orse RBC Red blood cell RISQI Reciprocal of the Insulin Square Root Index scFOS Short Chain Fructo Oligosaccharides


12 SSA Sulfosalicylic acid STZ Streptozoto cin TAG Triacylglycerol TB Thoroughbred TNF Tumor Necrosis Factor VLDL Very Low Density Lipoprotein VMRCM Virginia Maryland Regional College of Veterinary Medicine WSC Water Soluble Carbohydrate


13 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science INVESTIGATION OF CITRULLINE MALATE SUPPLEMENTATION ON PLASMA AMINO ACIDS AND GLYCEMIC AND INSULINEMIC RESPONSES IN HORSES By Leigh Ann Skurupey August 2012 Chair: Lori K. Warren Major: Animal Sciences Supplementation with a rginine has been shown to improve insulin sensitivity in several species Because it escapes splanchnic extraction, cit rulline has been shown to be more effective at increasing arginine availability than direct supplementation with arginine. Therefore, citrulline could provide a novel dietary intervention for the management of insulin resistance horses. The objectives of this study were to: 1) investigate citrulline malate ( CIT ) supple mentation and its effects on availability of other amino acids; 2) assess whether oral CIT will act as a dietary precursor for arginine; and 3) determine if oral CIT will alter glycemic and i nsulinemic response s to a starch rich meal in healthy horses. We hypothesized that supplementation of CIT would increase arginine availability and consequently alter insulin respon se to a meal. Twelve clinically normal horses ( mean SE, age 10.8 2.5 y; BW 552.0 31.2 kg ; body condition score 5 to 6 on scale of 1 to 9 ) we re randomly assigned to urea ( isonitrogenous control ; 25 mg/kg BW ) or citrulline malate ( CIT; 86 mg/kg BW ) supplementation for 14 d. I nsulinemic and glycemic responses to a meal (0.25% BW) of grain mix concentrate and the daily allotment of urea or CIT supplement were evaluated on d 14 following an


14 overnight fast. V enous blood samples were taken 30 min and immediately before the meal an d then every 30 min after the meal for 5 h Plasma was assayed for glucose, insulin and amino acids. Statistical analysis was a mixed model with repeated measures with time, treatment, and time by treatment as fixed effects. Plasma citrulline, arginine, or nithine and glutamate increased in response to a meal and were higher in horses supplemented with CIT compared to urea. Plasma urea, lysine, methionine and threonine were unaffected by CIT consumption. Glycemic response to a grain meal was similar between treatments ; however serum insulin was lower when horses consumed a meal containing CIT versus urea. Insulin sensitivity ( estimated by the reciprocal of the insulin square root index and homeostasis model assessment ) and cell responsiveness (e stimated by modified glucose to insulin ratio and homeostasis model assessment ) were not affected by CIT supplementation. Results demonstrate that CIT can be used to increase whole body arginine supply without negatively affecting amino acids that may be l imiting in equine diets. In addition, supplementation with CIT may be useful for maintaining glycemic control while reducing hyperinsulinemia in insulin resistant horses, but deserves further study.


15 CHAPTER 1 INTRODUCTION Veterinary r esearchers in Virginia reported that 51% of horses evaluated on farms were overweight and 19% were obese ( Thatcher et al., 2007 ) Furthermore, 18% of the overweight horses and 32% of the obese horses were hyperinsulinemic Insulin resistance has been implicated in several equine dis eases as a pathogenesis, including equine metabolic syndrome (EMS), pituitary pars intermedia dysfunction (PPID; ( Kronfeld et al., 2005a 2006 ; Treiber et al., 2006a ; Pratt et al., 2009 ; Valberg and Fishman, 2009 ; Zimmel and McFarlane, 2009 ) Thus, there is great interest in find ing ways to cont rol insulin resistance in the horse. Arginine is a potent secretag ogue of the endocrine system including insulin secretion by the pancreas ( Sener et al., 1989 ) A rginine supplementation has been reported to increase insulin sensitivity in hea l thy and diabetic subjects ( Jobgen et al., 2009 ; Clemmensen et al., 2011; Monti et al., 2012) I mprovement in insulin sensitivity appears to result from improved glucose tolerance with arginine supplemented subjects usually having lower glucose concentrations. The effects on circulating insulin concentrations have been shown to vary with arginine sup plementation Although the control have been attributed to enhanced production of nitric oxide ( Calver et al., 1992 ) and accumulation of polyamines in pancreatic islet cells ( Sener et al., 1989 ) Dietary arginine is rapidly absorbed by the intestine and degraded by arginase, contributing little to plasma arginine concentrations ( Bger and Bode Bger, 2001 ) Furthermore, o ral supplementation of citrulline has been shown to be more effic ient at increasing whole body arginine supply ( Schwedhelm et al., 2008 ) Therefore, citrulline


16 suppleme ntation may be more effective than arginine for improving insulin responsiveness. The objectives of this st udy were to: 1) investigate oral citrulline malate supplementation and its effects on the availability of other amino acids; 2) assess whether citrul line malate will act as a dietary precursor for arginine ; and 3) determine if citrulline malate will alter glycemic and insulinemic response to a starch rich meal in healthy horses. We hypothesized that supplementation of citrulline malate would increase arginine availability and consequently improve insulin response to a meal.


17 CHAPTER 2 LITERATURE REVIEW Insulin Resista nce Insulin is a natural hormone secreted from the cells of the pancreas. Insulin acts as a major regulatory hormone in glucose and fat metabolism, vascular function, inflammation, and tissue remodeling ( Treiber et al., 2006b ) In a normal horse, insulin will stimulate the uptake of glucose by skeletal muscle, adipose tissue and liver. I nsulin r esistance is simply the failure of these tissues to respond appropriately to insulin. The pancreas will continue to secrete insulin to compensate for a decrease in tissue or cell effectiveness. Therefore, the resting serum insulin levels will be high in ho rses with moderate to severe insulin resistance. In humans, insulin resistance typically develops with obesity and can eventually result in the onset of type 2 (non insulin dependent) diabetes ( Valberg and Fishman, 2009 ) Few horses are thought to be truly diabetic but they can be insulin resistant. Insulin resistance has been implicated in several equine diseases as a pathogenesis including equine metabolic syndrome (EMS), pituitary pars intermedia dysfunction ( PPID; adenoma, and forms of laminitis ( Kronfeld et al., 2005a 2006 ; Treiber et al., 2006a ; Pratt et al., 2009 ; Valberg and Fishman, 2009 ; Zimmel and McFarlane, 2009 ) Insulin resistance is also thought to play a role in diseases such as hyperlipemia ( Forhead, 1994 ) endotoxemia ( Tth et al., 2008 ) and osteochondritis dissecans in horses ( Valberg and Fishman, 2009 ) Insulin resistance happens over time as the horse loses its sensitivity t o insulin and is unable to utilize glucose from the diet as effectively. Insulin resistance has been observed in fasting, obese, and inactive horses ( Treiber et al., 2005a ) Moreover, insulin


18 resistance also influences reproductive efficiency and probably exercise tolerance ( Kronfeld et al., 2005a ) Insulin resistance especially when associated with PPID, can contribute to muscle loss, abnormal fat accumulation, laminitis excessive water consumption, frequent urination and incr eased susceptibility to disease ( Zimmel and McFarlane, 2009 ) Insulin Sensitivity versus Insulin Effectiveness Clarification of a few terms regarding i nsulin and its functions may be beneficial. mediated glucose transport into the cell of target tissues, such as adipose tissue and skeletal muscle, is referred to as insulin sensitivity ( Treiber et al., 2006c ; Pratt et al., 2009 ) Irrespective of the cause, insulin insensitivity is pertaining to reduced cell surface activity and glucose supply, whereas insulin ineffectiveness pertains to reduced intr acellular glucose utilization ( Kronfeld et al., 2005a ) Insulin resistance however, involves a decreased response to circulatin g insulin by insulin sensitive cells, which are primarily in skeletal muscle and adipose tissue, but also in liver ( Treiber et al., 2005a ) Insulin resistance alludes to insensitivity at the cell surface. The term resistance can refer to inefficient insulin signaling at the cell surface, which is low insulin sensitivity, or in the case of disruption of insulin signaling pathways within the i nsulin sensitive cells, such as insulin ineffectiveness ( Kronfeld et al., 2005a ; Treiber et al., 2005a ) Prevalence of Insulin Resistance in the current horse population Currently, there is limited information on the prevalence of insulin resistance in horses. The Virginia Maryland Regional College of Veterinary Medicine (VMRCM) along with the College of Agriculture and Life Sciences at Virginia Tech, conducted a perspective research study on the prevalence of obe se and insulin resistant horses


19 ( Thatcher et al., 2007 ) The study included 300 horses (4 to 20 years of age) from 114 far ms chosen randomly from 1,000 horses examined by the VMRCM field service. Horses were evaluated prior to any grain or concentrate consumption to reduce the chance of glucose and insulin levels being altered. Horses were evaluated based on two independent body condition scores (BCS) to assess the amount of fat cover and morphometric measurements were taken to calculate body weight and body mass index (BMI). The study examined each horse for signs of laminitis as well as measured blood glucose, insulin and other hormones, cytokines and oxidative biomarkers. Researchers found that 51% of the horses in the study were overweight and 19% were found to be obese. Furthermore, 18% of the overweight horses and 32% of the obese horses were hyperinsulinemic ( Thatcher et al., 2007 ) Interestingly, the researchers also suggests that equine obesity may result from natural grazing behavior in addition to the overuse of grains and other feed supplements. If these data are extrapolated to the 9.2 million horses reported to be in the United States ( AHC, 2012 ) an estimated 560,000 to 846,000 horses (6 to 9% of the total population) could be affected by insulin resi stance. Insulin mediated Glucose Transport The two primary variables involved in glucose homeostasis are: 1) the insulin secretory response of cells within the islets of Langerhans of the pancreas to increases in blood glucose; and 2) the sensitivity of skeletal muscle and adipose tissues to serum insulin concentrations ( Firshman a nd Valberg, 2007 ) Consumption of a meal containing starch and/or sugar will result in the absorption of glucose and an increase in circulating blood glucose. Elevated blood glucose subsequently triggers the secretion of insulin from the cells within the pancreas. Insulin mediates the transport and metabolism of glucose, as well as its storage, first in skeletal muscle then in adipose


20 tissue ( Gould and Holman, 1993 ) Insulin also both inhibits glu cagon secretion and lowers serum free fatty acid concentrations, consequently decreasing liver glucose production ( Shepherd and Kahn, 1999 ) Toxic effects on cel ls begin to result from prolonged elevation of blood glucose concentrations ( Yki Jarvinen, 1992 ) In addition, low blood glucose concentrations can result in seizures ( Valberg and Fishman, 2009 ) Therefore, maintaining blood glucose concentrations through hormonal regulation is extremely important. Glu cose transport, the initial step in glucose utilization, is considered rate determining in glucose utilization in muscle ( Kahn, 1 992 ) However, there evidence from clinical studies ( Yki Jrvinen et al., 1987 ; Kelley et al., 1996 ) and studies from cultured human muscle cells ( Jacobs et al., 1990 ) that glucose phosphorylation by hexokinase also affects the rate of glucose util ization, especially under high glucose concentrations or stimulation by insulin ( Perriott et al., 2001 ) Because vertebrate muscle has little glucose 6 phosphatase activity ( Su rholt and Newsholme, 1981 ) the phosphorylation of glucose is irreversible in this tissue Irreversibility is a feature of rate determining steps in metabolism and supports the notation that phosphorylation aids in determining the overall rate of glucos e utilization ( Perriott et al., 2001 ) The lipid bilayers in cell membranes are naturally impermeable to glucose; thus, glucose needs a special transport system to enter the cell. Facilitated diffusion down a glucose concentration gradient is mediated by transmembrane proteins known as glucose transporters (GLUT). Several G L UT isoforms, distinguished by their tissue distribution have been identified in most species and include GLUT 1, GLUT 2, GLUT 3, and GLUT 4 ( Gowrishankar et al., 2011 ) Not all GLUT are influenced by insulin. S keletal muscle,


21 myocardium, and adipose tissue express the GLUT 4 and GLUT 1 isoforms. In these tissues, GLUT 1 is largely responsible for basal glucose transport, because a significant amount of this transport er isoform is present at the cell surface in the absence of insulin ( Giorgino et al., 2000 ) In contrast, the majority of GLUT 4 localizes to intracellular t ubulovesicular structures clustered in the cytoplasm, often close to the plasma membrane ( Giorgino et al., 2000 ) In adipocytes, GLUT 4 is slowly exocytosed and rapidly endocytosed, and a very small percent of the total GLUT 4 is in the plasma membrane ( Blot and McGraw, 2008 ) The GLUT 4 transporter is sequestered intracellularly and only translocates to the cell membrane under the influence of either insulin or exercise ( Holman and Kasuga, 1997 ) Insulin increases the number of GLUT 4 transporters in the plasma membrane, resulting in substantial increases in both cell surface GLUT 4 levels and glucose transport rates ( Giorgino et al., 2000 ) Translocation of GLUT 4 to the cell membrane occurs via a complex process initiated as insulin binds to the GLUT 4 ( Valberg and Fishman, 2009 ) Insulin binding causes phosphorylation of the receptor and insulin receptor subst rate proteins ( Valberg and Fishman, 2009 ) The substrates produced form complexes with docking proteins in order to activate phosphoninositide 3 kinase, a major pathway for the mediation of insulin stimulated glucose transport and metabolism ( Shepherd et al., 1998 ) The functionally important targets further downstream in the phosphoinosit id e 3 kinase signaling cascade are still being studied and identified, but are thought to include proteins that help regulate the docking of GLUT 4 containing vesicles at the plasma membrane and their fusion to the membrane ( Rea and James, 1997 )


22 Insulin signaling involves numerous substances and affects the transport and utiliza tion of glucose, particularly in correlation to hexokinase, glycogen synthase, and other key enzymes in glucose and lipid metabolism ( Kronfeld et al., 2005a ) Tyrosine kinase is involved in the main insulin receptor on the cell membrane ( Kronfeld et al., 2005 a ) The activation of tyrosine kinase leads to translocation of glucose transporter, mainly GLUT 4 in muscle, from the interior to the cell surface ( Kronfeld et al., 2005a ) Two main chains of proteins link the tyrosine kinase receptor to the GLUT 4 transporters and therefore is critical to the biologic response of insulin ( Kronfeld et al., 2005a ) Other insulin receptor signals affect enzymes involved in glucose and lipid metabolism inside the cell in order for proper function ( Kronfeld et al., 2005a ) Therefore, the insulin receptor itself is then capable of integrating insulin sensitivity and effectiveness such as glucose tran sport into the cell and its subsequent intracellular utilization ( Kronfeld et al., 2005a ) Although insulin is the foremost stim ulus for glucose uptake into cells via GLUT 4, other stimuli, such as thyroid hormone and leptin, can also activate the translocation of GLUT 4 into muscle and fat cell membranes ( Valberg and Fishman, 2009 ) Non Insulin Mediated Glucose Uptake The GLUT 1 glucose tran sporter is constantly present in the plasma membranes of cardiac and skeletal muscle and adipocytes and provides basal, non insulin dependent uptake of glucose ( Gaster et al., 2001 ) The GLUT 3 transporter has a similar function to GLUT 1, but is expressed in neural tissue ( Taha et al., 1995 ) In co ntrast to GLUT 4 and GLUT 1, the GLUT 2 facilitative glucose transporter is found primarily in liver and pancreatic cells ( Giorgino et al., 2000 ) Also in contrast to GLUT 4 and GLUT 1, GLUT 2 in the liver has been shown both in vivo and in vitro to be


23 upregulated by glucose and downregulated by insulin ( Giorgino et al., 2000 ) Further, GLUT 2 has been suggested to serve as a glucose sensor in the liver portal vein, indicating its essential nature for the rapid response to glucose ( Giorgino et al., 2000 ) Regulation of GLUT 2 is at the transcriptional level and yet it permits a rapid, dose dependent response to glucose by the liver ( Giorgino et al., 2000 ) Exercise stimulates glucose transport through the use of pathways that are independent of phosphoinositide AMP activated kinase ( Shepherd and Kahn, 1999 ) Basal and insulin stimulated glucose uptake into muscle cells and adipocytes are increased by thyroid hormone, partially as a result of an increase in GLUT 4 expression ( Kahn, 1992 ; Abel et al., 2004 ) In horses, levothyroxine is known to improve insulin sensitivity and decrease blood lipid concentrations ( Frank et al., 2005 ) Adipocytes secrete leptin and in the brain it signals a response to change energy stores ( Berti et al., 1997 ) Leptin improves insulin response in skeletal muscle and i nsulin stimulated glucose disposal, which is largely attributed to an increase in fatty acid oxidation and a decrease in intramuscular triacylglycerol ( Stefanyk et al., 2011 ) Insulin Resistance : Obesity and Fatty Acids Insulin secreted after a meal acts as an anabolic hormone, promoting fuel stor age and favoring weight gain ( Pittas and Roberts, 2006 ) From an evolutionary standpoint, insulin hypersecretion in response to a meal may have conferred a survival advantage in increasing the efficiency of energy storage in adipose tissue when it was in abundance, which is also known as the thrifty gene hypothesis ( Southam et al., 2009 ) Few studies have examined the prevalence of obesity in horse and pony populations. The 1998 National Animal Health Monitoring System study estimated that 4.5% of the horse population in the United States was overweight or obese ( Geor, 2008 ) Thatcher


24 and coworkers (2007) more recently predict ed th at the prevalence of overweight (51%) and obese (19%) horses supersedes that of the National Animal Health Monitoring System in 1998, indicating that obesity in the equine industry in greatly increasing. sented with an abundance of readily available food and are kept in relative confinement. Some horse owners also lack understanding of the proper dietary management of horses and overfeed them. Collectively this has led to excessive weight gain and fat acc umulation. Some studies in humans have found an association between in sulin secretion and weight gain ( Hodge et al., 1996 ; Odeleyle et al., 1997 ; Sigal et al., 1997 ) whereas others have not found the same association ( Weyer et al., 2000 ; Mayer Davis et al., 2003 ; Silver et al., 2006 ) In horses, diet induced weight gain occurred concurrently with decreased insulin sensitivity that was effectively compensated for by an increase in insulin secretory response ( Carter et al., 2009 ) The study fed 200% of the h digestible energy requirements for maintenance for 16 wk to induce weight gain which resulted in hyperinsulinemia and hyperleptinemia. Th u s prevention of obesity is a potential strategy to help avoid insulin resistance, hyperinsulinemia, and hyper leptinemia in the horse s. Another study used obese geldings (BCS 7 to 8) to estimate glucose effectiveness and insulin sensitivity and found that these geldings were insulin resistant and seemed to rely primary on glucose effectiveness for glucose disposal ( Hoffman et al., 20 03 ) indicating they had reduced insulin sensitivity due to high serum insulin when compared to other non obese horses One mechanism that may explain the association between insulin hypersecretion and future weight gain is the development of hypoglycem ia in the post absorptive period, which has been shown


25 to result in a pattern of increased hunger, frequent snacking, and increased energy intake in humans ( Pittas and Roberts, 2006 ) This is thought to be the pri mary cause of weight gain in medical or iatrogenic conditions associated with hyperinsulinemia ( Sinha et al., 1996 ; Dizon et al., 1999 ) There is also evidence to suggest that relative hypoglycemia contributes to increased intake in healthy, non obese people ( Pitta s et al., 2005 ; Pittas and Roberts, 2006 ) In healthy subjects short term hypoglycemia increases morning food intake ( Schmid et al., 2008 ) Similar effects of hypoglycemia on increased voluntary intake are likely present in horses, but this has not been thoroughly investigated. It is well established that adipose tissue is not only involved in energy storage, but functions as an endocrine organ that secretes various bioactive substances such as adipokines ( Ouchi et al., 2003 ; Berg and Scherer, 2005 ) Ad ipose tissues in obese individuals and in animal models of obesity are infiltrated by a large number of macrophages, whose recruitment is linked to systemic inflammation and insulin resistance ( Weisberg et al., 2003 ; Xu et al., 2003 ) Functionally, M2 macrophages are associated with repair of injured tissues and resolution of inflammation ( Gordon, 2003 ) It has been suggested that M1 macrophages promote insulin resistance and M2 macrophages protect against obesity induced insulin resistance ( Odegaard and Chawla, 2011 ) Insulin resistance can be driven by a chronic, low grade inflammatory state characterized by elevated serum levels of pro inflammatory cytokines [ e.g interleukin ( IL ) 1, IL 6, IL 8, IL 12, and tumor necrosis factor (TNF )] chemokines ( e.g. monocyte chemotactic protein 1 and macrophage inflammatory protein 1) insulin resistance associated adipokines (e.g. r etinol binding protein 4 and resistin), and insulin


26 sensitivity associated adipokines (e.g. adiponectin, visfatin, omentin, and vaspin) ( Odegaard and Chawla, 2011 ) Free fatty acids (FFA) are important to the pancreatic cell for its normal function, its capacity to compensate for insulin resistance, and its failure in type 2 diabetes ( Nolan et al., 2006 ) D epriving pancreatic islets of fatty acids in the pancreas causes loss of glucose stimulated insulin secretion, a process that can be reversed by supplying exogenous FFA ( Nolan et al., 2006 ) In contrast, high FFA supply augments glucose stimulated insulin secr etion However chronic excess of saturated fatty acids especially coupled with elevated glucose, reduces insulin biosynthesis ( Poitou t et al., 2006 ) and secretion ( Prentki et al., 2002 ) and induce s cell apoptosis ( Lee et al., 1994 ; Prentki et al., 2002 ; El Assaad et al., 2003 ) Saturated and certain monounsaturated fats have been implicated as factors causing insulin resistance, whereas poly unsaturated fatty acids (PUFAs) and, particularly omega 3 fatty acids, appear to have no adverse effects or even fairly positive effects on the action of insulin ( Manco et al., 2004 ) Polyunsaturated fa t t y acids re gulate fuel partitioning within the cells b y inducing their own oxidation through the reduction of lipogenic gene expression and the enhancement of t he expression of those genes controlling lipid oxidation and thermogenesis ( Manco et al., 2004 ) Moreover, PUFAs prevent insulin resistance by increasing membrane fluidity and GLUT 4 transport ( Manco et al., 2004 ) In contrast, saturated fatty acids are stored in non adipocyte cells as triacylglyceride leading to cellular damage as a sequence of their lipotoxicity. Although it is not entirely clear how obesity produces insulin resistance or type 2 diabetes, elevated plasma FFA are thought to play a major role ( Boden, 2003 ) FFA


27 directly stimulate insulin secretion and decrease metabolic clearance of insulin ( Boden, 2005 ) In healthy, young women an increase in plasma FFA from approximately 0.5 to 1.1 mmol/L raised insulin secretion by 17% under euglycemic conditions without a change in insulin clearance ( Hennes et al., 1997 ) However, when blood glucose concentration was raised to approximately 7 mmol/L and plasma FFA to approximately 1.1 mmol/L, the subsequent rise in serum insulin was attributed to both increased insulin secretion and decreased metabolic clearance of insulin ( Hennes et al., 1997 ) When blood glucose was further increased to approximately 11 mmol/L, the rise in serum insulin was almost entirely due to a decrease in insulin clearance. Currently, data suggests that when healthy individuals h ave elevated levels of FFA it stimulate s long term insulin secretion precisely to the degree needed to compensate for the FFA induced insulin resistance ( Boden, 2005 ) In contrast, i n individuals genetically predispos ed to type 2 diabetes, FFA stimulation of insulin secretion is not sufficient to compensate for the FFA induced insulin resistance, which may lead to diabetes ( Boden, 2005 ) Mechanisms of Insulin Resistance re sistance to the stimulatory effect of insulin on glucose utilization is considered an important pathogenic feature of obesity, metabolic syndrome, and most human forms of type 2 diabetes ( Shepherd and Kahn, 1999 ) The precise mechanisms that cause insulin resistance are not known in either humans or horses. Several mechanisms may be involved in the gradual process of becoming insulin resistant. Four possible mechanisms are discussed below. The first mechanism that might be involved in insulin resistance involves aberrations in GLUT 4 trafficking. The GLUT 4 transporter is responsible for an insulin


28 mediated increase in glucose uptake by skeletal an d cardiac muscle and adipocytes Uptake of glucose by the cell is the rate limiting step for glucose metabolism in these cell types, and therefore the appropriate regulation of GLUT 4 is critical to maintenance of normal whole body glucose homeostasis. I n sulin stimulates the recruitment of GLUT 4 from intracellular compartments to the plasma membrane ; thus if GLUT 4 translocation is hindered, it may contribute to insulin resistance. Much effort has been expended on identifying the amino acid motifs in GLU T 4 that are responsible for its insulin regulated subcellular trafficking. A decrease in insulin stimulated glucose uptake in obese humans and obese mice was associated with the impairment of GLUT 4 translocation in both skeletal muscle and adipocytes ( Zierath et al., 1998 ; Miura et al., 2001 ) However, not all studies have found the insulin stimulated translocation mechanism to be impaired; therefore, the role of GLUT 4 trafficking in the pathogenesis of type 2 diabetes is not absolute ( Gaster et al., 2001 ) Under normal conditions, a greater amount of GLUT 4 present at the cell surface appears to largely account for the concomitant increase in glucose transport activity ( Song e t al., 2008 ) ; thus an adequate presence of GLUT 4 within the membrane is important for the correct biological response to insulin. However, GLUT 4 trafficking in the fasting and insulin stimulated states is still the subject of controversy. P ossible mu tations in GLUT 4 could also be a mechanism of insulin resistance Mutations of glutamic acids of the acidic cluster TELEY motif has been shown to affect GLUT 4 movement from the endosomal recycling pathway to a subcompartment of the trans Golgi network in both basal and insulin stimulated adipocytes ( Blot and McGraw, 2008 ) There are several motifs that regulate diffe rent steps of GLUT 4 traffic ( Blot and


29 McGraw, 2008 ) and if mutated or hindered it would affect insulin and whole body glucose homeostasis. T he second mechanism that may be involved in insulin resistance is the expression of GLUT 4 Reduced GLUT 4 expression on the cell surface limits the activation of phosphoinositide 3 kinase ( Zierath et al., 1998 ) which is the major pathway for the mediation of insul in stimulated glucose transport and metabolism ( Shepherd et al., 1998 ) M uscle GLUT 4 overexpression in transgenic animals ameliorates insulin resistance associated with obesity or diabe tes as it increases glucose uptake without high concentrations of insulin ( Zorzano et al., 2005 ) The third mechanism that may be involved in insulin resistance is the impairment of insulin stimulated glucose transport by circulating or paracrine factors ( Valberg and Fishman, 2009 ) Tumor necrosis factor (TNF) is a major pro inflammatory cytokine which has been implicated in metabolic disorders, such as obesity and insulin resistance ( Qin et al., 2008 ) Apolipoprotein B (apoB100) is a major protein component of plasma lipoproteins and is required for the synthesis and secretion of triacylglycerol rich circulating lipoproteins such as very low density lipoproteins (VLDL) ( Qin et al., 2008 ) Qin and coworkers (2008) provided evidence that TNF body insulin resistance and impairs he patic insulin signaling, which is accompanied by the overproduction of aopoB100 containing VLDL particles, an effect likely mediated via TNF receptor 2. The chronic elevation of serum FFA concentrations, such as those that occur in obese or diabetic humans or horses with equine metabolic syndrome, may also contribute to the decreased uptake of glucose into peripheral tissues ( Valberg and Fishman, 2009 ) Elevated FFA concentrations are linked with the onset of peripheral


30 and hepatic insulin resistance as the high FFA a nd intracellular lipid appear to inhibit insulin signaling, leading to a reduction in insulin stimulated glucose transport decreased muscle glycogen synthesis and glycolysis and liver hyperglycemia ( Boden and Shulma n, 2002 ) Lastly, the fourth mechanism that is a highly probable cause of insulin resistance is chronic hyperglycemia. Glucose in excess causes toxic effects on the structure and function of organs, including the pancreatic islets where insulin is secr eted from ( Yki Jarvinen, 1992 ) A potential mechanism for glucose toxicity is the formation of excess reactive oxygen species, which cause oxidative stress over time, defective insulin gene expression, and insulin secretion impairment ( Robertson, 2004 ) Other possible mechanisms of insulin resistance could pertain to the breakdown of insulin signaling receptors (such as tyrosine kinas e) which could reduce circulating insulin or downregulate insulin downstream ( Treiber et al., 2006c ) affecting downstream kinases such as phosph atidylinositol 3 kinase or serine/threonine kinase Tyrosine phosphorylation of insulin receptor substrate 1 (IRS 1) by the insulin receptor, promotes association of the docking protein with effector proteins, such as phosphatidylinositol 3 kinase ( Ozes et al., 2001 ) The phosphatidylinositol 3 kinase and its downstream target, Akt, promote insulin induced movement of GLUT 4 to the cell membrane, glucose uptake, gl ycolysis, glycogen synthesis, and protein synthesis ( Ozes et al., 2001 ) The most common causes of insulin resistance are thought to be alterations in signal transduction associated with decreased insulin receptor autophosphorylation decreased IRS 1 phosphorylation and reduced activation of phosphatidylinositol 3 kinase ( Treiber et al., 2006c ) Subsequently, the disruption of


31 intracellular glucose metabolism regulated by enzymes such as hexokinase and gly cogen synthase could reduce insulin mediated glucose uptake and storage ( Treiber et al., 2006c ) Another possible mechanism could be the decrease in insulin response due to rapid insulin degradation or possibly to the neutralization by antibodies ( Kronfeld et al., 2005a ) Interference at the insulin receptor on the cell surface, such as a glycoprotein associated with receptor tyrosine kinases could be a cause of insulin resis tance ( Kronfeld et al., 2005a ) as discussed above. Ultimately, a diminution in the intracellular capacity to utilize glucose in the muscle, liver, or adipose tissue, subsequently leads to insulin resistance, regardless of glucose transport and availability, where insulin can become partially or completely ineffective. Compensations for insulin insensitivity may be achieved by either an increase in the release and secretion of insulin from pancreatic cells or increasing concentration of plasma and extracellular glucose by an increase in glucose mediated gl ucose uptake ( Kronfeld et al., 2005a ) A ssessment of Insulin R esistance The amount of insulin that the pancreas secretes in res ponse to glucose and the sensitivity of skeletal muscle and adipose tissue to insulin, affects whole body insulin response. Therefore, both need to be assessed to provide an accurate measure of insulin resistance ( Firshman and Valberg, 2007 ) There are currently 7 different methods to measure insulin resistance in horses, which ar e described below. Oral glucose tolerance test The oral glucose tolerance test (OGTT) is used to assess small intestinal absorption of glucose, hepatic glucose uptake and, to a lesser extent, the endocrine


32 function of the pancreas and peripheral insulin r esistance ( Firshman and Valberg, 2007 ; Valberg and Fishman, 2009 ) The test requires the horse to fast overnight and then 1 g/kg body weight (BW) of glucose is administered orally via a nasogastric tube ( Valberg and Fishman, 2009 ) Blood glucose is measured before glucose administration and every 30 to 60 minutes thereafter ( Valberg and Fishman, 2009 ) Blood glucose leve ls generally peak 90 to 120 minutes after glucose administration then return to baseline around 240 to 360 minutes after glucose administration. A sustained high level of blood glucose in response to an OGTT might suggest reduced pancreatic function or pos sible insulin resistance ( Ralston, 2002 ) However, the test can be affected by the stress of intubation. Further, the test may be affected by variable rates of glucose administration, gastric emptying, and intestinal absorption ( Valberg and Fishman, 2009 ) and is therefore somewhat limited for evaluating insulin resistance. It also does not account for endogenous insulin secretion and therefore is limited in subjects with altered pancreatic function ( Kim, 2008 ) Intravenous glucose tolerance test An intravenous glucose tolerance test (IVGTT) is somewhat similar to an OGTT with the exception that the glucose is administered intravenously. This test is used to avoid the variable absorption of glucose by the intestinal tract as can occur with an OGTT ( Kronfeld et a l., 2005b ) Horses are fasted for 12 to 24 hours and then administered 0.5 g glucose per kg/BW intravenously over a 10 minute time period ( Valberg and Fishman, 2009 ) Blood glucose and insulin concentrations are determined at 0, 2, 15, 30, 60, and 90 minutes and t hen hourly for 5 to 6 hours after injection ( Valberg and Fishman, 2009 ) Half life of glucose disposal and the fractional turnover rate, which is a measure of glucose utilization and the peripheral insulin resistance can


33 then be calculated ( Valberg and Fishman, 2009 ) A normal horse will show an immediate increase in blood glucose concentration and will return to normal levels within one hour ( Valberg and Fishman, 2009 ) The insulin response curve should parallel the glucose response curve with a peak in insulin at approximately 30 minutes post glucose injection ( Ralston, 2002 ; Valberg and Fishman, 200 9 ) Horses with insulin resistance would potentially produce a higher peak in blood glucose and a consistent delay before returning to a baseline after 2 hours post injection ( Valberg and Fishman, 2009 ) Insulin concentrations must also be measured to determine if the pancreas secretion of insulin is impaired or if insulin stimulated glucose disposal is impaired ( Valberg and Fishman, 2009 ) If the pancreatic cell is impaired there could be a delay in both glucose clearance from circulation and the appearance of insulin ( Valberg and Fishm an, 2009 ) Although the IVGTT overcomes some of the limitations encountered with an OGTT, the IVGTT is still not a sensitive means to measure diminished pancreatic response ( Firshman and Valberg, 2007 ) Insulin tolerance test An insulin tolerance test (ITT) measures blood glucose response to an intravenous injection of insulin The amount of insulin administered to horses has been highly variable, ranging from 0.2 IU/kg to 0.6 IU/kg ( Valberg and Fishman, 2009 ) An ITT test is also used to assess the response of the horse to an insulin induced hypoglycemia ( Valberg and Fishman, 2009 ) Depending on the dose of insulin used for injection, blood glucose levels will drop to 50% of the original value within 20 to 30 minutes of injection, then return to fasting level within 1.5 to 2 hours ( Valberg and Fishman, 2009 ) An ITT test is usually used for an animal that is known to be insulin insensitive ; thus, it has limited use in the screening for insulin resistance. In an insulin resistant horse, blood


34 glucose levels will not fall as dramatically as a healthy horse and will return to basal levels more qui ckly compared to a healthy horse ( Valberg and Fishman, 2009 ) Frequently sampled intravenous glucose tolerance test The frequent sampling intravenous glucose tolerance (FSIGT) test uses the combination of intravenous injections of glucose and insulin to test both pan creatic insulin response to elevated blood glucose and peripheral insulin resistance ( Pratt et al., 2005 ; Treiber et al., 2005a ) With the FSIGT test, horses are not fasted. Blood is drawn prior to administration of glucose and then horses are rapidly administered 300 mg/kg of glucose solution intravenously ( Valberg and Fishman, 2009 ) Blood is then taken via a catheter at 0, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 1 4 16, and 19 minutes afterward ( Valberg and Fishman, 2009 ) Twenty minutes after glucose has been administered a small intravenous dose of insulin (20 mIU/kg) is given and blood is then taken at 22, 23, 24, 25, 27, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, and 180 minutes ( V alberg and Fishman, 2009 ) Glucose and insulin dynamics are evaluated via minimal model analysis described by the following equations: G (t) = G(t) x [Sg + X(t)] + Sg x Gb, where G (t) is the net rate of change in plasma glucose (mg dL 1 min 1 ) ( Treiber et al., 2005a ) Glucose effectiveness (Sg) describes one component of the plasma disposal r ate (min 1 ), which is the capacity of the cells to take up glucose without insulin mediation. The plasma glucose concentration (mg/dL) at time = t is G(t); Gb is the basal glucose concentration (mg/dL), maintained primarily by hepatic production. Insulin a ction ( X(t) ) represents the insulin mediated component of plasma glucose disposal rate via the acceleration of glucose uptake in response to an increment change in the insulin concentration This component is further described by X (t) = p 3 x [I(t) Ib] p 2 x X(t), where X (t) is the rate of change of the insulin action (min 1 ) p 3 describes the delivery of


35 insulin to the interstitium, and p 2 describes the disposal of insulin from the interstitial fluid, possibly reflecting hepatic extraction of plasma in sulin. Insulin sensitivity (SI ) is the ratio of the parameters: SI = p 3 /p 2 (L min 1 1 ) and represents the efficiency of insulin to accelerate glucose uptake by the cells Insulin sensitivity (SI) is estimated from the reciprocal of basal insulin concentration: SI = (7.93(1/( [insulin])) 1.03 ( Treiber et al., 2005b ; Valberg and Fishman, 2009 ) Responsiveness of cells to the glucose load is described by the acute response of insulin to glucose (AIR g ; mIU/[L min]), which is the increase in plasma insulin above basal concentration integrated from 0 to 10 minutes after the glucose dose ( Treiber et al., 2005a ) The product of ARI g (AIR g = (70.1 [MIRG]) 13.8) uses the modified insulin response to glucose (MIRG; calculated as 800 0.30[insulin 50] 2 )/ (glucose 30)) and SI to determine the disposition index (DI) or the app ropriateness of the cell response relative to the degree of insulin resistance in the tissue (DI = AIR g x SI). Unlike the glucose clamp, which is discussed below and depends on steady state conditions, the minimal model approach uses dynamic data. Valid minimal model analysis of the FSIGT requires several assumptions ( Muniyappa et al., 2008 ) Muniyappa and coworkers (2008) d escribe several assumptions to be considered: F irst assumption: instantaneous distribution of the glucose bolus in a monocompartmental space is assumed to occur S econd assumption: glucose disappearance in response to glucose or insulin is assumed to occur at a monoexponential rate Third assumption: glucose concentration at the end of the FSIGT is assumed to be identical to the beginning concentration Fourth assumption: insulin is assumed to act through extravascular (interstitial or extracellular space w here insulin directly exerts metabolic actions) activity to promote glucose disappearance Fifth assumption: the minimal model lumps together effects of insulin to promote glucose disposal in skeletal muscle and suppres sed hepatic glucose production


36 Sixth assumption: to obtain a valid estimate of S 1 the minimal model assumes that total insulin secretion (endogenous plus exogenous) during the FSIGT is above a certain threshold T he FSIGT and the minimal model analysis is generally easier to perform than the glucose clamp method because though both are labor intensive, the FSIGT it is slightly less labor intensive, steady state conditions are not required, and there are no intravenous infusions that require constant adjustment ( Muniyappa et al., 2008 ) In addition, i nformation on insulin sensitivity, glucose effectiveness, and cell function can be derived from a single dynamic test Moreover, the minimal model generates excellent predictions of glucose disappearance during t he FSIGT. A major limitation to the FSIGT/ minimal model approach is that it involves intensive blood sampling over a 3 h period making it labor intensive when compared to other model approaches. Further, the minimal model oversimplifies the physiology of actual glucose homeostasis and it lumps together the effects of insulin to promote peripheral glucose utilization and suppress hepatic glucose production Hype rglycemic and hyperinsulinemic c lamps The glucose clamp technique is a method for quantifying insulin secretion and resistance, which was developed in 1979 ( DeF ronzo et al., 1979 ) The hyperinsulinemic euglycemic clamp involves intravenous infusion of insulin at a constant rate to raise and maintain systemic insulin levels causing a hyperinsulinemic state ( Kim, 2008 ) The hyperglycemic clamp is used to fix plasma glucose at an acutely elevated level for two hours to suppress endogenous hepatic glucose production ( DeFronzo et al., 1979 ) Glucose intravenously infused at variable rates is used to maintain euglycemia. The rate of glucose infusion directly correlates with insulin sensitivity. This is accomplished by an intravenous glucose infusion consisting of two


37 phases: 1) a 15 min priming dose needed to raise the glucose level in plasma and extravascular compartments to the desired plateau and 2) a maintenance dose that is computed at 5 mi n intervals throughout the study ( DeFronzo et al., 1979 ) Perio dic adjustments in the glucose infusion rate made every 5 min are based on the negative feedback principle: if the actual glucose concentration is higher than the goal, the infusion is decreased and vice versa ( DeFronzo et al., 1979 ) In this manner, the glucose infusion rate becomes a measure of pancreatic insulin se cretion, allowing quantification of the pancreatic ( Valberg and Fishman, 2009 ) This procedure is followed by the hyperinsulinemic clamp which provides a supramaximal but steady state level of insulin ( Valberg and Fishman, 2009 ) Insulin is in fused to raise the plasma insulin concentration acutely to a new plateau and maintain it at that level ( DeFronzo et al., 1979 ) The rate of glucose infusion required to maintain euglycemia during the hyperinsulinemic clamp acts as a measure of insulin sensitivity for skeletal muscle and adipose tissues ( Valberg and Fishman, 2009 ) Incorporation of radioactive labeled glucose during a euglycemic clamp can further be used to measure glucose metabolism by individual organs. One advantage of this test is that it is a more sensitive measure of insulin action and can be used to asses s insulin sensitivity in individual organs. Another advantage of this test is that endogenous insulin secretion can be controlled and any potential fluctuations during the test that might alter glucose homeostasis are minimized ( Valberg and Fishman, 2009 ) A disadvantage of clamps is that they are ill suited for large studies because of the extensive requirements for cost, labor, and technical expertise ( Lee et al., 2011 ) Operator induced variability and site specific differences in clamp methods can pose a


38 problem as well. This test tends to require more technical expertise due to the constant infusion and mitigation of hormone levels at specific time s and doses in order to maintain goal plasma concentrations. Nonetheless, the clamping technique remains one of the most accurate means to measure sensitivity of tissues to hyperglycemia and hyperinsulinemia ( Firshman and Valberg, 2007 ) Basal glucose and insulin measurement Single fasting glucose and insulin measurements have be en commonly used by veterinarians to identify horses that are suspected to be insulin resistant ( Kronfeld et al., 2005a ; Treiber et al., 2006c ) However, the accuracy of a single fasting sample has been questioned due to the wide variatio n in both glucose and insulin concentrations noted within an individual horse over a short time period ( Treiber et al., 2005a ; Treiber et al., 2006c ) Proxies and reference quintiles for basal glucose and insu lin Proxies for insulin sensitivity and pancreatic cell responsiveness that are derived from basal glucose and insulin concentrations are commonly used to screen potential diabetic patients. They minimize sampling and stress to the animal and require les s labor and materials, yet are thought to be an improvement above fasting glucose and insulin concentrations for assessing insulin resistance. Treiber et al. (2005a) evaluated insulin sensitivity and responsiveness in a small number of normal and insulin resistant suspect horses and ponies via a FSGIT and compared the outcome to several proxies based on basal values of insulin and glucose that are routinely used in public health. The authors found that the proxies that best represented insulin sensitivity and insulin responsiveness according to the minimal model were the reciprocal of the insulin square root index using basal insulin


39 concentrations of m I U/L (RISQI; calculated as 1/ insulin) and the modified insulin response to glucose using basal glucose concentrations of mg/dL and basal insulin concentrations of mIU/L (MIRG; calculated as 800 0.30[insulin 50] 2 )/ (glucose 30)), respectively. The RISQI estimates the amount of insulin compensation required to chronically maintain glucose homeostasis, w hereas MIRG estimates the capacity of the pancreatic cells to increase insulin secretion and compensate for exogenous glucose. The MIRG capacity is limited by chronic basal insulin secretion, while chronic decompensation is indicated by increasing basal glucose levels. The combined use of RISQI and MIRG have enabled assessment of compensatory insulin secretion in apparently healthy horses and insulin signaling failure in hyperglycemic horses ( Treiber et al., 2005b ) In addition, these proxies have been used to document insulin resistance in ponies and predict the likelihood of developing laminitis ( Treiber et al., 2006c ) It should be noted that proxies are less accurate than the specific quant itative param eters they predict and can have resting blood sample variability as described by Pratt and coworkers (2009). However, they are superior to nonspecific indicators, such as basal hyperinsulinemia, glucose intolerance, or analogies to diseases in other specie s ( Treiber et al., 2005b ) The homeostasis model assessment (HOMA) is a test used to determine the degree to which insulin resistance and deficient cell function can be assessed from a ( Matthews et al., 1985 ) The calculation for HOMA is: (basal glucose (mg/dL) insulin (mU/L))/ 22.5 (Treiber et al., 2005b). The HOMA is used to take advantage of the reciproc al relationship between both basal insulin and basal glucose concentrations and to avoid ambiguity of low


40 insulin concentrations that could indicate high insulin sensitivity or insulin secretion failure ( Matthews et al., 1985 ) High HOMA scores denote low insulin sensitivi ty or insulin resistance ( Bonora et al., 2000 ) In humans, the HOMA index shows a correlation to the euglycemic hyperinsulinemic clamp technique and, to a lesser extent, the hyperglycemic clamp techniq ue ( Treiber et al., 2005b ) However, the estimation of the set of fasting insulin values by HOMA are unlikely to be precise, in part because the range over whic h insulin is measured is small and results depend on precision of the insulin radioimmunoassay ( Matthews et al., 1985 ) Equine pancreatic cells appear particularly resistant and in insulin resistant horses a high basal insulin concentration is usually seen ( Treiber et al., 2005b ) Therefore, the usefulness of HOMA in identifying insulin resistant horses may not be any more accurate than other proxies, such as MIRG and RISQI. Equ ine Metabolic Syndrome The term equine metabolic syndrome (EMS) is a label for horses whose physical examination and laboratory testing suggest a heightened risk for developing laminitis as a result of underl yi ng insulin resistance ( Johnson et al., 2010 ) Equine metabolic syndrome is not considered a disease, but rather a collection of clinical abnormalities that are combined to identify a patient with the likelihood of develop ing laminitis as opposed to individuals lacking EMS characteristics. An examination that shows positive characteristics of EMS is characterized by obesity, regional adiposity, insulin resistance, hypertriglyceridemia, and hyperleptinemia ( Frank, 2009 ) sy it was proposed that obesity, insulin resistance, and laminitis were all a component of a clinical syndrome recognizable in both horses and ponies ( Johnson, 2002 ) Usage of the term


41 EMS was adopted based on its similarities to the metabolic syndrome in humans, which is a collection of risk factors used to predict type 2 diabetes mellitus and the incidence of coronary artery disease ( Frank et al., 2010 ) Equine metabolic syndrome usually develops in horses less than 15 years of age, which differs from PPID that affects horses who are over 15 years of age ( McFarlane, 2011 ) However, both EMS and PPID share the commonality of underlying insulin resistance. Equine Metabolic Syndrome and Obesity Most horses diagnosed with EMS are obese with enlarged adipose tissue within the nuchal ligament of the neck, commonly referred to as a cresty neck ( Frank et al., 2010 ; Tadros and Frank, 2011 ) Fat pads will also develop around the tailhead and within the prepuce or mammary gland area in horses considered to have EMS ( Frank et al., 2010 ; Tadros and Frank, 2011 ) Occasionally, horses that are affected with EMS will also have subcutaneous adipose tissue deposits randomly distributed on their body ( Tadros and Frank, 2011 ) It should be noted that some obese and overweight horses and ponies do have normal insulin sensitivity. Not all EMS affected horses are obese and not all obese horses develop insulin resistance ( Johnson et al., 2010 ) Whether obesity induces insulin resistance or the insulin resistant horse is pred isposed to obesity has not been determined. In humans, the development of type 2 diabetes mellitus is thought to have a higher correlation with mesenteric and omental adipose tissues when compared to fat accumulation elsewhere ( Frank et al., 2010 ) T he fatty acids and adipokines released into portal circulation from these visceral sites have been shown to caus e a more profound effect on hepatic metabolism and insulin clearance. Whether a similar


42 difference exists between adipose tissue from the neck crest or abdomen and fat tissues collected from other locations in the horse is currently under investigation The two primary theories linking insulin resistance and obesity are: 1) the down regulation of insulin signaling pathways induced by adipokines and cytokines produced in adipose tissue; and 2) the accumulation of intracellular lipids in insulin sensitive tissues such as skeletal muscle, which is also known as lipotoxicity ( Frank et al., 2010 ) A typical equine diet is low in fat; however, excess dietary carbohydrate can be converted to fat via de novo lipogenesis ( Frank et al., 2010 ) Fats are stored in adipose tissue cells as triacylglycerol and are used for energy. Once the storage of the cells have reached capacity, fats are repartitioned towards nonadipose tissues su ch as skeletal muscle, liver, and pancreas. These tissues will attempt to utilize the fats by increasing oxidation. Consequently, if too much lipid accumulates in these tissues it alters normal cellular functions, including insulin signaling. Equine Meta bolic Syndrome and Laminitis With EMS usually comes the onset of some form of laminitis. Laminitis literally means the inflammation of the laminae in the hoof, the interdigitating plates that lock the hoof wall to the third phalangeal bone ( Kronfeld et al., 2006 ) The lamellar structure confers weight carrying strength through a combination of rigidity and resilience. In horses with EMS, laminitis appears to occur spontaneously without a history of a bacterial disease, retained placenta, or grain overload ( Tadros and Frank, 2011 ) Insulin resistance represents a risk factor for laminitis; consequently the development of laminitis is used to support the diagnosis of EMS in horses ( Johnson et al., 2010 ) Although the physical or radiographic appearance of the hoof indicates laminitis in an EMS horse, the owner may report that lameness or pain has not been


43 evident. Therefore, structural changes in the hoof lamellar interface may occur in the absence of laminitic pain in EMS horses. The insulin resistance component of EMS most likely predisposes a horse to laminitis, because insulin possesses vasoregulatory properties ( Frank, 2009 ) Activation of the insulin receptor stimulates t wo different signaling pathways within the vascular endothelial cell. One of the pathways is mediated by phosphatidylinositol 3 kinase (PI3K), which promotes vasodilation, whereas the second pathway is the mitogen activated protein kinase (MAPK) pathway, w hich promotes vasoconstriction ( Frank, 2009 ) These two pathways are critical in the regulation of blood flow to the hoof, especially the sensitive laminae. The vasodilatory effects of insulin and insulin dependent stimulation of glucose uptake are both mediated by the PI3K pathwa y, which becomes disrupted when insulin resistance develops ( Tadros and Frank, 2011 ) Consequently, vasoconstriction is then promoted in an insulin resistant animal because the MAPK pathway still remains fully functional. PI3K is one of the most important regulatory proteins involved in different signaling pathways and controlling key functions of the cell ( Krasilnikov, 2000 ) The PI3K pathway leads to the phosphorylation and activation of phosphoinositide dependent kinase 1 (PDK 1), which in turn phosphorylates and activates Akt (also known as protein kinase B) ( Kim et al., 2006 ) The Akt will directly phosphorylate endothelial nitric oxide (NO) synthase (eNOS), which results in increased eNOS activity and NO production for vasodilation that regulates vascular tone ( Iwakiri et al., 2002 ; Kim et al., 2006 ) Although insulin is a key regulator to induce the PI3K pathway, various cytokines and growth factors have also been shown to induce the PI3K and subsequent


44 phosphorylation of eNOS ( Iwakiri et al., 2002 ) Because the PI3K pathway is the one that stimulates glucose uptake, this pathway is likely to be compromised in an animal that is insulin resistant ( Frank, 2009 ) The MAPK pathw ay is the second pathway of insulin signaling that generally regulates growth and mitogenesis and controls the secretion of endothelin 1 (ET 1) in the vascular endothelium which leads to vasoconstriction ( Kim et al., 2006 ) Insulin resistance is associated with hyperinsulinemia that has been linked to the activation of the endot helin system ( Yang and Li, 2008 ) Insulin induced elevation of ET 1 contributes to abnormal cell growth, atherosclerosis and hypertension in insulin resistant humans. In 2007 an inc rease in plasma ET 1 concentration was detected in blood collected from the digital veins 12 hours after carbohydrate was administered to induce laminitis in healthy horses ( Eades et al ., 2007 ) These findings suggest that digital vessels undergo vasoconstriction as a result of carbohydrate overload in horses, which may contribute to the development of laminitis. Vasoconstriction is already promoted in horses with chronic insulin resi stance and may be more likely to develop laminitis ( Tadros and Frank, 2011 ) Hyperinsulinemia and Vascular D ynamics Laminitis can be induced by prolonged hyperinsulinemia in healthy young, lean ponies, independent of changes in blood glucose concentration of insulin sensitivity ( Asplin et al., 2007 ; Nourian et al., 2009 ) This evidence suggests that the laminae of the hooves have a sensitivity to the effects of elevated glucose or insulin concentrations. In horses, hyperinsulinemia is defined by a serum/plasma insulin concentration of greater than 20 / mL ( U/mL) ( Frank et al., 2010 ; Tadros and Frank, 2011 ) Experimentally induced laminitis has been performed in healthy ponies and


45 Standardbred horses by inducing hyperinsulinemia ( Asplin et al., 2007 ; De Laat et al., 2010 ) In both studies, glucose and insulin were infused intravenously according to the euglycemic hyperinsulinemic clamp procedure, with mean serum insulin concentrations exceeding 1000 U/mL. Mean time to onset of Obel grade 2 laminitis was 46 h in horses and 55 h in ponies ( Asplin et al., 2007 ; De Laat et al., 2010 ) A n increase in the temperature of the hoof surface in response to the insulin infusion was noted, suggesting that hyperinsulinemia actually induces laminitis through a mechanism involving vasodilation. Hyperinsulinemia induced vasodilation would then overcome the MAPK driven vasoconstriction, which is promoted by insulin resistance ( Tadros and Frank, 2011 ) Also proposed is that the hyperinsulinemia in duced vasodilation causes an increase in glucose delivery to the hoof tissues, consequently causing local glucotoxicity ( De Laat et al., 2010 ) Management of Equine Metabolic S yndrome and Insulin R esistance Key strategies for managing insulin resistance and EMS may be differ based on the presence or absence of la minitis However, whether laminitis has been noticed or not promoting weight loss in obese or overweight horses and improving insulin sensitivity through dietary management and exercise should be taken seriously ( Tadros and Frank, 2011 ) Horses and ponies that are affected with EMS seem to be pa rticularly sensitive to ingested non structural carbohydrates (NSC) ( Johnson et al., 2010 ) Diet Diets that are high in NSC such as a high grain diet or ad libitum access to very lush pasture tend to exacerbate insulin resistance because the diet further stimulates the production of insulin ( Frank, 2008 ) In fact, the term pasture associated laminitis is becoming more common and is given to horses that develop laminitis whilst grazing


46 pasture without any other known concurrent disease or pro blem to have caused the onset of laminitis. Grazing lush pasture is the most common cause for laminitis recognized by practicing veterinarians ( Johnson et al., 2010 ) Exposure to pasture forage high in NSC (15 to 20% DM) exacerbated hyperinsulinemia in insulin resistant ponies and this diet associated alteration in insulin dynamics may contribute to increased risk of laminitis ( Treiber et al., 2005b ; Bailey et al., 2008 ) The total carbohydrate content of pasture grass is characterized by the carbohydrates that constitute the cell wall structure of plant cells (structural carbohydrates or fiber, such as cellulose and hemicellulose), which are indigestible by mammalian enzymes ( Johnson et al., 2010 ) The NSC (starch es soluble sugar s and fructans) in pasture grass can be high, and when ingested can cause both glycemic and insulinemic spiking, which seems to be associated with aggravated laminit is. It is also possible that certain types of fructans undigested by the small intestine may cause perturbations in hindgut microflora leading to colonic acidulation, increased epithelial permeability, and the absorption of other laminitis triggers such as endotoxins and vasoactive amines. The horse with EMS is similar to a person with diabetes, so excessive sugar should be avoided as it may exacerbate insulin resistance. Due to the quantity consumed, p asture grass is usually one of the largest sources of s ugar in the diet of a horse ( Frank, 2008 ) The carbohydrate content of a past ure varies between regions and depends on soil type, climate, hours of sunlight, and grass species ( Frank, 2008 ) Therefore, horses with EMS and/or insulin resistance should be restricted from pasture, depending on the severity of their condition.


47 A forage based diet, with hay that has a NSC content of less than 10% (on a dry matt er (DM) basis) has been recommended for an insulin resistant horse ( Tadros and Frank, 2011 ) Pratt and coworkers (2006) found that physical conditioning lessened the impact of a high NSC diet on insulin sensitivity. In addition to NSC, intake of water soluble carbohydrates (WSC), which comprise t he simple sugars (glucose, fructose and sucrose) and more complex oligo or polymeric fructans need to be restricted ( Longland et al., 2011 ) It is believed that WSC constituents promote the development of laminitis through potentiation rapid fermentation of the fructan fraction. It has been suggested that diets for horses prone to insulin resistance or laminitis should contain diets low in WSC with approximately 100 g WSC/kg DM as a maximum for such animals. Longland and coworkers (2011) demonstrated the variability of different hays when soaked for 0, 20, and 40 min and 3 and 16 h in water. Results from this this study concluded that soaking of hay is not a reliable way to lower the water soluble carbohydrate (WSC) content as previously thought. Overweight and obese horses should be placed on a weight management diet composed of hay at 1.5% BW (e.g., 7.5 kg for a 500 kg horse) and a protein/vitamin/mineral supplement ( Tadros and Frank, 2011 ) B ody condition score (BCS) should be assessed and recorded. If the horse does not begin to lose weight after one month on this diet, the amount of hay fed should be low ered to 1% of kg horse). A reduced meal should be fed until an ideal BCS of 5 or 6 is reached. Any type of grain should be eliminated from the diet and access to pasture should be eliminated or strictly limited, o r horses may be equipped with a grazing muzzle while the horse is trying to lose weight ( Tadros and Frank, 2011 )


48 There has been very limited exploration of the effects of insulin secretion on weight loss response to hypocaloric diets in humans ( Pittas and Roberts, 2006 ) or horses. Weight loss is historically difficult to achieve and maintain in humans and can also be difficult in horses. There is animal and human evidence that suggests that the dynamics of insulin sensitivity and secretion play a role i n body weight regulation ( Pittas and Roberts, 2006 ) Therefore, the parameters may affect individual responses to hypocaloric or low calorie diets. Moreover, specific dietary factors that influence these parameter s may interact with subject specific characteristics of insulin dynamics to influence the effect of the hypocaloric diet with varied macronutrient composition on weight loss and maintenance. Initial evidence for an important role of insulin secretion in e nergy balance and weight loss comes from pharmacologic studies in which insulin secretion was assessed ( Pittas and Roberts, 2006 ) In one study obese participants received a hypocaloric diet and then randomized to either placebo or diazoxide, a K + (ATP) channel agonist that decreased the secretion of insulin and is used in the medical management of insulinomas ( Alemzadeh et al., 1998 ) Compared with the placebo group, the diazoxide group lost more weight (4.6 vs. 9.5 kg, respectively) while on the hypocaloric diet. This evidence suggests an important role for insulin secretion in modifying weight loss in response to caloric restriction. In a mor e recent pharmacologic study insulin secretion was suppressed in obese individuals by octreotide LAR, a somatostatin analog used in various endocrine hypersecretory con ditions ( Velasquez Mieyer e t al., 2003 ) For the entire cohort, significant insulin suppression was achieved with


49 accompanied weight loss and decreased self reported carbohydrate craving, without a concomitant lifestyle intervention. In a post hoc analysis, participants who lost the most weight exhibited the highest suppression of pancreatic cell activity and the highest reduction in carbohydrate cravings and intake. In summary, the contribution of insulin secretion to weight and response to hypocaloric diets, supplements, inclu ding those that vary in macronutrient composition, is still controversial. As an example, a current study examined insulin resistance in unrelated ponies where the variation between their insulin sensitivity was reduced by feeding a diet moderately high in NSC diet with increased amounts of free glucose for 32 days ( Tinworth et al., 2011 ) These ponies remained clinically health y with no significant changes in body weight and laminitic effects were not found, suggesting possible adaptation or different tolerance thresholds to high NSC diets my vary between horses. The effects are also diff icult to isolate from insulin resistance. Exercise Weight management alone may not be effective enough to improve insulin resistance. Weight management combined with exercise is a healthier and more effective option to reduce weight on a horse. Long term e xercise programs have been shown to improve insulin sensitivity and reduce fasting and glucose stimulated plasma insulin levels in obese humans ( Brown et al., 1997 ) However, intensive training is not required to improve insulin sensitivity. Studies have shown that low intensity exercise can improve insulin sensitivity and increase glucose uptake into the muscle in obese horses even in the absence of weight loss ( Powell et al., 2002 ) In one study, 31 stabled horses underwent three different exercise regimes: turnout, light exercise, and moderate exercise, while being fed a diet


50 containing 60% concentrate ( Turner et al., 2011 ) Blood was s ampled monthly and analyzed for insulin. Insulin sensitivity was assessed and compared across months ; the study conclud ing that insulin was high er during periods of moderate and light physical activity when compared with turnout. Moderate exercise intensit y performed 5 days a week seemed to benefit insulin sensitivity, even in insulin sensitive horses in this study. R esults indicate that turnout alone may not be adequate to improve insulin sensitivity in horses fed high amounts of concentrates. A program t o increase physical activity should be implemented to promote weight loss, which has been shown to be effective therapeutic intervention to increase insulin sensitivity in obese insulin resistant people ( Frank et al., 2010 ) It has been shown that exercise increases glucose uptake from the plasma with and without insulin mediation ( Treiber et al., 2006a ) In addition, exercise decreased insulin secretion, which could promote mobilization and utilization of fatty acids as an additional energy source in order to induce weight loss. Hyperinsulinemic obese ponies subject to training had improved insulin sensitivity after only 2 weeks ( Ralston, 2002 ) Another study looked at age related training on insulin sensitivity as insulin sensitivity declines with age in horses ( Malinowski et al., 2002 ) and found that exercise improves pancreatic cell function and insulin sensitivity in old and young ho rses ( Liburt et al., 2011 ) It has been suggested that an exercise regime should include at least 200 minutes of moderate intensity exercise per week ( Frank et al., 2010 ) The exercise can start with 2 to 3 exercise sessions per week of either riding or longeing for at least 20 to 30 minutes per session with a gradual increase in intensity and duration building t o 5 sessions per week. It is thought that exercise improves insulin sensitivity by 3


51 mechanisms: 1) reduced muscle and liver glycogen; 2) increased binding of insulin to insulin receptors; and 3) increased glucose uptake by muscle cell glucose transporters ( Sternlicht et al., 1989 ; Powell et al., 2002 ) Pharmacologic i ntervention Medical management can also be used through pharmaceutical products. Weight loss can be induced and insulin sensitivity improved through the administration of levothyroxine sodium ( Frank et al., 2010 ) Metformin is a drug used in human medicine to increase insulin sensitivity as it reduces glucose concentrations by improving hepatic and peripheral tissue insulin sensitivity without affecting insulin secretion ( Dunn and Peters, 1995 ) H yperinsulinemic horses have had positive responses to metformin ( Frank et al., 2010 ) Dietary Supplements Dietary supplementation may improve ins ulin sensitivity instead of having to control the health problem with a drug; however, there is a lack of scientific proof establishing significance of supplementation improvement of EMS or insulin resistance. Common supplements containing leucine, cinnamo n extract or fish oil are believed to improve insulin resistance and EMS. However, studies have shown that leucine does not affect plasma glucose or insulin ( Etz et al., 2011 ) and neither cinnamon extract nor fish oil supplementation affected insulin sensitivity of mares of known reduced insulin sensitivity ( Earl et al., 2011 ) If supplements are proven in humans or rats to work to improve insulin sensitivity, such as cinnamon extract ( Ander son, 2008 ) does not mean that this crosses over and works in the horse as well. A recent study found that supplement ation with short chain fructo oligosaccharides had no effect on plasma glucose, but moderately improved insulin sensitivity in


52 overweigh t Arabian geldings ( Respondek et al., 2010 ) P syllium supplementation affected glycemic and insulinemic responses as it resulted in lower mean postprandial blood glucose and insulin concentrations ( Moreaux et al., 2011 ) Ps y llium fed daily for 60 d altered post prandial glycemia and insulinemia in normal, nonobese, and unexercised horses According to studies performed in humans, the mechanism of action of psyllium is related to its ability to increase the viscosity of digest a, which results in altered transit time and absorption of nutrients ( Sierra et al., 2001 2002 ) A study in endurance horses supplemented with p syllium concluded that the psyllium lowered pack cell volume and the authors hypothesized this was due to the availability of sequestered water in the gastrointestinal tract ( Cinotti et al., 1997 ) The water retaining capacity of feeds and potentially the rate of gastric emptying is related to the ratio of insoluble to soluble fiber entering the stoma ch of mammals ( Bach Knudsen, 2001 ) The v i s cosity and passage rate of digesta throu gh the small intestine of human s and swine is affected by soluble dietary fiber. It has been suggested that the viscosity and rate of digesta flow through the small intestine of the horse alter uptake of nutrients ( Ellis and Hill, 2005 ) Small intes tinal luminal pH also affects the active transport of nutrients across the intestinal mucosa ( Hoffman, 2003 ; Ellis and Hill, 2005 ) which is suggested to contribute to altered tr ansit time of psyllium Arginine arginine was needed to create urea, a waste product produced when toxic ammonia is removed from the body ( Mayo Clinic, 2010 ) Arginine is known by several names such as (S) 2 Amino 5 arginine with a molecular formula of C 6 H 14 N 4 O 2


53 Arginine is a basic amino acid and one of the 20 most common, naturally occurring amino acids in nature. Arginine is also considered among the 10 essential amino acids for the horse and other mammals ( NRC, 2007 ; Wu et al., 2009 ) Arginine is a component of dietary protein and body fluids ( Wu et al., 2009 ) In animals and humans, the amino acid fulfills versatile physiological functions that are critical to normal regulatory properties. The requirements of arginine in most mammal ian species are frequently met through either dietary intake or endogenous synthesis ( Deusdelia et al., 2002 ) In the Based on nitrogen balan ce or growth and the functional needs beyond tissue protein synthesis, arginine is nutritionally essential for: 1) development of the mammalian conceptus; 2) neonates; 3) birds, cats, ferrets, and fish; and 4) adults under certain conditions, such as intes tinal resection or dysfunction, burns, or renal dysfunction associated with NO deficiency ( Wu et al., 2009 ) In comparison to other species, there has been no research on arginine requirements in nonruminant herbivores, such as the horse. Dietary Arginine Supply Several foods are naturally rich in arginine, including seafood, water melon juice, nuts and seeds, algae, meats, rice protein concentrate, and soy protein ( Wu et al., 2009 ) In feedstuffs provided to livestock and horses, those with a higher protein content will generally have a higher amount of arginine. For example, alfalfa has a higher percentage of arginine (0.77% of DM) compared to a warm season grass such as bermudagrass (0.04%) ( NRC, 2007 ) Grains such as oats and corn have approximately


54 0.85% and 0.17% arginine, respectively. Within common oilseed meals, cottonseed meal has by far the most arginine ( 5.01 %) whereas canola meal has the least (2.42%). Arginine tends to be low in the milk of most mammals including cows, humans, and pigs ( Wu et al., 2009 ) However, stage of lactation may influence milk arginine content. In a comparative study, arginine was 38% lower in milk than colostrum in humans, but 29% higher in milk than colostrum in the horse ( Davis et al., 1994 ) Compared to milk from the cow and sheep (34 mg /L ), llama ( 36 mg /L ), pig (44 mg /L ) or the elephant (48 mg /L ), arginine seems to be the hi ghest in milk from the horse (60 mg /L ) and the cat (64 mg /L ). This suggests that neonates of some species may have a higher requirement for arginine than others, perhaps due to reduced or delayed ability to synthesize arginine endogenously. Role of Arginin e in the Body Arginine is one of the most versatile amino acids as it serves as a precursor for the synthesis of protein, NO, urea, polyamines, proline, glutamate, creatine, and agmatine ( Guoyao and Morris, 1998 ) Furthermore, arginine has been shown to stimulate the secretion of different hormones, such as insulin, growth hormone, glucagon and prolactin. Thus, arginine availability might affect the hom eostasis of different biochemical pathways in the horse and other mammals due to its important role in the body. Arginine has several metabolic functions including involvement in the transport, processing and excretion of nitrogen, and urea synthesis in the liver ( McConell, 2007 ) Arginine is critical to the detoxification of ammonia, which is highly toxic to the central nervous system ( Wu et al., 2009 ) In addition, experimental and clinical trials have produced evidence that arginine regulates interorgan metabolism of energy substrates


55 and t he function of multiple organs, and is nutritionally essential for spermatogenesis, embryonic survival, fetal and neonatal growth, as well as for the maintenance of vascular tone and hemodynamics. More recent studies provide evidence that dietary supplemen tation or intravenous administration of arginine improves reproductive, cardiovascular, pulmonary, renal, gastrointestinal, liver, and immune functions, as well as facilitates wound healing, enhances insulin sensitivity, and maintains tissue integrity. Ar ginine Metabolism A prominent feature of arginine metabolism is the complex differential expression of relevant enzymes within the major organs ( Wiesinger, 2001 ) In essence, t here is not a single organ or cell type that expresses all the enzy mes in volved in arginine metabolism ; thus, many of the various aspects of arginine synthesis and degradation occur in site specific tissues Subcellular compartmentalization and expression of various isoenzymes further complicates arginine metabolism. Specific aspects of arginine metabolism are discussed in the following sections. It is worth noting that most studies on arginine metabolism have been conducted in humans, pigs, sheep, and rodents While arginine metabolism is presumably similar in horses, further research is needed in this species. Endogenous Synthesis of Arginine In mature animals, the synthesis of endogenous arginine involves the intestinal renal axis ( Wu and Morris, 1998 ) (Figure 2 1A) The process begins in the small intestine, with the conver sion of glutamine, glutamate and proline into citrulline in the mitochondria of enterocytes ( Wu et al., 2009 ) Citrulline is then released from the small intestine into circulation and taken up primarily by the kidneys. Within the kidneys,


56 citrulline is converted into arginine and the arginine is released back into circulation ( Guoyao and Morris, 1998 ; Sidney, 2004 ) Biosynthesis of arginine differs between mature animals and the neonate ( Figure 2 1 B) G lutamine serves as the main precursor for citrulline formation in the mitochondria of enterocytes in adults Adults r elease citrulline into the circulation so it can be taken up by the kidney to be converted to arginine, which is different from the neonate where citrulline is converted into arginine locally in the cytosol of the enterocyte s ( Wu et al., 2007b ) Studies in neonat al rats have shown that the enzymes responsible for metabolizing citrulline to arginine are located only in the upper part of the small intestinal villi, whereas the enzymes involved in the synthesis of citrulline from glutamine are concentrated within the crypts of the intestine ( Marini et al., 2010a ) The absence of arginase (an arginine degrading enzyme) in neonatal enterocytes allows maximal output of arginine from the small intestine into the portal circulation, as was demonstrated in pre weaning pigs ( Wu et al., 2007b ) These meta bolic strategies are thought to help maximize the supply of arginine from the mother to fetus and from maternal milk. Independent of age the small intestine is essential for arginine uptake and/or biosynthesis. C itrulline derived from glutamine, glutamat e and proline in the gut is equally effective as dietary arginine as a source of arginine for the body ( Wu et al., 2007b ) pyrroline 5 carboxylate synthase (P5CS), which is a nexus between the tricarboxylic acid and urea cycles ( Bertolo and Burrin., 2008 ) Because of these pathways, both amino acid s serve as dietary precursors for arginine and urea synthesis. The metabolic


57 pathway for arginine synthesis in mammals is via P5CS and proline oxidase ( Wu and Morris, 1998 ) Some of the enzymes in this pathway are present in a variety of cell types, while others are highly restricted. Phosphate dependent glutaminase ornithine aminotransferase (OAT), argininosuccinate synthase (ASS), argininosuccinate lyase (ASL) and aspartate aminotransferase are widely distributed in animal tissues. Whereas, carbamoyl phosphate synt hase 1 (CPS 1), ornithine carbamoyltransferase (OCT), and N acetylglutamate synthase (NAGS) are restricted to the liver and intestinal mucosa. Proline oxidase is mainly in the small intestine, liver, and kidneys, but PCS is located almost exclusively in th e intestinal mucosa, with only trace amounts in other tissues. Enteric synthesis of citrulline from the amino acid precursors is catalyzed by P5CS and NAGS in the small intestine ( Wu et al., 2009 ) Proline oxidase and OAT allow proline to serve as a precursor for citrulline synthesis in the intestine ( Bertolo and Burrin., 2008 ) As a result of citrulline synthesis in the intestine, citrulline has been used as a biomarker of intestinal failu re in infants and adults ( Rhoads et al., 2005 ; Crenn et al., 2008 ) Interestingly, chickens, cats, and ferrets cannot produce citrulline from glutamine and glutamate due to a lack of P5 C synthase in enterocytes ( Wu and Morris, 1998 ) .The enzyme NAGS is important in regulating citrulline production in enterocytes as it is an allosteric activator of both P5CS and CSP 1 ( Wu et al., 2004 ) Thus, when dietary levels of arginine are high, intestinal syn thesis of citrulline from glutamine and glutamate may be inhibited, thereby sparing these amino acids for use in other metabolic pathways ( Wu et al., 2009 ) Synthesis of arginine in the liver depends on the presence of OCT, which together with CPS 1 is located in the mitochondrial matrix of the liver ( Wiesinger, 2001 ; Wraith,


58 2001 ) However, expression of both OCT and CPS 1 enzymes are restricted to the periportal hepatocytes in liver, the epithelial cells in the mucosa of the small intestine and to a smaller extent, the colonocytes of the large intestine ( Wiesinger, 2001 ) Citrulline released from the small intestine has not been shown to be extracted by the liver to any great extent and is instead utilized for arginine synthesis by extrahepatic tissues ( Wu et al., 2007b ) Curis and coworkers (2005) explain that the main rea son for citrulline metabolism split between the intestine and kidney is related to the efficacy of the capture of arginine by the liver. Without metabolic adaptation, all of the arginine from dietary supply would be withdrawn from the portal blood by the l iver, leaving very low amounts of available arginine for other organs. Curis and coworkers (2005) further explain that as arginine is a positive regulator of ureagenesis, other amino acids could be inappropriately degraded Citrulline is considered s solution to this problem as it acts as a masked form of arginine that essentially bypasses the liver. Citrulline is then converted to arginine by the kidney and released into the blood to make it available for the whole body. It was found that nearly 10 0% of the citrulline and 90% of the arginine derived from the gut, bypass the liver in pigs (Wu et al., 2007c). Citrulline is readily converted into arginine in nearly all cell types, including adipocytes, endothelial cells, enterocytes, macrophages, neuro ns, and myocytes ( Wu and Morris, 1998 ) The kidney has perhaps the highest rate of arginine synthesis from endogenous and exogenous citrulline compared to other tissues. Studies with macrophages and endothelial cells demonstrated that citrulline is transported into the cells by the N system, which is selective for amino acids with a side chain amide group (e.g., glutamine and


59 asparagine) ( Wu et al., 2009 ) Once inside the cell, the only pathway for citrulline utilization is through the conversion of citrulline to arginine via ASS and ASL Arginine and the Urea Cycle disposal of surplus nitrogen, thus providing a means of detoxification of ammonia ( Wiesinger, 2001 ) (Figure 2 2) The liver is the main organ that has a full complement of enzymes necessary to convert ammonia and aspartate to urea ( Walker, 2009 ) ; however, there is evidence for a low level of urea cycle activity in enterocytes of post weaning pigs ( Wu, 1995 ) The substrates in the urea cycle are ammonia, bicarbonat e and aspartate ( Walker, 2009 ) With each turn of the cycle, two atoms of nitrogen (N) are converted to urea. N acetylglutamate is synthesized from glutamate and acetyl Co enzyme A by the hepatic mitochondrial enzyme NAGS ( Mew and Caldovic, 2011 ) Carbamyl phosphate from NH 4 + ATP, and bicarbonate help to form CPS 1 in the mitochondrial matrix ( Jackson et al., 1986 ) The initial step in the urea cycle is localized in the mitochondria where CPS 1 catalyzes a reaction with NAGS as an allosteric activator ( Morris, 2002 ) The next step in the mitochondrial matrix is when OCT catalyzes the synthesis of citrulline from ornithine and carbamoyl phosphate, and then citrulline is shuttled into the cytosol ( Jack son et al., 1986 ) The mitochondrial aspartate glutamate carrier (AGC1; citrin) is an important isoform that catalyzes an exchange between intra mitochondrial aspartate and cytosolic glutamate ( Napolioni et al., 2011 ) The AGC1 is exclusively mitochondrial ( Ramoz et al., 2004 ) and is a component of the malate aspartate shuttle transferring glutamate and aspartate between the cytosol and mitochondrial matrix in order to proceed with the urea cycle ( Napolioni et al., 2011 ) Next ASS catalyzes the


60 condensation of aspartate and citrulline in th e cytoplasm to form argininosuccinate with the concomitant hydrolysis of ATP to AMP and PP i ( Jackson et al., 1986 ) This step in the cytoplasm is followed by argininosuccinate being converted to arginine and fumaric acid by the enzyme AS L. Also in the cytoplasm, the next step is when arginase catalyzes the cleavage of arginine to ornithine and urea. Ornithine is then transferred back into the mitochondria via the mitochondrial ornithine transporter, which has specificity of only the for ms of ornithine ( Monn et al., 2012 ) Ornithine is the n used again to condense with O C T to form citrulline. There is a common misconception that arginine is formed in the liver and released into circulation. Although arginine can be synthesized in the liver, there is no net synthesis of arginine via the hepatic urea cycle ( Wu and Morris, 1998 ) This is because the liver contains high levels of the enzyme arginase, which hydrolyzes arginine into urea and or nithine. It is the input of ornithine, the ability to convert ornithine into citrulline, and the catabolism of arginine (mainly by arginase and NOS) that determine the role of an organ or cell as an arginine producer or consumer ( Wiesinger, 2001 ) Arginine Degradation Arginine uptake by cells involves the system y + (a high affinity, Na + independent transporter) and Na dependent transporters in a cell specific manner ( Wu et al., 2009 ) Once arginine is inside the cell there are multiple pathways for arginine degradation to produce NO, ornithine, urea, polyamines, glutamine, creatine, and/or agmatine ( Wu and Morris, 1998 ) Many of these byproducts have biological importance. Arginine degradation is initiated by either arginase I or II the three isoforms of NOS, arginine:glycine amidinotransferase, and arginine decarboxylase ( Wu et al., 2009 ) There are two distinct isoforms of mammalian arginase (arginase I and II), which are


61 encoded by different genes and differ in molecular and immunological properties, tissue distribution, subcellular location, and reg ulation of expression ( Wu and Morris, 1998 ) Arginase I (a cytosolic enzyme) is highly expressed in the liver and to a much lesser extent in a few other cell types, whereas expression of arginase II (a mitochondrial enzyme) is widespread ( Wu et al., 1997 ; Morris, 2002 ) Arginine turns over rapidly in mammals, with a half life i n circulation of 1.06, 0.75, and 0.65 h for adult, pregnant, and neonatal pigs, respectively ( Wu et al., 2007a ) In short, arginine degradation in mammals involves multiple organs and complex compartmentalization at cellular and systemic levels ( Wu and Morris, 1998 ) The understanding of arginine catabolism is still limited due to its complexity. Regulation of Arginine Metabolism Arginine metabolism is regulated by multiple factors that include nutrients such as lysine, manganese, and omega 3 fatty acids; hormones such as glucocorticoi ds, growth hormone, leptin; cytokines; endotoxins; and endogenously generated substances such as creatine, lactate ornithine, P5CS, and methylarginines ( Wu et al., 2009 ) Lysine competes with arginine for entry into cells and also inhibits arginase activity ( Wu and Morris, 1998 ) Lysine was also shown to reduce insulin stimulated NO producti on by inhibiting arginine transport ( Kohlhaas et al., 2011 ) Therefore, the dietary arginine:lysine ratio is a critical factor to cons ider when supplementing with arginine ( Wu et al., 2009 ) Based on data in pigs the total amount of arginine in the diet should not exceed 150% than that of lysine ( Wu et al., 2009 ) Glucocorticoids play a major role in upregulating arginine degradation via the arginase pathway in many cell types, particularly hepatocytes and enterocytes ( Wu et al., 2009 ) These hormones also inhibit NO generation by suppressing NOS expression.


62 Cytokines such as interleukin 4 and interferon nflammatory stimuli such as lipopolysaccharides, and cyclic adenosine monophosphate (cAMP) can greatly stimulate the expression of arginase I, arginase II, and ornithine decarboxylase in many cell types. As a result concentrations of arginine in plasma ar e reduced markedly in response to infection or inflammation. Castillo and colleges (1993) studied the kinetics of arginine metabolism in healthy adult young men and established that arginine homeostasis is achieved by coupling the net rate of arginine degr adation to the intake of arginine. Deusdelia and colleagues (2002) reported that whole body arginine catabolism is decreased in arginine deprived adult mice. Inadequate intake of dietary arginine has been associated with several health issues, such as impa ired spermatogenesis, alterations in the urea cycle causing orotic aciduria and transitory hyperammonemia, and reductions in tissue and circulating levels of arginine and ornithine ( Castillo et al., 1993 ; Deusdelia et al., 2002 ) Citrulline Citrulline is a nonprotein amino acid, and therefore is not commonly found in food ( Curtis et al., 2007 ) As a result there is little to no citrulline present in the diet of most mammals, with the exception of watermelon. In fact, citrulline received its name from the Latin word for watermelon, Citrullus vulgaris which contains large amounts of this amino acid ( Curis et al., 2005 ) Interestingly, red flesh watermelons have slightly less citrulline than yellow or orange flesh watermelons (7.4, 28.5, and 14.2 mg/g DM respectively) ( Rimando and Perkins Veazie, 2005 ) In addition, t he rind contained more citrulline than flesh of the watermelon (24.6 and 16.7 mg/g DM respectively) ( Rimando and Perkins Veazie, 2005 ) It has been proven that plasma concentration of arginine can be increased through intake of citrulline from watermelon (780 g watermelon juice


63 per d for 3 wk) ( Collins et al., 2007 ) Remarkably, citrulline is one of the most potent scavengers of hydroxyl radical, and the watermelon accumulates citrulline simply as the plant has no other way to allow the spec ific decomposition of the free radical ( Moinard and Cynober, 2007 ) Therefore, unless watermelon is a part of the diet, citrulline found in the body is almost entirely of endogenous origin ( Marini et al., 2010a ) Role of Citrulline in the Body Citrulline primary role is as precursor for endogenous arginine synthesis in the small intestine partic ularly as part of the urea cycle ( Curis et al., 2005 ) Pioneering studies have shown that citrulline is the end product of intestinal glutamine metabolism, and accounts for 27.6% of the metabolized glutamine nitrogen ( Windmueller an d Spaeth, 1981 ) Citrulline used in a very important reaction with aspartate as nucleophilic compound; the reaction produces argininosuccinate and constitutes a step in the urea cycle ( Curis et al., 2005 ) Citrulline can form peptide bonds; hence it can therefor e be present in proteins. However, there is no known codon in the genetic table for this amino acid and thus must result from post transcriptional modification of the protein. Furthermore, the supplementation of citrulline can lead to a dramatic improvemen t of nitrogen balance and protein status ( Moinard and Cynober, 2007 ) role in the body, it has been studied for its potential health benefits. Citrulline at 3 g/kg d has been shown to accelerate the clearance of ammonium and lactate from plasma, possibly contributing to the improvement of muscle function during exercise ( Giannesini et al., 2011 ) Additionally, citrulline supplementation (6 g/d) in young normotensive men resulted in reduced brachial a nd aortic blood pressure ( Figueroa et al., 2010 ) C itrulline was also fed to malnourished rats (5 g/kg d) as a means to increase whole body nitrogen status after dietary restriction and was


64 reported to increase protein synthesis and protein content in skeletal muscle ( Osowska et al., 2006 ) Citrulline Biosynthesis Citrulline was originally thought to be mainly produced from glutamine in the small intestine. More recent research in mice and other species has indicated that arginine and ornithine likely serve as the main precursors for citrulline synthesis in the small intestine ( Marini et al., 2010b ; Marini, 2012 ) Marini and coworkers (2010a) concluded that dietary glutamine is a poor carbon skeleton precursor for the synthesis of citrulline ; h owever, this is still under investigation. Additional sources of endogenous c itrulline include turnover of citrllinated proteins and the action of NOS on arginine and dimethylarginine dimethlaminohydrolase on dimethylarginine. The metabolism of free citrulline can be classified into three pathways ( Curis et al., 2005 ) The first metabolic pathway is arginine biosynthesis, which involves citrulline exchange at the whole body level. The second pathway is the NO cycle, which can involve local recycling of citrulline. The third pathway is the complete urea cycle, taking place in the liver. The use of citrulline for arginine biosynthesis was discussed previously in the section on arginine metabolism. The other metabolic fates of citrulline are discussed in more detail below. Citrulline is produced when arginine is acted upon by any of the NOS enz ymes, a reaction that occurs in all tissues. Within the NOS family, there are three different enzymes that differ in their level of expression within different tissues ( Curis et al., 2005 ) The naming of the NOS enzymes is based on where these enzymes are most prev alent; where nNOS is mainly present in neural cells, iNOS in macrophages and eNOS in endothelial cells ( Curis et al., 2005 ) All these enzymes share a common


65 outcome, whereby citrulline is generated from arginine with the release of NO. The reaction also requires n icotinamide adenine dinucleotide phosphate (NADP + ), flavin mono nucleotide and biopterin as cofactors ( Meulemans, 2000 ) There is currently no known citrulline specific transporter for cellular uptake of citrulline ( Curis et al., 2005 ) However, various types of cells appear able to take up or release citrulline, with several studies demonstrating that citrulline can be transported b y common, generic amino acid transporters. The nervous system may employ a citrulline specific transport, but the mechanism of citrulline extraction by neural cells has not yet been fully elucidated. The uptake of citrulline by endothelial aortic cells has been suggested to have a different transport system than the y + system used for arginine, since this system does not carry citrulline. In rat aortic smooth muscle cells, citrulline transport appears to be partially Na + dependent and pH insensitive. In mac rophages, there are two transport systems; one is a saturable system for neutral amino acid transport, while the other presents a competitive inhibition of arginine transport by citrulline. The uptake of citrulline by enterocytes appears to require sodium. A citrulline carrier in the kidney, where a large proportion of citrulline is converted to arginine, has yet to be identified. Citrulline degradation is catalyzed via the urea cycle and within the small intestine by ASS and ASL in a reversible ATP depende nt condensation of citrulline with aspartate to form argininosuccinate ( Husson et al., 2003 ) However, ASS and ASL are in low concentrations within the intestine ( Osowska et al., 2004 ) Argininosuccinate is the immediate precursor of arginine leading to the production of urea in the liver and that of NO in many other cells ( Husson et al., 2003 ) The ASS enzyme is almost ubiquitous to


66 all cells, with high levels found in the liver and kidney and the lowest levels found in the intestine in adults ( Curis et al., 2005 ) The expression of ASS can be limited to certain cell subpopulations or certain regions ( Husson et al., 2003 ) The effects of citr ulline loading on anabolic hormones were determined in an interesting study, which found that renal arginine synthesis become saturated with high dosages of citrulline Eight fasting males underwent four separate oral loading tests (2, 5, 10, or 15 g CIT) in random order ( Moinard et al., 2008 ) Blood was drawn 10 times over an 8 h period. Plasma insulin in this study was not affected By comparison, c itrulline seemed to be cleared extremely rapidly from plasma. The phar macokinetic parameters suggest that saturation of CIT begins to occur at a load of 15 g, which was confirmed b y the increase in urinary arginine excretion and the decrease in both citrulline retention percentage and fractional reabsorption rate. Citrulline Malate Malate is an intermediate of the tricarboxylic acid cycle (TCA) and its supplementation has been shown to enhance energy production ( Giannesini et al., 2009 ) Recently, the combination of citrulline and malate (i.e., citrulline malate) has been promoted as a performance enhancing supplement. Results from a limit ed number of studies in humans and rats have indicated that citrulline malate supplementation improved muscle performance by reducing muscle fatigability and weakness ( Giannesini et al., 2009 2011 ) It has also been shown that oxidative stress was reversed and the antioxidative defense system strengthened by dietary supplementation with malate ( Wu et al., 2008 )


67 Arginine and Citrulline Supplementation a nd Insulin Resistance Arginine and Metabolic Disease Obesity in humans and animals occurs because of a chronic imbalance between energy intake and expenditure ( Wu et al., 2009 ) Not only is obesity a crisis worldwide in humans, but also becoming more prevalent in the equine industry. Growing evidence in humans indicates that arginin e supplementation may be a novel therapy for obesity and metabolic syndrome. Dietary supplementation of arginine decreased plasma levels of glucose, homocysteine, fatty acids, and triglycerides, and improved insulin sensitivity in chemically induced diabet ic rats ( Kohli et al., 2004 ) Kohli and coworkers (2004) found arginine hydrochloride (HCl ) (1.51 % ) or alanine (isonitrogenous control, 2.55%) added to drinking water for nondiabetic rats and arginine (0.43%) and alanine (0.73%) in the drinking water for diabetic rats (which consumed more water) for 14 d reduced plasma glucose concentration and resulted in the loss of BW in streptozotocin (STZ) induced diabetic rats, regardless of food intake. arginine HCl (0.62 g/kg BW) to diabetic hamsters ( Popov et al., 2002 ) induced diabetes ( Mendez and Balderas, 2001 ) also reduced plasma glucose levels by 65% Kohli and coworkers (2004) also found that arginine supplemented to nondiabetic and diabetic rats increased plasma co ncentrations of insulin in both groups compared to isonitrogenous supplementation with alanine The underlying mechanisms for the decrease in plasma glucose and BW in diabetic rats are unknown, but may involve an increase in insulin release. Arginine has been shown to stimulate the secretion of insulin cells ( G uoyao and Morris, 1998 ; Adeghate et al., 2001 ; Flynn et al., 2002 ; Kohlhaas et al., 2011 )


68 Interestingly, both diabetic rats ( Bronsnan et al., 1983 ; Pieper and Dondlinger, 1997 ) and humans ( Grill et al., 1992 ) have markedly decreased concentration s of plasma arginine. One of the hallmarks of diabetes mellitus is endothelial dysfunction, which may result from a deficiency of NO ( Kohli et al., 2004 ) Kohli and coworkers (2004) found that the intracellular concentration of arginine (0.48 mmol/L) in diabetic rats was substantially lower than in nondiabetic rats This finding suggests that endothelial NO synthase was saturated with intracellular arginine in both diabetic an d nondiabetic rats and that arginine was not a limiting substrate for endothelial NO synthesis. Kohli and coworkers (2004) did find that dietary arginine supplementation markedly increased in vitro NO production in the coronary endothelial cells of both diabetic and nondiabetic rats. Further findings were that arginine treatment normalized endothelial NO synthesis in STZ diabetic rats to the values of nondiabetic rats. Therefore, these results suppo rt that intracellular or extracellular arginine concentrations are indeed critical for endothelial NO production ( Wu and Meininger, 2002 ) Endothelial cells synthesize endothelium arginine ( Pollock et al., 1991 ) The pharmacological and biochemical properties of EDRF are mimicked by NO or NO containing compounds, which activate soluble guanylyl cyclase to increase the second m essenger of NO, cyclic guanosine monophosphate (cGMP). In turn, cGMP causes relaxation of vascular smooth muscle. Both clinical and experimental studies have shown beneficial effects of arginine administration improving vascular function in diabetic subjec ts. Intravenous arginine, the precursor of NO, was measured in 10 male type 1 diabetic patients and 10 nondiabetic patients and was found to increase cGMP and citrulline and decreased blood pressure


69 in both ( Smulders et al., 1994 ) A more recent crossover clinical trial on 6 patients with type 2 diabetes mellitus and mild hypertension were given 3 g of arginin e per hour, for 10 hours on either d 2 or d 3 of the three day trial ( Huynh and Tayek, 2002 ) Results from this study showed an increase in plasma citrulline, which may reflect an increase in the con version of arginine into NO and citrulline Systolic blood pressure was reduced from 135 7 to 123 8 mmHg These data suggest that oral arginine may increase endothelial NOS to increase vascular NO to reduce blood pressure. S erum insulin, mean blood glu cose, cortisol, epinephrine, norepinephrine, glucagon, growth hormone (GH), or insulin like growth factor 1 (IGF 1) concentrations were not affected by arginine supplementation in this study The supplementation of citrulline or arginine has shown positiv e treatment effects in overweight subjects. The supplementation of arginine (2.0% in drinking water) retarded the progression of atherosclerosis induced by a high fat diet in obese rabbits and improved NO dependent vasodilator function ( Bger et al., 1997 ) I n rabbits fed a high cholesterol diet endothelium dependent vasorelaxation in isolated thoracic aorta and blood flow of the ear artery in vivo wer e impaired ( Hayashi et al., 2005 ) Rabbits administered a rginine or citrulline, alone or in combination w ith antioxidants, improved endothelium dependen t vasorelaxation and blood flow, with the most notable increase in the ingestion of arginine, citrulline and antioxidants. Supplementing either 0% or 1.0% arginine into conventional diets for growing finishin g pigs for 46 days reduced body fat accretion, enhanced muscle gain, and improved the metabolic profile ( He et al., 2009 ) He and coworkers (2009) also found concentrations of low density lipoprotein (LDL), VLDL, and urea were lower in the


70 arginine supplemented pigs and concentrations of lipid signaling molecules were reduced. These findings suggest that dietary arginine supplementation alters catabolism of fat and amino acids in the whole body. Another study in pigs found similar results with the same level ( 1.0% of DM) of arginine supplementation ( Tan et al., 2009 ) were arginine was shown to beneficially promote muscle gain and red uce body fat accretion in growing finishing pigs. These findings suggest arginine may be a potential therapy to treat obesity. A distinct advantage of arginine or citrulline over drugs is that dietary arginine supplementation reduces adiposity, while imp roving insulin sensitivity. Fu and coworkers (2005) found that arginine HCl (1.51%) increased serum concentrations of arginine and NO were in arginine supplemented rats compared to control rats. Body weight was 6, 10, and 16 % lower at wk 4, 7, and 10 in ar ginine supplemented rats in comparison to control rats. A rginine reduced abdominal and epidiymal adipose tissues (45 and 25%, respectively) when compared to control rats. Arginine treatment enhanced NO production (71 to 85%), lipolysis (22 to 24%), and oxi dation of glucose (34 to 36%). Howeve r arginine did not increase serum levels of insulin or growth hormone in the ZDF rats, which is in contrast to results observed for STZ induced diabetic rats ( Kohli et al., 2004 ) These results indicate that the response of ZDF rats to dietary manipulation likely depends on target tissues of individual nutrients. The supplementation of watermelon pomace juice was fed as it is high in arg inine and citrulline, to ameliorate the metabolic syndrome in ZDF rats ( Wu et al., 2007c ) In this study, the drinking water containe d 0% or 0.24% arginine HCl, 63% watermelon pomace juice, 0.01% lycopene, or 0.05% citrus pectin for 4 weeks. The diets with arginine or watermelon pomace juice


71 increased serum concentrations of arginine, reduced fat accretion, lowered serum concentrations of glucose, free fatty acids, and enhanced vascular relaxation. However, s imilar to Fu and coworkers (2005), serum concentrations of insulin and growth hormone did not differ between the groups of rats in this study ( Wu et al., 2007c ) Jobgen and coworkers (2009) tested the effectiveness of arginine in diet induced o besity. In this study 4 wk old m ale Sprague Dawley rats were fed a high fat (40% energy) or a low fat (10% energy) diet for 15 wk resulting in an 18% high er BW gain and 74% greater weight of major white fat pads (retroperitoneal, epididymal, subcutaneous, and mesenteric adipose tissue). At 19 wk of age rats in each dietary gr oup were supplemented for 12 wk with 1.51% arginine HCl or 2.55% alanine (isonitrogenous control) in drinking water. Despite similar energy intake, weights of white fat pads increased by 98% in control rats over a 12 wk period, but only 35% in arginine sup plemented rats. Arginine reduced relative weights of white fat pads by 30% and enhanced those of soleus muscle by 13%, extensor digitorum longus muscle by 11%, and brown fat by 34% compared with control rats. Arginine seems to regulate the repartitioning o f dietary energy to favor muscle over fat gain in the body. Also in agreement with Fu et al. (2005) and Wu et al. (2007c), Jobgen and coworkers (2009) found no difference in the serum concentrations of insulin or growth hormone between arginine supplemented rats and controls but did observe lower serum glucose in response to arginine supplementation The possible un derlying mechanisms for the effect of arginine may involve multiple NO dependent pathways that favor whole body oxidation of fatty acids and glucose ( Wu et al., 2009 )


72 Interestingly, there are contradicting effects of whether arginine is actually the cause of increased insulin secretion Studies suggest that arginine increase d seru m insulin concentrations ( Guoyao and Morris, 1998 ; Adeghate et al., 2001 ; Flynn et al., 2002 ; Kohlhaas et al., 2011 ) and other studies have found no increase in serum insulin concentrations with arginine supplementation ( Huynh and Tayek, 2002 ; Fu et al., 2005 ; Wu et al., 2007c ; Jobgen et al., 2009 ) The studies discussed suggest that arginine or citrulline supplementation may provide novel and effective target therapy for obesity, diabetes, and metabolic syndrome. The beneficial effect of arginine in treating many developmental and health problems is unique among amino acids. Arginine has been previously reported and thought to increase insulin secretion ( Guoyao and Morris, 1998 ) possibly through the increase of NO, as NO is known to stimulate the release of anabolic hormones such as insulin ( Jobgen et al., 2006 ) Arginine Mechanisms that Improve Insulin Sensitivity Arginine has been shown to i ncrease insulin secretion from cells and improve insulin sensitivity in tissues via enhanced production of NO ( Calver et al., 1992 ) In another study, 10 normal subjects underwent euglycemic hyperinsulinemic clamp procedures after adm inistration of arginine (3 g consumed three times/d) for 1 month which was considered the lowest dosage possible in orde r to create endothelial effects without changing insulin secretion ( Piatti et al., 2001 ) This study found an increased NO availability induced by arginine which resulted from an increase in cGMP. However, even if arginine treatment normalized NO activity, it was not able to completely overcome the defect of insulin sensitivity in type 2 diabetic patients. This suggests that insulin resistance is multifacto rial. The mechanisms of the insulinotropic


73 action of arginine and of the glucose/arginine interaction are largely unknown ( Pueyo et al., 1994 ) It has been suggested that the accumulation of arginine, a positivel y charged cells leads to depolarization of the plasma membrane and eventually to insulin secretion as purely biophysical effect ( Blachier et al., 1989 ) Arginine has been shown to depolarize the plasma membrane in a way which is potentiated by glucose ( Hermans et al., 1987 ) and to stimulate Ca 2+ influx through voltage sensitive Ca 2+ channels ( Hermans et al., 1987 ; Smith et al., 1997 ; Weinhau s et al., 1997 ) Other data have questioned membrane depolarization a s th e sole mechanism in arginine cells showing in rat islets that insulin secretion persists by depolarized high K + ( Blachier et al., 1989 ) suggesting that arginine in addition to stimulation of Ca 2+ influx may stimulate insulin secretion by other mechanisms ( Blachier et al., 1989 ; Sener et al., 1990 ) Kohli and coworkers (2004) suggested they increased insulin sensitivity with supplementation of arginine through increased NO production since they found increased plasma concentrations of insulin in rats fed arginine A possible mechanism on insulin sensitivity is through selective overexpression of GLUT 4 in adipose tissue, which enhances glucose uptake by adipocytes thereby improving whole body insulin sensitivity ( Shepherd et al., 1993 ) Another possibl e mechanism for arginine to increase insulin sensitivity is a study that found that in the presence of arginine (0.52 mg/kg 1 min 1 ), steady state plasma glucose was lowered significantly ( Wascher et al., 1997 ) However, in this same study arginine did not elicit an increase in blood flow in response to insulin suggesting that arginine does not stimulate intact vascular effects of insulin


74 Advantage of Citrulline o ver Arginine Supplementation Citrulline supplementation may be a better vehicle for increasing arginine delivery, as substantial amounts of orally administered arginine never enter the systemic circulation in adult humans, pigs and rats ( Wu et al., 2009 ) Over 40% of dietary arginine is degraded by the small intestine in first pass metabolism ( Wu et al., 2009 ) Studies have shown that oral supplementation of citrulline is more efficient than giving arginine orally ( Curtis et al., 2007 ) Oral citrulline supplementation has proven to raise plasma arginine concentration and augment NO dependent signaling in a dose dependent manner in a cross over study with 20 healthy volunteers ( Schwedhelm et al., 2008 ) This randomized, dou ble blind, placebo controlled cross over study gave participants either 0.75 1.5 or 3 g of citrulline twice daily 1.0 g of immediate release arginine three times a day or 1.6 g of sustained release arginine twice daily and compared it to a placebo. The researchers found that citrulline given at 0.74 g increased plasma arginine to the same extent as 1.6 g of sustained release arginine and 1.0 g immediate release arginine. Both h igher doses of citrulline elevated plasma arginine This strongly suggests th at oral citrulline is at least as efficient as arginine supplementation for increasing plasma arginine concentrations. Bendahan and coworkers (2002) and Sureda and coworkers (2009) found that 6 g/d of citrulline increased plasma arginine in men Supplement ation of citrulline, rather than arginine may be a more efficient and safer way of increasing plasma and tissue levels of arginine in mature animals and such supplementation may be particularly appropriate in horses who are obese or have EMS. By comparison arginine supplementation could cause a problem as arginine is taken up and metabolized by the liver to yield urea, which raises questions about the safety of


75 arginine supplementation ( Curis et al., 2005 ) However, a study with artificially rear e d piglets fed argi nine ( 0 .2, or 0.4%) in milk replacer showed a decrease in plasma concentrations of ammonia (20 and 35%, respectively) and urea (19 and 33% respectively) ( Kim et al., 2004 ) Unfortunately eightfold the dose of arginine was fatal for a 21 m onth old girl who die d from cardiac arrest and myeli n ol ysis ( Garard and Luisiri, 1997 ) This may not be the s ame reaction in mature mammals. Arginine supplementation has also been associated with naus ea and vomiting ( Boyd and Olin, 1984 ) and abdominal cramping and bloating in humans ( Kattwinkel et al., 1972 ) Arginine is liable to cause excessive urea production, since it acts a s a catalyst for ureagenesis ( Curis et al., 2005 ) This effect on urea production is not generally observed with citrulline supplementation. The effects of citrulline loading on anabolic hormones was determined in a study with e ight fasting males who underwent 4 se parate oral loading tests (2, 5, 10, or 15 g CIT) in random order ( Moinard et al., 2008 ) Blood was drawn 10 times over an 8 h period. None of the volunteers suffered nausea or diarrhea or any other side effect with any dosage of citrulline Citrulline M alate to Ameliorate Insulin Resistance Although strong epidemiological data are not available, the prevalence of obesity horse owners, where provision of fo od is equated with care and concern, regretfully is a very poor and unsafe choice for the horse. Further, improvements in plant breeding to support faster tissue gains in cattle have led to forages rich in NSC that are f some equines. Ongoing research is investigating the link between obesity and the onset of glucose intolerance or insulin resistance in horses, similar to that observed with type 2 diabetes in humans. An insulin resistant


7 6 horse has fat deposits due to the inappropriately stores the extra glucose as fat. The high level of insulin secreted also suppresses fat metabolism and supports fat deposition ( Frayn et al., 2004 ) Arginine and/or citrulline may provide novel and effective therapies in the horse for health problems such as obesity, insulin resistance, and blood circulation issues. The effect of arginine and citrulline in treating different developmental and health related issues is unique among amino acids, which can offer great promise for the improved health and wellbei ng of horses. Understanding the relationship between arginine and citrulline may lead to a more defined and scientifically based nutritional intervention for horses with EMS, insulin resistance or laminitis issues. Most arginine research has been performe d in mice, pigs and humans, with very little research being conducted in investigated in horses. C itrulline malate supplementation may be a useful means to manage horses with E MS or to mitigate insulin resistance and future bouts of laminitis. Therefore, citrulline malate could be used as a potential therapeutic target to decrease the continuous secretion of insulin as it tries to compensate for a decrease in tissue cell effecti veness in those horses found to be insulin resistant and overweight. Simultaneously, citrulline malate may induce a weight loss effect, since a lower level of insulin will not promote the same level of fat storage. Although pharmacologic agents have been successfully used to suppress insulin secretion and elicit weight loss in overweight horses with EMS, there are usually side effects associated with chronic drug administration. Arginine or citrulline


77 supplementation may offer a safer, less expensive alter native to drugs for suppressing insulin secretion and/or improving insulin sensitivity. Further, supplement induced suppression of pancreatic cell activity in the horse may induce weight loss. However, the ability of arginine or citrulline to suppress i nsulin production in horses is unknown, and the approach must be cautious to avoid starving cells of glucose. It is possible that the careful regulation of insulin through the supplementation of citrulline will reduce the risk of horses becoming less sensi tive to insulin due to the overproduction of insulin. Suppressing the pancreatic cell activity may not only induce weight loss but also keep the pancreatic cell cells from being exhausted. The objectives of this st udy were to: 1) investigate citrulline malate supplementation and its effects on the availability of other amino acids; 2) assess whether oral citrulline malate will act as a dietary precursor for arginine; and 3) determine if oral citrulline malate will alter glycemic and insulinemic response s to a starch rich meal in healthy horses. We hypothesized that supplementation of citrulline malate would increase arginine availability and consequently improve insulinemic response to a meal.


78 A. B. Figure 2 1. Arg inine synthesis. A) In the adult citrulline is synthesized from glutamin e (GLN) glutamate (GLU) proline (PRO) and arginine (ARG) in the small intestine and released to the portal vein where it bypasses the liver and is taken up by the kidney to be converted to arginine. B) In neonates arginine is synthesized predominantly from proline in the small intestine and released into the portal vein The kidney of neonates is not capable of synthesizing arginine from citrulline (CIT) and thus relies on intestinal synthesis and su pply Other a bbreviations: o rnithine (ORN); pyrroline 5 carboxylate synthase (P5C); tricarbox ylic acid cycle (TCA). ( Adapted from Brosnan and Brosnan ( 2004 ) and Bertolo and Burrin ( 2008 ) ).


79 Figure 2 2. Metabol ism of citrulline and arginine in the hepatic urea cycle and related pathways. Abbreviations: ASL, argininosuccinate lyase; ASS, argininosuccinate synthetase; CPS 1 carbamoylphosphate synthetase 1; OAT, ornithine aminotransferase; ORNT1, the mitochondrial ornithine transport er (SLC25A1 5); OT C, ornithine transcarbamylase (also called ornithine carbamoyltransferase ) ; P5C = 1 pyrroline 5 carboxylate synthase ( Adapted from Mandel et al. ( 2005 ) and Walker ( 2009 ) ).


80 CHAPTER 3 MATERIALS AND METHODS Horses Twelve mature, non gravid mares (6 Thoroughbreds and 6 Quarter Horses) with a mean SE body weight (BW) of 552.0 31.2 kg and age of 10.8 2.5 y were utilized in this study. All horses had a BCS of 5 to 6 ( Henneke et al., 1983 ) Horses were group housed on a 4 ha pasture at the Institute of Food and Agricultural Sciences (IFAS) Equine Sciences Center (ESC) in Ocala, Florida. Pastures were equipped with outdoor, 3 m x 3 m pens that allowed individual feeding of concentrate and supplements once daily I nsulinemic response to a grain meal was performed with horses confined indoors in individual 3.7 m x 3.7 m stall s. All horses received routine healthcare, including vaccination, anthelmintic treatment and hoof care established in the standard operating procedures for the IFAS ESC. All animal protocols and procedures were reviewed and approved by the IFAS Animal Care and Use Committee at the University of Florida. Dietary Treatments After blocking for age and breed, horses were randomly assigned to one of two dietary treatments: urea supplementation (CON; n = 6) or citrulline malate supplementation (CIT; n = 6). Citru lline malate (99.1%; UniChem Enterprises, Inc., Neward, NJ) was supplemented at a rate of 86 mg/kg BW. The level of CIT supplementation was chosen based on studies in humans where 6 g CIT/d was shown to increase plasma arginine concentration ( Bendahan et al., 2002 ; Sureda et al., 2009 ) Feed grade urea was supplemented at a rate of 25 mg/kg BW and served as an isonitrogenous control. To facilitate ease of sample collection, horses were further divided into 3 groups of 4 horses, with 2 horses from each dietary treatment


81 represented in each group. Dietary treatments were initiated in one group of horses per day, over 3 consecutive days. As intended, this arrangement also staggered each data collection over a 3 d period. Horses received their re spective dietary treatments for 14 days. Horses were fed a basal diet consisting of ad libitum access to Coastal Ocala, FL) fed at 0.5% BW/d. The CIT and CON supplements were h and mixed into the concentrate and fed individually once daily at 0730 h. Although horses were housed on pasture during the supplementation period, the study was conducted in February; thus, the majority of the pasture forage was dormant and intake was ass umed to be minimal. Nutrient analysis of the hay and grain mix making up the basal ration is provided in Table 3 1. Insulinemic Response to a Meal G lycemic and insulinemic responses to a grain meal containing CIT or CON were evaluated on d 14 after an overnight fast. Horses were transferred from pasture housing to indoor, individual stalls on the afternoon of d 13 to facilitate fasting and sample collection. Body weights were obtained prior to the start of supplementation and on d 13 u sing a livestock scale accurate to 0.5 kg (MP800, Tru test, Inc., Mineral Wells, TX). On d 14 at 0700 h, a 16 ga x 9 cm catheter (Abbocath T, Hospira, Inc., Lake Forest, IL) was placed in the right jugular vein under local anesthesia using aseptic techni que. At 0800 h, horses were fed 0.25% BW of the same grain mix concentrate used in their basal ration, along with the daily allotment of their respective dietary treatment. The time it took the horse to consume the meal was recorded with the clock stoppin g when the entire meal was consumed Blood samples were obtained 30 min ( 0:30) and


82 immediately (0:00) before the meal was offered, and every 30 min for 5 h after the meal was consumed. Patency of catheters and extension tubing was maintained by flushing w ith heparinized (2,000 U/L) saline (0.9% NaCl) after each blood sample was collected. Blood was placed into tubes (Vacutainer, Becton Dickinson Co., Franklin Lakes, NJ) containing sodium heparin (for plasma amino acids), EDTA (for plasma glucose) or no ant icoagulant (for serum insulin). Samples with anticoagulant were immediately placed on ice and were processed within 2 h of collection by centrifugation at 2000 g for 15 min at 4C. Samples with no anticoagulant were kept at room temperature (approximately 20C) for 5 h to permit clot formation prior to centrifugation. Serum and plasma were harvested and stored in polypropylene cryogenic vials in 1.0 mL aliquots at 80C until analyses were performed. Sample Analyses Plasma Glucose Plasma glucose concentrat ion was determined using a commercially available kit The kit determined g lucose concentration based on an oxidase peroxide reaction. Briefly, gluconolactone wi th flavin adenine dinucleotide (FAD) dependent glucose oxidase. The reduced form of glucose oxidase, glucose oxidase FADH 2 was converted back to glucose oxidase FAD form by adding oxygen, yielding hydrogen peroxide. Finally, with horseradish peroxidase as a catalyst, hydrogen peroxide reacted with 3,5 dichloro 2 hydroxybenzenesulfonic acid and 4 aminoantipyrine to generate a pink dye with an optimal absorption at 514 nm. Glucose concentration in plasma samples was determined colorimetrically using a BioTek PowerWave XS (BIO TEK Instruments, Inc., Winooski, VT) plate reader at a wavelength


83 of 500 nm. Samples were analyzed in duplicate and compared to a set of glucose standards ranging from 0 to 250 mg/dL. Intra and interassay coefficients of variation (CV) of pooled samples were 1.5 and 2.8 %, respectively. Glycemic response to a grain meal was evaluated as plasma glucose concentration over time, area under the curve (AUC), time to peak glucose, and peak glucose concentration. Serum Insulin Serum insulin was measured by radioimmunoassay using a commercially available kit (Coat A Count Insulin, Siemens, Los Angeles, CA) previously validated for use in horses ( McGowan et al., 2008 ) Each sample was analyzed in duplicate. Detecti on limits for the insulin assay was approximately 1.53 to 371 IU/L. Intra and interassay CV of pooled samples were 1.6 and 11.4%, respectivel y. Insulinemic response to a meal was evaluated as serum insulin concentration over time, AUC, time to peak insulin, and peak insulin concentration. In addition, proxies based on baseline (fasting) glucose and insulin concentrations were used for screenin g insulin sensitivity (SI) and pancreatic cell responsiveness. As described for horses by ( Treiber et al., 2005b ) the reciprocal of the insulin square root index (RISQI), calculated as 1/ insulin (with insulin as mU/L), was used to reflect the amount of insulin required to chronically maintain basal glucose homeostasis and served as a proxy for insulin sensitivity. The modified insulin response to glucose (MI RG), calculated as (800 0.30[insulin 50] 2 ) / glucose 30 (with insulin as mU/L and glucose as mg/dL), was used to estimate the capacity of pancreatic cells to increase insulin secretion and compensate for exogenous glucose (Treiber et al., 2005b). The homeostasis model assessment (HOMA) was used to derive an estimate of insulin sensitivity from the


84 mathematical modeling of fasting plasma glucose and insulin concentrations ( [ basal glucose (mg/dL) insulin (mU/L) ]/ 22.5 ) where a higher HOMA indicates i nsulin resistance ( Ma tthews et al., 1985 ) The glucose to insulin ratio was used to estimate the effect of insulin on glucose concentrations (or insulin sensitivity of peripheral tissues), as it is positively correlated with insulin sensitivity ( Firshman and Valberg, 2007 ) Plasma Amino Acids, Urea and Ammonia Plasma citrulline and arginine in response to a grain meal with CIT or CON supplementation were of primary interest in this study, as well as amino acids that are linked with their metabolism, including glutamate, ornithine, and proline. To verify the effe ct of CIT or CON supplementation on amino acids most likely to be limiting in equine diets, plasma lysine, methionine, and threonine were also evaluated. Plasma urea and ammonia concentrations were simultaneously assessed with amino acid analysis. Plasma samples were prepared for analysis by precipitating amino acids with 35% sulfosalicylic acid (SSA). Aminoethyl cysteine hydrochloride (AEC) was included in the SSA solution as an internal standard and used to calculate the efficiency of amino acid recover y. Samples were incubated at 4 C for 20 min followed by centrifugation at 11,000 g for 10 min at 4 C. Supernatant was harvested and filtered using a 0.22 m syringe filter (Fisherbrand, MCE membrane, Fisher Scientific, Houston, TX), and then diluted with 0 .02 N hydrochloric acid. Precipitates were then placed in 2 mL clear glass vials (Snap Plasma amino acid concentrations were determined using classic ion exchange separation followed by post column derivatization with ninhydrin using an L 8900 Amino Acid Analyzer (Hitachi High Technologies, Pleasanton, CA). The 60 mm X 4.6 mm (i.d.) chromatographic column consisted of polystyrene cross linked by divinylbenze, with


85 sulfone (SO 3 ) group s as active exchange sites and was equipped with a 40 mm X 4.6 mm (i.d.) guard column. Column temperature was 57 C and the reactor temperature was 135 C. While awaiting injection, sealed sample vials were maintained at 4 2 C in the autosampler rack. All buffers and the ninhydrin reagent were purchased from Hitachi High Technologies (Pleasanton, CA). Amino acid detection was by spectrophotometry at 570 and 440 nm with the ninhydrin reaction. Based on AEC concentrations, mean SE recovery of amino acids w as 111 3%. Statistical Analyses Differences in plasma glucose, amino acids, urea, and ammonia and serum insulin were analyzed using the PROC MIXED procedure in SAS with repeated measures (version 9.3, SAS Institute, Inc. Cary, NC). Dietary treatment, t ime, and time by treatment were included in the model as fixed effects and horse within treatment was included as a random variable. Based on Information Criterion, heterogeneous autoregressive covariance structu re was used for plasma glucose and serum insulin and the autoregressive covariance structure was used for all plasma amino acids. Type 3 fixed effects, least squared means, and differences of least squared means were used to determine statistical significa nce and the slice function of the LSMEANS statement was used as a means separation technique. Glucose and insulin data from o ne horse in the CON group was incomplete (missing at the 2.5 and 3 h time points ) due to a labeling mistake during collection Ther efore, the Kenward Rodger denominator degrees of freedom method (ddfm=kr) was used to evaluate the unbalanced data sets for unknown distributions ( Kenward and Rodger, 1997 ; Gomez et al., 2005 ) performed with PROC GLM to determine heterogeneous variance in insulin concentrations and error variances were


86 greater for CON than CIT Therefore, i nsulin concent rations were not normally distributed and were consequently normalized with logarith mic transformations in SAS (log[INS]) prior to evaluation The trapezoidal rule was used to estimate the area under the plasma glucose and serum insulin level time curve. The formula used for calculating AUC was ((m 2 + m 1 )*t 1 )/2 + ((m 3 + m 2 )*t 2 )/2 + ((m 4 + m 3 )*t 3 )/2 + ((m 5 + m 4 )*t 4 )/2 + ((m 6 + m 5 )*t 5 )/2 + ((m 7 + m 6 )*t 6 )/2 + ((m 8 + m 7 )*t 7 )/2 + ((m 9 + m 8 )*t 8 )/2 + ((m 10 + m 11 )*t 11 )/2 + ((m 12 + m 11 )*t 11 )/2, where m = repeated measures over time and t = time interval between measures ( Pruessner et al., 2003 ) Differences in plasma glucose and serum insulin AUC were analyzed using the PROC GLM procedure in SAS. Treatment effects were evaluated among all time points collectively, as well as accumulated AUC for hours 1, 2, 3, and 4 after meal consumption. Differences between treatments in p eak glucose and insulin concentrations time to peak glucose and insulin RISQI, MIRG HOMA, glucose to insulin ratio meal consumption time, and BW were analyzed by ANOVA using the PROC MIXED function of SAS. All data are expressed as least square means SE. Differences were considered significant at P for significance acknowledged at P 0.10.


87 Table 3 1. Nutrient composition of feeds included in the basal ration Nutrient 1,2 Bermudagrass hay Grain mix DE, Mcal/kg 1.90 3.24 Crude protein, % 10 .4 20.3 Crude fat, % 1.7 3.8 NDF, % 72.4 23.1 ADF, % 38.2 13.5 Starch, % 2.4 27.0 ESC, % 4.6 7.6 Ca, % 0.3 1 0.89 P, % 0.2 2 0.66 Cu, mg/kg 6 62 Zn, mg/kg 2 9 224 Arginine, % 0. 40 1.22 Citrulline, % 0.0 0 0.0 0 Glutamic Acid, % 0.86 3.19 Lysine, % 0.40 1.03 Methionine, % 0.13 0.27 Ornithine, % 0.01 0.01 Proline, % 0.55 1.02 Threonine, % 0.35 0.69 1 DE = digestible energy, NDF = neutral detergent fiber, ADF = acid detergent fiber, ESC = ethanol soluble carbohydrates 2 All values are presented on a 100% DM basis.


88 CHAPTER 4 RESULTS All horses remained in good health throughout the observational period and willingly consumed both the urea and citrulline malate supplements. Body weight of horses did not differ among treatments before supplementation began ( 546 13 kg CON, 557 13 kg CIT) nor after 14 d of treatment with CIT (570 13 kg) or CON (563 13 kg) The amount of time it took the horses to consume the grain meal containing CIT ( 14.5 2.7 min ) or CON ( 15.3 2.7 min ) did not differ between treatments and there were no feed refusals in response to the meal As expected, plasma glucose concentration was affected by sampling time ( P = 0.0001) in response to the meal (Figure 4 1). Across treatments, plasma glucose i ncreased within 30 min after the meal wa s consumed and r eturned to baseline values by 4 .5 h. Plasma glucose was not affected by CIT supplementation ( P = 0. 80 ) or the time*treatment interaction ( P = 0. 63 ) (Figure 4 1). In addition, treatment had no effect on glucose AUC ( P = 0.43 ) (Table 4 1). Although the time to peak glucose concentration following a grain meal was not affected by CIT supplementation ( P = 0.79) there was a trend ( P = 0.09) for peak glucose concentration to be higher in CON vs. CIT (Table 4 1). Serum insulin in response to a grain meal containing CON or CIT, also changed with respect to time ( P < 0.0001) and mirrored the rise in plasma glucose (Figure 4 1). There was a trend for s erum insulin to be affected by dietary treatment ( P = 0.06 ) but not the time*treatment interactio n ( P = 0.86 ). Serum insulin increased above baseline ( P < 0.0 1 ) within 30 min of meal cessation in CON and CIT and was lower ( P < 0.05 ) in CIT at 2.5 3, 3.5, 4 and 4.5 h postprandially compared to CON (Figure 4 1). Serum


89 insulin returned to baseline conce ntration at 4.5 h in C IT, but failed to return to baseline during the 5 h observation period in CON. As a result, insulin AUC was also lower ( P = 0.05) in CIT than CON (Table 4 1). Although the time to peak insulin concentration was similar among treatments, there was a trend for pe ak insulin concentration to be higher ( P = 0.09 ) in CON than CIT (Table 4 1). Proxy measurements for insulin sensitivity (RISQI, P = 0.330 ; HOMA, P = 0.92 2 ) pancreatic cell responsiveness (MIRG, P = 0.345) and the glucose to insulin ratio ( P = 0.384) were not affected by 14 d of CIT supplementation (Table 4 1). Plasma citrulline and arginine in response to a grain meal containing CON or CIT are presented in Figure 4 2. The concentration of citrulline in plasma was affected by time ( P < 0.0001), treatment ( P < 0.0001), and the time*treatment interaction ( P < 0.0001). Plasma citrulline increased ( P < 0.0001) in horses supplemented with CIT, with co ncentrations remaining higher in CIT than CON horses from 1 to 5 h after meal consumption ( P < 0.0001). Additionally, plasma citrulline remained elevated above baseline in CIT at the e nd of the 5 h observation period ( P < 0.0001). In contrast, plasma citru lline remained unchanged in response to the grain meal in CON horses. Plasma arginine was similarly affected by time ( P < 0.0001), treatment ( P < 0.0001), and the time*treatment interaction ( P = 0.0094) (Figure 4 2). Plasma arginine concentration had incre ased ( P < 0.0001) above baseline values 1 h postprandially in both treatments. It continued to increase in CIT horses at 2 h ( P = 0.0001 ) and plateaued thereafter through 5 h post meal consumption. By comparison, plasma arginine gradually declined from 2 t o 5 h in CON horses and was no different than baseline concentration 5 h after meal consumption ( P = 0.094) As a result, plasma


90 arginine concentration was higher ( P < 0.0001) in CIT horses at 2, 3, 4 and 5 h after meal cessation (Figure 4 2). Changes in p lasma ornithine, proline and glutamate in response to a grain meal containing CIT or CON are presented in Figure 4 3. Plasma ornithine concentration was affected by time ( P < 0.0001) and treatment ( P = 0.0119), but not the treatment*time interaction ( P = 0 .0659). Plasma ornithine was elevated above baseline within 1 h after meal cessation in CIT horses ( P = 0.0001 ), but not until 2 h postprandially in CON horses ( P = 0.001). In CON and CIT, plasma ornithine remained above ( P < 0.01) baseline concentrations through the 5 h observation period. P lasma ornithine concentration was higher from 1 to 5 h after meal cessation when horses were supplemented with CIT compared to CON ( P < 0.0001 ). Plasma proline concentration was affected by time ( P < 0.0001), but not tr eatment ( P = 0.7686) or treatment*time ( P = 0.1012) (Figure 4 2). Across treatments, plasma proline increased gradually above baseline concentration through 3 h post meal consumption ( P < 0.0 001 ), and then sharply declined below baseline concentration at 4 h ( P < 0.0001 ), where it remained through 5 h following the grain meal ( P < 0.0001 ). Plasma glutamate concentration was affected by time ( P = 0.0037) and treatment ( P = 0.029 0 ), but not the treatment*time interaction ( P = 0.4991 ). In CIT horses, p lasma g lutamate was elevated above baseline at 1 h ( P = 0 .0188 ) and 2 h ( P = 0.0134 ) after meal cessation, but had return ed to baseline concentration 3 h ( P = 0.1441 ) after the meal had been consumed By comparison, in CON horses plasma glutamate remained elevat ed above baseline concentrations from 1 to 4 h postprandially ( P < 0.01). P lasma


91 glutamate concentration was greater at 1, 3, and 4 h after meal cessation in CON than CIT ( P < 0.05) Figure 4 4 shows the response of plasma lysine, methionine and threonine to a grain meal containing CIT or CON. Plasma lysine ( P < 0.0001), methionine ( P < 0.0001), and threonine ( P < 0.0001) were affected by time. Plasma lysine was elevated above ba seline concentration from 1 to 4 h following meal consumption ( P < 0.0 0 1), b ut had returned to baseline by 4 h ( P = 0.2289) Plasma methionine was elevated 1 h after meal consumption ( P < 0.0001 ), but declined thereafter such that plasma concentrations were below baseline measurements at 4 and 5 h postprandially ( P < 0.0 087 ). Plasma threonine was elevated 1 h after meal consumption ( P < 0.0001) and was no different than baseline concentrations by 5 h ( P = 0.7186 ). Supplementation with CIT had no effect on plasma lysine ( P = 0.3607), methionine ( P = 0.2855), or threonine ( P = 0. 2200) Plasma urea concentration in response to a grain meal containing CON or CIT was unaffected by time ( P = 0.1224 ) or treatment ( P = 0.9175) (Figure 4 5). Plasma NH 3 concentration was highly variable among horses, but was also unaffected by time ( P = 0.3348 ) or treatment ( P = 0.8803) (Figure 4 5).


92 Table 4 1. Measures of glucose and insulin response to a grain meal containing citrulline malate (CIT) or an isonitrogenous amount of urea (CON). Treatment Measurement 1 CON CIT SEM P value Basal insulin, IU/mL 5.27 4.08 0.91 0.38 Basal glucose, mmol/L 4.68 4.65 0.37 0.95 AUC Glucose, mmol/L 3 4.17 31.27 2.50 0.43 Insulin, IU/mL 276.57 151.38 40.21 0.05 Peak glucose Concentration, mmol/L 9.64 8.43 0.45 0.09 Time to peak, h 2 .1 2 .1 0.17 0 .79 Peak insulin Concentration, IU/mL 95.37 55.28 15.24 0.09 Time to peak, h 2. 7 2.5 0.32 0.58 RISQ, mIU/L 0.5 0.44 0.57 0.09 0.33 MIRG, (mU insulin 2 /[10 L mg glucose ]) 4.12 3.19 0.66 0.34 HOMA, (mg/dL glucose mU/L insulin )/22.5 3.57 3.53 0.30 0.92 Glucose:I nsulin ([mg/dL]/ [ mU/L ]) 18.48 28.17 7.47 0.38 1 AUC = area under the curve; RISQ = reciprocal of the square root of insulin index ; MIRG = modified insulin response to glucose ; HOMA = homeostasis model assessment


93 A. B. Figure 4 1. Plasma glucose and serum insulin res ponses before (0:00 h) and after horses consumed a grain meal containing citrulline malate (CIT) or an isonitrogenous amount of urea (CON). A) Plasma glucose: overall effect of time ( P = 0. 0001 ), treatment ( P = 0.8036 ), and time*treatment ( P = 0.6361 ). B) Plasma insulin: overall effect of time ( P < 0.0001 ), treatment ( P = 0.0635 ), and time*treatment ( P = 0.8593 ). An a sterisk (*) indicates a difference between treatments at a specific time point ( P < 0.05 ).


94 A. B. F i gure 4 2. Plasma citrulline and arginine concentrations before (0:00 h) and after horses consumed a grain meal containing citrulline malate (CIT) or an isonitrogenous amount of urea (CON). A) Plasma citrulline: overall effect of time ( P < 0.0001), treatme nt ( P < 0.0001), and time*treatment ( P < 0.0001). B) Plasma arginine: overall effect of time ( P < 0.0001), treatment ( P < 0.0001), and time*treatment ( P = 0.0094). An a sterisk (*) indicates a difference between treatments at a specific time point ( P < 0.01 ).


95 A. B. C. F igure 4 3. Plasma ornithine, proline, and glutamate concentrations before (0:00 h) and after horses consumed a grain meal containing citrulline malate (CIT) or an isonitrogenous amount of urea (CON). A) Plasma ornithine: overall effect of time ( P < 0.0001), treatment ( P = 0.0119), and time*treatment ( P = 0.0659). B) Plasma proline: overall effect of time ( P < 0.0001), treatment ( P = 0.7686), and time*treatment ( P = 0.1012). C) Plasma glutamate : overall effect of time ( P < 0.0037 ), treatment ( P = 0.0290 ), and time*treatment ( P = 0 .4991 ). An a sterisk (*) indicates a difference between treatments at a specific time point ( P < 0.01).


96 A. B. C. Fi gure 4 4. Plasma lysine, methionine, and threonine concentrations before (0:00 h) and after horses consumed a grain meal containing citrulline malate (CIT) or an isonitrogenous amount of urea (CON). A) Plasma lysine: overall effect of time ( P < 0.0001), treatment ( P = 0.3607), and time*treatment ( P = 0.5834). B) Plasma methionine: overall effect of time ( P < 0.0001), treatment ( P = 0.2855), and time*treatment ( P = 0.3234). C) Plasma threonine : overall effect of time ( P < 0.0001) treatment ( P = 0.2200 ), and time*treatment ( P = 0.3327 ).


97 A. B Figure 4 5. Plasma urea and NH 3 concentrations before (0:00 h) and after horses consumed a grain meal containing citrulline malate (CIT) or an isonitrogenous amount of urea (CON). A) Plasma urea: overall effect of time ( P = 0.1224), treatment ( P = 0.9175), and time*treatment ( P = 0.1500). B) Plasma NH 3 : overall effect of time ( P = 0.3348), treatment ( P = 0.8803), and time*treatment ( P = 0.4040).


98 CHAPTER 5 DISCUSSION The key findings of this study were: 1) CIT supp lementation increased the pool of citrulline, arginine and ornithine in plasma; 2) CIT supplementation did not appear to interfere with availability of lysine and other potentially limiting amino acids in equine diets; 3) glycemic control in response to a grain meal was accomplished in CIT supplemented horses with a lower circulating insulin concentration; and 4) short term supplementation with CIT had no effect on insulin sensitivity, as estimated by RISQI and MIRG With the notable exception of watermelon which is not commonly fed to horses, there is typically little to no citrulline present in the diet ( Rimando and Perkins Veazie, 2005 ; Curtis et al., 2007 ; Marini et al., 2010a ). Thus, circulating citrulline is believed to be almost entirely of endogenous origin ( Curtis et al., 2007 ; Marini et al., 2010a ) Hepatic uptake of citrulline from portal circulation has been shown to be minimal ( Wu et al., 2009 ) questioned (van de Poll et al., 2007). In the current study, plasma citrulline was elevated 3 to 5 fold by CIT supplementation. Although synthesis of citrulline from ornithine, proline and glutamine can occur in enterocytes and other tissues ( Marini, 2010 ) the dramatic increa se in plasma citrulline was likely attributed to the direct absorption of supplemented CIT, rather than a high level of de novo synthesis. This is further supported by the observation that plasma citrulline in the urea fed horses remained unchanged during the 5 h period following the grain meal. In the current study, plasma citrulline remained elevated through the 5 h observation period in horses supplemented with CIT. By comparison, plasma citrul line


99 appears to be cleared fairly rapidly from plasma in humans ( Barr et al., 2007 ; Moinard et al., 2008 ) The h alf life of citrulline was calculated to be approximately 60 m inutes in humans (Barr et al., 2007). The prolonged elevation of plasma citrulline in horses in the current study compared to humans and other species supplemented with CIT could be due to difference s in digestion and rate of passage between species S tudies have shown that diabetic patients have slower gastric emptying when compared to non diabetic controls ( Horowitz et al., 1991 ) Additionally the o ral vectors in which citrulline is supplemented to subjects are likely to influence rate of passage and subsequently citrulline absorption For example, several rat and mice studies use d water as a means of oral citrulline supplementation whereas CIT was included in a meal matrix in the current study It has been shown that liquid meals empty the stomach quicker than solid meals ( Collins et al., 1991 ) Metabolism of citrulline generally follows one of three pathways: 1) arginine biosynthesis; 2) nitric oxide (NO) cycle; or 3) urea cycle in the liver (Curis et al., 2005). Citrulline is converted to arginine mainly in the kidney via a partial urea cycle involving argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL) (Curis et al., 2005). The newly made arginine is released into general blood circulation where it can be used for protein sy nthesis, NO production, and ureagenesis, among other functions. In the current study, CIT supplementation resulted in a sustained increase in plasma arginine that superseded that observed in horses fed urea, in both concentration and duration. Similar incr eases to the arginine pool were reported with intravenous citrulline infusion ( Lassala et al., 2009 ) oral citrulline supplementation ( Bendahan et al., 2002 ; Osowska et al., 2006; Schwedheim et al., 2007; Sureda et al., 2009 ), and watermelon

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100 consumption ( Mandel et al., 2005 ; Collins et al., 2007 ; Wu et al., 2007c ) which likely reflects the conversion of citrulline to arginine in the kidney and other extrahepatic tissues. Thus, CIT supplementation appears effective at inc reasing arginine availability in horses. In other species, intravenous or oral administration of citrulline has been shown to be at least, if not more effective at increasing arginine supply to the body than direct supplementation with arginine (Schwedheim et al., 2007; Lassala et al., 2009). W hen sheep received an intravenous bolus dose of citrulline (155 mol/kg BW) or the same dose of arginine HCl on d 135 of gestation, citrulline was more effective in achieving and sustaining a prolonged increase in arg inine concentration in the fetal circulation ( Lassala et al., 2009 ) Lassala and coworkers (2009) also found that the T 1/2 of citrulline in maternal plasma was twice that of arg inine in pregnant ewes This may be explained by a higher arginase activity which breaks down arginine compared to the activities of argininosuccinate synthase and argininosuccinate lyase which metabolize citrulline ( Wu and Morris, 1998 ) Additionally, the lower r ate of transport of citrulline by animal cells (e.g. endothelial cells and macrophages) and th e faster rate of transport of arginine also reduces the T 1/2 of arginine when compared to citrulline ( Wu and Flynn, 1993 ; Li et al., 2001 ) These factors help to explain why only half the amount of cit rulline compared to arginine supplementation was needed to raise plasma arginine in humans (Schwedhelm et al., 2008) Plasma ornithine was also increased in response to a meal with CIT in the present study. Ornithine is an immediate precursor for citrulline biosynthesis in the intestine and other tissues (Marini, 2010). Subsequent metabolism of arginine by arginase yields ornithine and urea (Wu et al., 200 9). Ornithine then precedes citrulline i n the urea

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101 cycle, which is con verted into arginine by ornithine transcarbamylase, ASS, and ASL. Thus, a rginine is a major positive ureagenesis regulator ( Shigesada and Tatibana, 1971 ) In the current study, the increase in arginine availability in CIT supplemented horses may have resulted in greater conversion of arginine to o rnithine, thereby raising circulating ornithine concentrations. However, this did not appear to reflect an increase in ureagenesis, as plasma urea and ammonia remained unchan ged in response to a CIT meal when compared to horses fed a meal containing urea. Oral supplementation with L citrulline in humans (Schwedhelm et al., 2007) or watermelon pumice in diabetic rats (Wu et al., 2007c) also resulted in increased plasma ornithine, but not to the same magnitude as that seen with arginine supplementation. Thus, although CIT supplementation elevated circulating arginine and ornithine concentrations in the present study, it may not have been sufficient to trigger upregulation of the urea cycle in the liver Increased ornithine availability may have benefits of its own, including serving as a substrate for production of polyamines and proline, which are important in cellular proliferation, tissue grown, and wound repair ( Albina et al., 1988 ) Ornithine supplementation has also been evaluated for its ability to improve nitrogen balance in various acute and chronic malnutrition states ( Cynober, 1991 ) and as a therapeutic treatment for hepatic encephal opathy, a serious complication of cirrhosis in humans ( Ong et al., 2011 ) Individual amino acid requirements, with the exception of lysine in growing horses, have not been established for the horse ( NRC, 2007 ) Threonine is suggested to be the second limiting amino acid in growing horses ( Graham et al., 1994 ) Because of reported antagonisms between arginine and other amino acids such as lysin e ( Ball et

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102 al., 2007 ) the current study evaluated the impact of CIT supplementation on p lasma lysine, threonine, and methionine. These amino acids are the most limiting for protein synthesis in equine diets (NRC, 2007). In pigs, lysine has been shown to compete with arginine for entry into cells and also inhibits arginase activity ( Wu and Morris, 1998 ) Therefore, the dietary arginine:lysine ratio is a critical factor to consider when supplementing swine diets with arginine. In swine diets, it is recommended that dietary arginine should not exceed 150% than that of lysine ( Wu et al., 2009 ) An older study evaluated arginine supplementation ( 0 .94, 1.29, and 1.63%) in pigs to deter mine the effect of excess arginine on growth ( Hagemeier et al., 1983 ) This study found no effect s on plasma lysine levels, but found that plasma threonine and methionine levels were reduced by excess arginine. In the current study, inclusion of 86 mg/kg BW of citrulline malate in a grain meal had no effect on plasma lysine, threonine or methionine co ncentrations. These findings suggest that citrulline supplementation may provide a means to increase arginine supply while avoiding potential antagonisms with lysine and other key amino acids. Emerging evidence, from both human and animal studies indicate s that arginine supplementation may be a novel therapy for metabolic related disorders ( McKnight et al., 2010 ) Arginine is well known as a potent secretagogue for i nsulin, via NO production ( Calver et al., 1992 ) or the accumulation of polyamines in the pancreatic islets cells ( Sener et al., 1989 ) However, the effect of arginine suppl ementation on circulating insulin has been mixed, with studies finding an increase ( Guoyao and M orris, 1998 ; Adeghate et al., 2001 ; Flynn et al., 2002 ; Kohli et al., 2004 ; Kohlhaas et al., 2011 ) a decrease ( Lucotti et al., 2006 ) or no effect on serum insulin ( Huynh and

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103 Tayek, 2002 ; Fu et al., 2005 ; Wu et al., 2007c ; Bertolo and Burrin., 2008 ; Jobgen et al., 2009 ) Presumably, citrulline supplementation would have similar effects on insulin via conversion to arginine, thereby increasing arginine availability for NO and polyamine production Schwedheim et al. (2007) reported that oral L citrulline supplementation augmented NO dependent signaling in a dose dependent manner and was at least as effective in this regard as L arginine supplementation. In the current study, serum insulin was lower in response to a moderate sized grain meal when horses were supplemented with 86 mg /kg BW citrulline malate compared to a meal with an isonitrogenous amount of urea These results are in contrast to Wu and coworkers (2007c) who observed no effect on serum insulin wh en 0.63% watermelon pomace (with 2.014 g/L citrulline) was provided in the drinking water of Zucker diabetic fatty rats for 4 wk Osowska and coworkers (2006) reported a lower basal plasma insulin concentration after 1 wk of citrulline supplementation ( 5 g 1 ) ) in malnourished rats. Reasons for the discrepancies between studies are likely multifactorial, including the level of arginine or citrulline supplementation, route of supplementation (e.g., intravenous vs. oral), length of supplementation, an d the health status of the subjects it was evaluated in. Furthermore, most st udies have evaluated basal (or fasting ) insulin responses whereas few studies have evaluated actual insulin secretion and removal in response to a glucose and/or insulin challeng e. Although the lower insulinemic response observed in CIT supplemented horses in the current study is difficult to explain, it may have useful application in the management of insulin resistant horses where hyperinsulinemia is thought to be responsible fo r a significant number of aberrant effects (Fr ank, 2008, 2009). The u pper limit of resting serum insulin concentrations is >

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104 in horses ( Frank, 2008 ) Pain and stress with laminitis markedly elevate resting serum insulin concentrations in EMS patients ( Frank, 2008 ) Resting serum insulin concentrations can often range from 100 ies with clinical laminitis ( Frank, 2008 ) In regards to the noted upper limi t concentrations, horses in the present study show ed no signs of insulin resistance or laminitis according to these suggestive concentrations The malate portion of the citrulline malate used in this study could have affected energy metabolism ( Bendahan et al., 2002 ) thereby contributing to the effects observed on serum insulin. Malic acid supplementation was shown to increase both plasma glucose and serum insulin in dair y cattle ( Wang et al., 2009 ) ; however, ruminants exhibit different glyce mic control than monogastrics such as the horse. Because a malate only supplemented group of horses was not included in the current study, the potential impact of malate (instead of citrulline) on serum insulin cannot be excluded. A hallmark of insulin res istance is a disturbance in glucose homeostasis. Upper resting glucose concentrations suggestive of an insulin resistant horse are > 100 mg/dL or 5.5 mmol/L ( Frank, 2008 ) Glucose homeostasis is primarily influenced by pancreatic cell response to glucose and the sensitivity of the body tissues to insulin. In the current study, although insulinemic response to a meal was reduced, short term supplementation with CIT had no effect on insulin sensitivity or cell responsiveness, as e stimated by RISQI MIRG, HOMA and glucose to insulin ratio. These proxies are calculated from fasting plasma glucose and serum insulin values, as opposed to dynamic responses to glucose and insulin evaluated with minimal model analyses of a frequently sam pled glucose insulin tolerance test (FSGIT) or evaluation of insulin

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105 sensitivity and pancreatic responsiveness to glucose using the euglycemic hyperinsulinemic clamp technique. Nonetheless, the RISQI and MIRG proxies were developed as predictors for minima l model outcomes from an FSGIT (Treiber et al., 2005b). Together, they identify apparently healthy individuals that are compensating for low insulin sensitivity with increased cell activity. Further, these proxies provide a means to determine changes in glucose tolerance that may occur between plasma insulin and its target action at the level of the tissue or whether changes resulted from altered cell responsiveness. However, care should be taken when using RISQI and MIRG to determine insulin sensitivit y and glucose tolerance as large daily variation and poor repeatability has been demonstrated in ponies and horses ( Pratt et al., 2009 ) It should be noted that all horses in the present study were clinically normal and none had a history of laminitis. Additionally, all horses exhibited fasting serum insulin concentrations < 20 uU/ mL indicating they were not hyperinsulinemic, as well as RISQI 0.33 mIU/L 0.5 indicating they were not insulin resistant (Pratt et al., 2009). Further, MIRG was 5.5 (mU insulin 2 /[10 L mg glucose ]) in all horses, indicate adequate glucose tolerance ( Pratt et al., 2009 ) Because many of the studies noting improvements in insulin sensitivity with arginine supplementation were performed in subjects with glycemic or pancreatic dysfunction, results with citrulline may be different in diabetic or insulin resistant subjects fr om those observed here. Elevated levels of NO have been shown to reduce hepatic glucose production and increase glycogen disposal (e.g., via increased glycogen synthesis in skeletal muscle, liver and adipose tissues), as well as increase blood flow into ti ssues ( Piatti et al., 2001 ; Kingwell et al., 2002 ; Wu et al., 2007c ; Jobgen et al., 2009 ; Clemmensen et al., 2011 ;

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106 Monti et al., 2012 ) In diabetic rats, arginine supplementation was suggested to increase insulin sensitivity by increasing the secretory response of the remaining pancreatic cells via enhanced production of NO, resulting in a concurrent reduction in plasma glucose ( Kohli et al., 2004 ) Similar to findings for insulin, the effects of arginine on glucose have varied with some studies showing a decrease in plasma glucose ( Wu et al., 2007c ; Jobgen et al., 2009 ; Clemmensen et al., 2011 ; Monti et al., 2012 ) or no change in plasma glucose ( Huynh and Tayek, 2002 ) Oral ingestion of citrulline is also known to increase NO production ( Hayashi et al., 2005 ) ; thus, we hypothesized citrulline malate supplementation would behave similarly to arginine in regards to glycemic contr ol. Supplementation with a citrulline rich watermelon pomace resulted in lower fasting blood glucose concentrations in Zucker diabetic fatty rats (Wu et al., 2007c). In the current study, there was a trend for a slight reduction in peak glucose concentrati on when horses were fed a meal containing CIT compared to urea. However, the overall glycemic response to a meal was not different between CIT and urea supplemented horses, despite reductions in circulating insulin concentrations. In conclusion, CIT suppl ementation appeared to modify the insulinemic response to a high starch meal while maintaining glycemic control. These data should be confirmed with a more stringent evaluation of insulin sensitivity and pancreatic responsiveness, such as minimal model an alysis with a FSGIT or euglycemic hyperinsulinemic clamp. Insulin resistant horses usually have hyperinsulinemia, particularly in response to starch and sugar consumption as the pancreas tries to compensate for a decrease in tissue cell effectiveness ( Zimmel and McFarlane, 2009 )

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107 Citrulline supplementation could be used as a potential therapeutic agent to reduce excessive secretion of insulin while maint aining glycemic control.

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108 A PPENDIX A PLASMA GLUCOSE ASSAY Materials Timer Gloves Microcentrifuge rack 56 Microcentrifuge vials ( per assay plate ) Four 20 mL scintillation vials (per kit ; used for reconstituting kit enzyme mixture ) 50 mL sterile centrifuge conical ( ) Kimwipes Parafilm Bucket of ice to keep supplies at 4C UltraPure water (approximately 5 mL) Incubator (set at 37 C) Plate reader Transfer pipettes Two 1 mL syringes for UltraPure water One 12 mL syringe Si x small gauge needles (20g works well) 20 Pipette man 100 Pipette man 200 Pipette man 1000 Pipette man Pipette tips for each individual pipette man size Glucose Assay Kit ( Cayman Chemical Company, Ann Arbor, MI ) containing: o One bottle of Glucose Assay Standard o One vial of Glucose Assay Buffer o Four vials of lyophilized Glucose Enzyme Mixture o Two 96 well plates o NOTE: one 96 well plate can analyze 39 samples, 1 internal control, and 8 standards (all run in duplicate). Thus, one Glucose Assay Kit can run 78 study samples However, the company sometimes skimps on the amount of enzyme provided, reducing the number of samples per kit to ~56. Before Beginning the A ssay (the day before) : T ha w samples and glucose kit in the refrigerator ( 4C ) overnight before using Turn on the i ncubator and make sure it is set for 37C. All reagents, samples and UltraPure water should be equilibrated to 4C before the start of the assay. Take 96 well plates f rom glucose kit and let sit at room temperature overnight ALWAYS v ortex samples immediately prior to pipetting to make sure they are mixed well.

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109 Preparation of the I nternal C ontrol NOTE: Inclusion of an internal control on each plate provides quality control for the assay, ensuring you/machine/reagents are all operating properly and consistently each time the assay is completed. It will also be used to calculate interassay variation. 1. To make a pooled internal control sample, pipette 250 from several study plasma sample s into a 50 mL sterile conical vial 2. Vortex the vial for 10 seconds to mix. 3. Dispense desired amount into microcentrifuge vials ( 0 250 mL in each ) for storage. These i nternal control samples can be stored at 20 C if not needed on the day of preparation (similar to how plasma samples are stored long term). Preparation of the Glucose S tandards 1. To prepare the concentrated glucose standards begin by labeling 8 microcentrifuge vials A though H on the top and place vials in a rack. 2. well prior to pipetting. 3. Using a 20 p ipette Stan (according to the table below). Note the unique pipetting needed for vials F G and H ; c heck off the boxes provided in the table to maintain accuracy. 4. Add the appropriate amount of ose A ssay B uffer to each vial according to the table below. Table A 1. Preparation of glucose standards. Vial Assay Buffer FINAL Glucose Concentration (mg/dL) Pipetteman A 0 200 0 B 2.5 197.5 12.5 C 5 195 25 D 10 190 50 E 20 180 100 F 30 170 150 G 40 160 200 H 50 150 250 Assay Part I Pipetting Standards and Samples 1. Label 8 new microcentrifuge vials A through H for your working standards 2. Label 39 microcentrifuge vials 1 through 39 for each plasma sample. 3. Label 1 microcentrifuge vial for your internal control (pooled) sample.

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110 4. Immediately prior to pipetting any standa rd or sample, v ortex the sample to ensure it is mixed well. 5. Using the 20 L Pipetteman, pipette 5 of each concentrated standard to its respective, newly labeled microcentrifuge vial (created in step #1) 6. Using the 20 L Pipetteman, pipette 5 of plasma sample to microcentrifuge vials 1 39 (created in step #2) 39. 7. Using the 20 L Pipetteman, pipette 5 of the internal control sample to microcentrifuge vial # 40 from the pooled plasma prepared earlier. Assay Part II Enzyme Mixture P reparation NOTE: The following steps #1 5 should be performed for each of the 4 Enzyme vials in the kit. 8. Add 500 of 4C UltraPure water to enzyme vial. Mix well by gentle inversion. 9. Transfer the reconstituted enzyme solution to a scintillation vial using a transfer pipette. 10. Add 6 mL of A ssay B uffer to the reconstituted enzyme solution in the scintillatio n vial 11. Take an additional 6 mL of the Assay Buffer and rinse any residual solution from the original Enzyme vial, and then transfer this solution to the scintillation vial containing the reconstituted enzyme solution using a transfer pipette In total, 12. Cover top of the scintillation vial with Parafilm and invert 9 times to make sure mixture is completely dissolved and mixed. NOTE: The reconstituted enzyme solution is stable for at least one hour when stored at 4C. NOTE: Two vials of reconstituted enzyme solution will be needed for every plate. However, sometimes the company fails to provide adequate enzyme to run two full plates/kit. Assay Part I I I Incubation and Plating 13. Add 50 0 of the enzyme mixture forcefully down the side of your standard internal control and plasma sample microcentrifuge vials 14. Vortex vials to mix thoroughly. 15. Place vials in a 37C incubator for 10 minutes 16. Using the 200 L Pipetteman, pipette 150 from each microcentrifuge vial to the 96 well plate. Pipette standards, samples and the internal control into duplicate wells. 17. Check for bubbles in the plate P op bubbles with a needle when present Make sure you wipe the needle with a Kimwipe after EACH bubble is popped to avoid cross contamination of samples 18. Read the absorbance at 500 520 nm using a plate reader.

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111 APPENDIX B SERUM INSULIN ASSAY Day 1 Procedures: Preparation of Radioactive Tracer and Insulin Standards NOTE: Make sure radioactive material ( 125 I t racer ; also called Hotstuff ) is in the correct location within the lab oratory (HAZARDOUS MATERIAL). 1. D ilute the 125 I tracer with 100 mL of distilled or deionized water and mix by gentle inversion (ALWAYS WEAR GLOVES) a. Set tracer in radioactive tr a y until needed b. This will be stable at 2 8 C for 30 days after preparation or until expiration date. Once finished with it, place it back in the refrigerator. 2. Do not place anything that is NOT labeled radioactive within the radioactive tray. 3. Take serum samples and insulin standards out of the refrigerator and allow them to warm to room temperature. You will need 200 uL of each sample. 4. Reconstitute insulin standards (lyophilized ; processed in nonhuman s erum ) a. Fill a 250 mL beaker with distilled or deionized water. b. Locate a 10 mL volumetric pipette (preferred) or serological pipette for reconstituting insulin standards. c. Add 6.0 mL of distilled or deionized water to Standard A (zero calibrator A), mix by g entle swirling. d. Add 3.0 mL of distilled or deionized water to standards B through G Mix by gentle swirling. e. Standards will be stable at 20 C for 30 days after reconstitution. f. Aliquot, if necessary to avoid repeated freezing and thawing. Tube Labeling NO TE: Make sure all components are at room temperature before use (15 20 C) 5. Set up tubes (always use two for each ( duplicates ) unless otherwise specified) a. Four tubes use c lear, uncoated polypropylene plastic tubes (5 mL, 12 x 75 mm) ; note that these do not come with the insulin kit. Two, Total Count (TC) tubes : label tubes 1 & 2 Two, None Specific Binding (NSB) tubes: label tubes 3 & 4 b. 24 Standard and Control tubes use green, Insulin Antibody Coated Tubes (come with kit). Two, Reference ( REF ) tubes : label tubes 5 & 6. Two, A Standard 0 : label tubes 7 & 8. Two, A1 Standard 1.525 /mL : label tubes 9 & 10.

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112 Two, A2 Standard 3.05 /mL : label tubes 11 & 12. Two, B Standard 6.1 /mL : label tubes 13 & 14. Two, C Standard 16.5 /mL : label tubes 15 & 16. Two, D Standard 55 /mL : label tubes 17 & 18. Two, E Standard 103 /mL : label tubes 19 & 20. Two, F Standard 200 /mL : label tubes 21 & 22. Two, G Standard 371 /mL : label tubes 23 & 24. Two, known low sample Quality Control ( QC Low ): label tubes 25 & 26. Two, known high sample Quality Control ( QC High ): label tubes 27 & 28. c. Double check your tube labeling with Table B 1; they SHOULD match! d. Sample tubes: These are the horse serum samples. Use green, Insulin Antibody Coated Tubes (come with kit). Label two tubes per sample (ie, in duplicate) ; continue the numbering system used with the standards above (ie, the first horse sample would be tubes 29 & 30 etc) when th ey are renamed by numbers. Radioimmunoa ssay Procedure 6. Step 1: a. Gently swirl each standard before use b. Add standards to appropriate tubes according to Step 1 in Table B 1. EACH tube should have a total volume 200 L when you are done Use 200 L pipette for this process Always pipette directly in to the bottom of the tube. Change pipette tips between pipetting samples! ALWAYS check labels before pipetting into them! 7. Step 2: a. Add 1000 L (1.0 mL ) of insulin 125 I tracer ( Hotstuff) to each tube (Table B 1). Use Eppendorf repeating pipette with 12.5 mL total volume and 250 L increments. Set dial to 4 (4 x 250 uL = 1000uL) This is radioactive! Use gloves and be in the correct radioactive area of your lab IMPORTANT : Do not let more than 40 minu tes elapse between the addition of the first sample and the completion of the tracer!!! Further, do not forget to keep track of how many tubes you use per bottle of t racer!

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113 8. Once all samples have the 1000 L of insulin 125 I, make sure everything is labeled with your last name and date. 9. Cover tubes with saran wrap and let incubate at room temperature for 18 24 hours a. Always note the time in which you completed process. b. Always note which kit you used if you have more than one and how many tubes were u sed. 10. RECORD HOW MUCH RADIOACTIVE MATERIAL WAS USED!! a. Each bottle of 125 I has 3.0 uci of radioactive material. Make sure you label within the lab. b. Record: 1) Date 2) Activity removed (3 uci/100 tubes = 0.03 *_____tubes used for day) 3) Remaining activity amount in bottle (3.0 uci number from # 2) 4) 5) Final disposal: WASTE 6) Provide the initials of your name for the day.

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114 Table B 1. Preparation of standards and samples for the serum insulin radioimmunoassay. Contents Tube Label # of tubes Tube Type Step 1 b Step 2 b Total 1,2 2 Clear Plastic Nothing insulin I 125 NSB 3,4 2 Clear Plastic Nothing insulin I 125 Ref 5,6 2 Green Coat a count A standard insulin I 125 A standard IU/mL 7,8 2 Green Coat a count A standard insulin I 125 A1 Standard IU/mL (extra standard) 9,10 2 Green Coat a count A standard insulin I 125 B standard A2 Standard IU/mL (extra standard) 11,12 2 Green Coat a count A standard insulin I 125 B standard B standard IU/mL 13,14 2 Green Coat a count B standard insulin I 125 C standard IU/mL 15,16 2 Green Coat a count C standard insulin I 125 D standard IU/mL 17,18 2 Green Coat a count D standard insulin I 125 E standard IU/mL 19,20 2 Green Coat a count E standard insulin I 125 F standard IU/mL 21,22 2 Green Coat a count F standard insulin I 125 G standard IU/mL 23,24 2 Green Coat a count G standard insulin I 125 Sample QC Low 25,26 2 a Green Coat a count sample insulin I 125 Sample QC High 27,28 2 a Green Coat a count sample insulin I 125 All other samples (horse serum) 29,30 ..etc. 2 a Green Coat a count sample insulin I 125 a Depends on pipetting skills. Duplicates were run in this study, but with good pipetting skills a single sample can be run. b Empty boxes in Step 1 and Step 2 can be used as check boxes during pipetting, to ensure completion of those steps. c Calculations f or insulin concentration in standards were based on 1 IU of insulin = 455 ng, so 1 IU should = 0.455 ng. (e.g., if you had 10 IU it would be 4.55 ng).

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115 Day 2 Procedures : Decant Samples 1. Cut a piece of absorbent paper big enough to fold in half to turn your tubes upside down on and place in radioactive tray. 2. Transfer all tubes EXCEPT the Total Count ( TC ) tubes to a green foam r ack. Make sure they are in inserted firmly in the green foam rack. 3. Decant (dump liquid) into specified radioactive tub locat ed under the fume hood. Then place the tubes inside the foam rack upside down on the absorbent paper you cut in step one, within the radioactive area and strike the tubes a few times to remove all visible moisture and residual droplets. This will enhance p recision. Allow them to dry for 2 3 minutes. 4. Transfer tubes (including the Total Count ( TC ) tubes, which should still have liquid in them) to plastic racks in order to carry them to the Auto Gamma Counter 5. Place racks with tubes on radioactive tray and g rab extra gloves. Make sure when carrying radioactive material to another lab you are only wearing ONE glove to carry th e tray, and the other hand is b ar e to open doors as necessary. Carry Tray to the gamma counter. Packard Cobra Auto Gamma Counter F igure B 1. Packard Cobra Auto Gamma Counter 1. Insert a floppy disk (yes, it is very old school!) into the machine so your work will be saved upon completion of a run 2. Record your name, date, time, isotope, and number of tubes in the gamma counter log book 3. Grab the correct ID for the Equine Insulin Protocol ( which is 50 ) from drawer. This goes on the side of the black cassettes that hold 20 samples (machine will automatically read this ID strip) Be sure to put all tubes in the cassettes from the right (spot 1) to the lef t (spo t 20) Actual Counter KEYBOARD 20 samples Stop cartridge First Rack with # 50 on it for Protocol The gamma counter will feed racks to other side 20 samples 20 samples 20 samples

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116 Keep your order of the tubes the same! Your first cassette should be the one that has your TC, NSB, Ref., and all standards within it labeled 50 4. Place tube racks into the gamma counter in order ( see diagram above ) The TC tubes should be on the ri ght near the actual gamma counter, at the front in spots 1 and 2 (labeled on cartridge) ) STOP will tell the machine that your samples have all been run 5. On the counter/keyboard: press any key to start Hit F1 see a screen that shows different protocols for things like insulin, cortisol, estrogen, P4, etc.) Hit F3 (Protocol for Edit) and enter 50 for the Equine Insulin Protocol. Hit F2 (to get to Commands) Hit F6 Next protocol, which will then START the assay Swipe Protocol A swipe MUST be made within seven days of an assay being run according to the Division of Environmental Health and Safety to ensure no radioactive material has been spread. 1. Obtain 10 clear plastic tubes (5 mL 75 x 12 mm tubes) 2. Obtain a radioactive swipe sheet from the front pocket of the binder to fill out (bind er is labeled RALGO ) 3. Put the clear tubes in a rack and go to the radioactive (hot) room 4. In the top drawer to the left is the filt er paper needed to make swipes (i n a little green box with the # 41 on it ) 5. Follow the instructions on the sheet regarding the particular swiping areas. Each area is labeled 1 through 10. Put gloves on, take a piece of the filter paper and swirl it around on the surface of each numbered area. Once completed, fold filter paper up and put it into the tube according to the corre ct number 1 through 10. To prepare to take it to the gamma counter, remove and dispose of one lab coat.

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117 Figure B 2. Swipe protocol. 6. Grab the plastic background cassette with the plastic tubes already in it from the top, left drawer by the gamma counter. These are the tubes for measuring the environment and should have nothing in them. This should be labeled with the clip number 30 7. Grab the clip number 29 and load your swipe samples in the bla ck cassette from right to left 8. Always make sure you place the plastic tube holder with the STOP clip on it, behind your samples. 9. Press F2 to initiate the protocol to run 10. Press F6 11. Once you have printed results you need to log this in the binder you initially got the paper for the swipes out of (RALGO binder). 12. Completely fill out the paper and put it in the binder with results you printed 13. Calculate the background first by averaging your CPM for the background (first tube holder labeled 30 ), the first print out with 5 tubes. 14. Calculate the swipes by taking the number found in 13, and subtracting all A:CPM from your swipe 15. Take your number from 14 and multiply it by 0.70 for the 70% efficiency for each swipe, 1 through 10. Your resulting numbers should NEVER be above 100. If so, notify someone immediately. Door to room 1 2 3 Floor 4 5 6 7 Floor 8 5 = In the sink 8 = In front of the Hood 9 9 =The front of fridge 10 = The fridge handle 10 Floor

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118 A PPENDIX C AMINO ACID, UREA, AND AMMONIA ANALYSIS Materials Needed 1. 2 mL microcentrifuge vials Eppendorf safe lock, 2 mL; hinged lid; clear vials Fisherbrand: 500 pk ( Part number: 05 402 7 ) 2. Syringe and needle 3cc, 20g with 1 inch needles Fisherbrand: 100 pk ( Part number: 1484061 ) 3. Syringe filters Nylon membrane; pore size: 0.22 um, Diameter: 25mm, sterile Fisherbrand: 50 per box ( Part number: 09 719C ) Low hold up volume helps you recover virtually your entire sample after filtering Tested for 100% membrane and housing integrity, eliminating concerns about fluid loss. 4. Vial, autosampler; Snap Cap, wide opening, 12 mm dia. X 32 mm, 2 mL Clear glass vial Fisherbrand: 100 pk ( Part number: 03 395C or C4011 5 ) 5. Closures for Glass Vials Pre slit blue PTFE/Silicone Septa; Clear Fisherbrand: 100 pk ( Part number: 03 396X or C4011 55 ) 6. 100 or 200 L pipette tips 7. 1000 L pipette tips Reagents for Physiologic Fluid Analysis of Amino Acids 1. 35% (w/v) sulfosalicylic acid (SSA): 5 Sulfosalicylic Acid Dihydrate SIGMA ALDRICH: 100G ( Part number: S2130 100G ) 2. Aminoethyl cystine (AEC): S (2 Aminoethyl) L cysteine hydrochloride 98% (TLC) SIGMA ALDRICH: 250MG ( Part number: A2636 250MG ) 3. 0.02 N HCL hydrochloric acid F.W.: 36.46, 1 hydrogen; (36.46g 0.02 = 0.7292g/L)

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119 Recipes for SSA, HCL, AEC 35% (w/v) SSA Recipe 1. Measure 350 g of sulfosalicylic acid (% = (g/ mL ) 100) ) 2. Fill to 1 L with ddH 2 O 0.02 N HCL Recipe 1. Calculate equivalent mass (E mass = FM/# H)( FW = formula weight, #H = number of hydrogen donors) 2. Grams = (N desired)(E mass ) (Volume in L) Or for liquids use: (Volume 1 ) (Concentraiton 1 ) = ( Volume 2 )(Concentration 2 ) AEC/SSA Solution 1. 126 mg AEC/ 250 mL 35% SSA. 2. Use 50 L of this AEC/SSA solution per 0.5 mL of plasma, with usual preparation of samples for analysis. 3. Used to provide a percent recovery of amino acids (a measure of extraction efficiency). 4. Recommended by Dr. Thomas P. Mawhinney Director of the Agricultural Extension Service Chemistry Laboratory at the University of Missouri (, 573 882 2608). Calculat ing Percent R ecovery of AEC Need 1 part AEC/SSA solution to 10 parts plasma Thus, 1/11 part AEC/SSA which is mixed into 1 part what? to 1 part 0.02 N HCL Therefore, (1/11) (1/2) = 1/22 is used to account for dilutions

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120 NOTE: There is a 2.2 multiplier factor on the amino acid an alyzer. Thus, 114 x 2.2 = 250 nmol/mL NOTE: Molecular Weight (MW) of AEC = 200.69 NOTE: For this study, which had 72 samples that used 50 L of the AEC/SSA solution per sample, a total of 3.6 mL of AEC/SSA solution was needed. Preparation of Plasma Samples for Amino Acid Analysis 1. Thaw plasma samples in the refrigerator prior to amino acid extraction 2. Keep samples on ice at ALL TIMES once you take them out of the fridge. 3. Vortex each plasma sample prior to pipetting. 4. Pipette 0.5 mL ( 500 L ) of plasma in to a 2 mL microcentrifuge vial 5. Add 50 L of AEC/35% SSA solution to each microcentrifuge vial containing plasma 6. Vortex each vial to mix well. 7. Incubate the vortexed plasma and AEC/SSA solution at 4 C for 20 minutes 8. Centrifuge the microcentrifuge vials a t 4 C for 10 minutes at 11,000 g 9. Draw up 0.5 mL of supernatant with syringe from centrifuged samples. Do not disturb the precipitated pellet on the bottom of the microcentrifuge vial. NOTE: Record the amount of supernatant recovered. S ometimes it may on ly be 0. 2 or 0 3 mL. If it is less than 0.5 mL you will need to a d just the amount of HCl added in step #11 below to ensure a 1:1 ratio between supernatant and HCl. E .g. if you only have 0.3 mL of supernatant, then you need 300 L of HCl. 10. Filter the recovered supernatant through a 0.2 m pore filter into a glass Snap It vial 11. Add 0.5 mL ( 500 L ) of 0.02 N HCl into the same Snap It vial and place the cap on the vial Make sure you have adjusted for your supernatant. See NOTE in step # 9. 12. Vortex the Snap It vial to mix well. 13. Keep samples on ice! 14. Samples are now ready for the amino acid analyzer 15. Samples can be r un immediately or store d at 4 C for no longer than 3 days 16. Refer to the manual (located in the lab) for the how t o utilize the Hitachi L 8900 Amino Acid Analyzer.

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121 Table C 1 Composition of buffers used in Hitachi L 8900 Amino Acid Analyzer Name PF 1 PF 2 PF 3 PF 4 PF RG Vessel (buffer) B1 B2 B3 B4 B5 Lithium concentration(N) 0.09 0.255 0.721 1.00 0.20 Distilled water (approx.) 700 mL 700 mL 700 mL 700 mL 700 mL Lithium citrate (4H 2 O) 5.73 g 9.80 g 8.79 g 9.80 g -----Lithium chloride 1.24 6.36 26.62 38.15 -----Citric acid (H 2 O) 19.90 g 12.00 g 11.27 g 3.30 g -----Lithium hydroxide --------------------8.40 g Ethanol 30.0 mL 30.0 mL 100.0 mL -----30.0 mL Thiodiglycol 5.0 mL 5.0 mL ---------------Benzyl alcohol ----------3.0 mL ----------Brij 35 4.0 mL 4.0 mL 4.0 mL 4.0 mL 4.0 mL pH (nominal) 2.8 3.7 3.6 4.1 -----Total (adjust) 1.0 L 1.0 L 1.0 L 1.0 L 1.0 L Caprylic acid 0.1 mL 0.1 mL 0.1 mL 0.1 mL 0.1 mL (Note*) indicate solution that was made in lab, all other solutions were purchased

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122 LIST OF REFERENCES Abel, E. D., P. R. Shepherd and B. B. Kahn. 2004. Glucose transporters and pathophysiologic states. Diabetes Mellitus: A Fundamental and Clinical Text 3rd Edition. 918 938. Adeghate, E., A. S. Ponery, T. El Sharkaway and H. Parvez. 2001. L arginine stimul ated insulin secretion for the pancrease of normal and diabetic rats. Amino Acids. 21: 205 209. AHC (American Horse Council) 2012. National Economic Impact of the U.S. Horse Insustry. Accessed May 13, 2012. economic impact us horse industry Albina, J. E., C. D. Mills, A. Barbul, C. E. Thirkill, W. L. H. Jr, B. Mastrofrancesco and M. D. Caldwell. 1988. Arginine metabolism in wounds. Am. J. Physiol. 254: E459 E467. Alemzadeh, R., G. Langley, L. Upchurch, P. Smith and A. E. Slonim. 1998. Benificial effect of diazoxide in obese hyperinsulinemic adults. J. Clin. Endocrinol. Metab. 83: 1911 1915. Anderson, R. A. 2008. Chronium and polyp henols from cinnamon improve insulin sensitivity. Proc. Nut. Soc. 67: 48 53. Asplin, K. E., M. N. Sillence, C. C. Pollitt and M.C McGowan. 2007. Induction of laminitis by prolonged hyperinsulinaemia in clinically normal ponies. Vet. J. 174: 530 535. Bach Feed Science and Technology. 90: 3 20. Bailey, S. R., H. L. Habershon Butcher, K. J. Ransom, J. Elliott and N. J. Menzies Gow. 2008. Hypertension and insulin resistance in a mixed breed population of ponies predisposed to laminitis. Am. J. Vet. Res. 69: 122 129. Ball, R. O., K. L. Urshel and P. B. Pencharz. 2007. Nutritional consequences of interspecies differences in arginine and lysine metabolism. J. Nutr. 137: 1626S 1641S. Barr, F. E., R. G. Tirona, M. B. Taylor, G. Rice, J. Arnold, G. Cunningham, H. A. B. Smith, A. Campbell, J. A. Canter, K. G. Christian, D. C. Drinkwater, F. Scholl, A. Kavanaugh McHugh and M. L. Summar. 2007. Pharmacokinetics and safety of in travenously administered citrulline in children undergoing congenital heart surgery: Potential therapy for postoperative pulmonary hypertension. J. Thoracic and Cardiovascular Surgery. 134: 319 326.

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143 BIOGRAPHICAL SKETCH Leigh Ann Skurupey was b orn in Williston, North Dakota, yet she spent most of her childhood in Parker, Colorado and later graduated from Ponderosa High School in 2002. Immediately following graduation, she moved to Sterling, Colorado to attend Northeastern Junior College wh ere s onors and received an Associat e of Applied Science Degree in equine m anagement. After her hiatus from school and training horses for two years she decided to continue her education in Cheyenne, Wyoming at Laramie Co unty Community College wh ere she graduated in 2008 with honors d istinction, and received Associate of Science Degree in a griculture. Immediately following, she transferred to Colorado State University in Fort Collins Colorado where she was awarded a Bach elor of Scienc e Degree with a first major in equine science, a second major in animal science, and a minor in business a dministration. She graduated cum laude from Colorado State University in 2010. She i s currently pursuing her Master of Science at the Un iversity of Florida in Gainesv ille, Florida, specializing in equine n utrition with the intention of graduation in summer 2012. Her passion for horses has its root in working with her mother raising miniature horses in addition to visi ting her father in North Dakota where she would ride horses with her aunts every chance she could get Leigh Ann, only three years of age, would for of her pants for constant support in efforts to break the p onies to ride. Her love for sports dealing with horses had excided her to perform and test her talents. Her college accolades abounds most of her experiences and Leigh Ann successfully competed in college on the rodeo t eam, the stock horse team, and made it to Intercollegiate Horse Show Association Nationals for her college equestrian team in

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144 2007. She also successfully competed on the intercollegiate horse judging team at Colorado State University where she was sixth hi gh individual overall at the All American Quarter Horse Congress competition, and finished on the reserve champion team. Further, she was ninth high individual overall at the American Quarter Horse World Show judging contest, and was also on the reserve ch ampion team. Her passion for horse judging awarded her the opportunity to attend the Unive rsity of Florida for her Master of Science on an a judging team. Upon completion of her Master of Science p rogram, she will begin her fall semester at the University of Florida as a doctoral candidate in the Department of Animal Scie nces, with a specialization in equine nutrition and a minor in exercise p hysiology. She hopes to be able to use the information an d experience gained through her education to continue researching ways for innovative therapies and interventions to improve the well being and health of horses.