Advanced glycation endproducts in horses with insulin-induced laminitis

Advanced glycation endproducts (AGEs) have been implicated in the pathogenesis of cancer, inflammatory conditions and diabetic complications. An interaction of AGEs with their receptor (RAGE) results in increased release of pro-inflammatory cytokines and reactive oxygen species (ROS), causing damage to susceptible tissues. Laminitis, a debilitating foot condition of horses, occurs in association with endocrine dysfunction and the potential involvement of AGE and RAGE in the pathogenesis of the disease has not been previously investigated. Glucose transport in lamellar tissue is thought to be largely insulin-independent (GLUT-1), which may make the lamellae susceptible to protein glycosylation and oxidative stress during periods of increased glucose metabolism. Archived lamellar tissue from horses with insulin-induced laminitis (n=4), normal control horses (n=4) and horses in the developmental stages (6 h, 12 h and 24 h) of the disease (n=12) was assessed for AGE accumulation and the presence of oxidative protein damage and cellular lipid peroxidation. The equine-specific RAGE gene was identified in lamellar tissue, sequenced and is now available on GenBank. Lamellar glucose transporter (GLUT-1 and GLUT-4) gene expression was assessed quantitatively with qRT-PCR in laminitic and control horses and horses in the mid-developmental time-point (24 h) of the disease. Significant AGE accumulation had occurred by the onset of insulin-induced laminitis (48 h) but not at earlier time-points, or in control horses. Evidence of oxidative stress was not found in any group. The equine-specific RAGE gene was not expressed differently in treated and control animals, nor was the insulin-dependent glucose transporter GLUT-4. However, the glucose transporter GLUT-1 was increased in lamellar tissue in the developmental stages of insulin-induced laminitis compared to control horses and the insulin-independent nature of the lamellae may facilitate AGE formation. However, due to the lack of AGE accumulation during disease development and a failure to detect an increase in ROS or upregulation of RAGE, it appears unlikely that oxidative stress and protein glycosylation play a central role in the pathogenesis of acute, insulin-induced laminitis.

The impact of prolonged hyperinsulinaemia on glucose transport in equine skeletal muscle and digital lamellae

REASONS FOR PERFORMING STUDY An increased incidence of metabolic disease in horses has led to heightened recognition of the pathological consequences of insulin resistance (IR). Laminitis, failure of the weight-bearing digital lamellae, is an important consequence. Altered trafficking of specialised glucose transporters (GLUTs) responsible for glucose uptake, are central to the dysregulation of glucose metabolism and may play a role in laminitis pathophysiology. OBJECTIVES We hypothesised that prolonged hyperinsulinaemia alters the regulation of glucose transport in insulin-sensitive tissue and digital lamellae. Our objectives were to compare the relative protein expression of major GLUT isoforms in striated muscle and digital lamellae in healthy horses and during hyperinsulinaemia. STUDY DESIGN Randomised, controlled study. METHODS Prolonged hyperinsulinaemia and lamellar damage were induced by a prolonged-euglycaemic hyperinsulinaemic clamp (p-EHC) or a prolonged-glucose infusion (p-GI) and results were compared to electrolyte-treated controls. GLUT protein expression was examined with immunoblotting. RESULTS Lamellar tissue contained more GLUT1 protein than skeletal muscle (p = 0.002) and less GLUT4 than the heart (p = 0.037). During marked hyperinsulinaemia and acute laminitis (induced by the p-EHC), GLUT1 protein expression was decreased in skeletal muscle (p = 0.029) but unchanged in the lamellae, while novel GLUTs (8; 12) were increased in the lamellae (p = 0.03), but not skeletal muscle. However, moderate hyperinsulinaemia and subclinical laminitis (induced by the p-GI) did not cause differential GLUT protein expression in the lamellae vs. control horses. CONCLUSIONS The results suggest that lamellar tissue functions independently of insulin and that IR may not be an essential component of laminitis aetiology. Marked differences in GLUT expression exist between insulin-sensitive and insulin-independent tissues during metabolic dysfunction in horses. The different expression profiles of novel GLUTs during acute and subclinical laminitis may be important to disease pathophysiology and require further investigation.

Equine hyperinsulinemia: Investigation of the enteroinsular axis during insulin dysregulation

Compared to other species insulin dysregulation in equids is poorly understood. Hyperinsulinemia causes laminitis, a significant and often lethal disease affecting the pedal bone/hoof wall attachment site. Until recently, hyperinsulinemia has been considered a counter-regulatory response to insulin resistance (IR), but there is growing evidence to support a gastrointestinal etiology. Incretin hormones released from the proximal intestine, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide, augment insulin secretion in several species, but require investigation in horses. This study investigated peripheral and gut-derived factors impacting insulin secretion by comparing the response to intravenous (IV) and oral D-glucose. Oral and IV tests were performed in 22 ponies previously shown to be insulin dysregulated, of which only 15 were classified as IR (IV test). In a more detailed study, nine different ponies received four treatments: D-glucose orally, D-glucose IV, oats and Workhorse-mix. Insulin, glucose and incretin concentrations were measured before and after each treatment. All nine ponies showed similar IV responses, but five were markedly hyper-responsive to oral D-glucose and four were not. Insulin responsiveness to oral D-glucose was strongly associated with blood glucose concentrations and oral glucose bioavailability, presumably driven by glucose absorption/distribution, as there was no difference in glucose clearance rates. Insulin was also positively associated with active GLP-1 following D-glucose and grain. This study has confirmed a functional enteroinsular axis in ponies which likely contributes to insulin dysregulation that may predispose them to laminitis. Further, IV tests for IR are not reliable predictors of the oral response to dietary non-structural carbohydrate.

Metabolic dysfunction and laminitis

Objective: This review focuses on laminitis that develops as a result of metabolic dysfunction and aims to provide a concise assessment of the current state of knowledge on this form of the disease. Outline: The most prevalent form of laminitis is associated with metabolic or endocrinopathic diseases, such as Equine Metabolic Syndrome and pituitary pars intermedia dysfunction, and the feeding of high-energy diets, particularly those rich in non-structural carbohydrates. Insulin dysregulation is the key hormonal imbalance implicated in causing this form of laminitis and hyperinsulinaemia is an important risk factor for the disease. Hyperinsulinaemia can occur in association with insulin resistance, obesity, regionalised adiposity, dysregulated cortisol metabolism and may also be related to other factors, such as breed and genetic predisposition. Recognition of hyperinsulinaemia is best achieved by using a dynamic oral glucose test that can be performed relatively easily under field conditions. Insulin produces a unique pathological lesion in the lamellae and the features of this lesion have informed investigations on the pathogenesis of the disease. Research into the mechanism of disease is continuing so that more targeted therapies than are currently available can be developed. However, dietary restriction and exercise remain effective management strategies for metabolic disease. Conclusions: Although the pathogenic mechanism/s of metabolic and endocrinopathic forms of laminitis remain the subject of intense research, ample data on risk factors for the disease are available. Efforts focussed on preventing the disease should aim to identify metabolic disease and reduce obesity and insulin resistance in at-risk individuals.

AICAR administration affects glucose metabolism by upregulating the novel glucose transporter, GLUT8, in equine skeletal muscle

Equine metabolic syndrome is characterized by obesity and insulin resistance (IR). Currently, there is no effective pharmacological treatment for this insidious disease. Glucose uptake is mediated by a family of glucose transporters (GLUT), and is regulated by insulin-dependent and -independent pathways, including 5-AMP-activated protein kinase (AMPK). Importantly, the activation of AMPK, by 5-aminoimidazole- 4-carboxamide-1-D-ribofuranoside (AICAR) stimulates glucose uptake in both healthy and diabetic humans. However, whether AICAR promotes glucose uptake in horses has not been established. It is hypothesized that AICAR administration would enhance glucose transport in equine skeletal muscle through AMPK activation. In this study, the effect of an intravenous AICAR infusion on blood glucose and insulin concentrations, as well as on GLUT expression and AMPK activation in equine skeletal muscle (quantified by Western blotting) was examined. Upon administration, plasma AICAR rapidly reached peak concentration. Treatment with AICAR resulted in a decrease (P < 0.05) in blood glucose and an increase (P < 0.05) in insulin concentration without a change in lactate concentration. The ratio of phosphorylated to total AMPK was increased (P < 0.05) in skeletal muscle. While GLUT4 and GLUT1 protein expression remained unchanged, GLUT8 was increased (P < 0.05) following AICAR treatment. Up-regulation of GLUT8 protein expression by AICAR suggests that this novel GLUT isoform plays an important role in equine muscle glucose transport. In addition, the data suggest that AMPK activation enhances pancreatic insulin secretion. Collectively, the findings suggest that AICAR acutely promotes muscle glucose uptake in healthy horses and thus its therapeutic potential for managing IR requires investigation.

Degradation, cyclic adenosine monophosphate production, insulin secretion, and glycemic effects of two novel N-terminal Ala(2)-substituted analogs of glucose-dependent insulinotropic polypeptide with preserved biological activity in vivo

Glucose-dependent insulinotropic polypeptide (GIP) has significant potential in diabetes therapy due to its ability to serve as a glucose-dependent activator of insulin secretion. However, its biological activity is severely compromised by the ubiquitous enzyme dipeptidylpeptidase IV (DPP IV), which removes the N-terminal Tyr(1)-Ala(2) dipeptide from GIP. Therefore, 2 novel N-terminal Ala(2)-substituted analogs of GIP, with Ala substituted by 2-aminobutyric acid (Abu) or sarcosine (Sar), were synthesized and tested for metabolic stability and biological activity both in vitro and in vivo. Incubation with DPP IV gave half-lives for degradation of native GIP, (Abu(2))GIP, and (Sar(2))GIP to be 2.3, 1.9, and 1.6 hours, respectively, while in human plasma, the half-lives were 6.2, 7.6, and 5.4 hours, respectively. In Chinese hamster lung (CHL) cells expressing the cloned human GIP receptor, native GIP, (Abu(2))GIP, and (Sar(2))GIP dose-dependently stimulated cyclic adenosine monophosphate (camp) production with EC50 values of 18.2, 38.5, and 54.6 nmol/L, respectively. In BRIN-BD11 cells, both (Abu(2))GIP and (Sar(2))GIP (10(-13) to 10(-8) mol/L) dose-dependently stimulated insulin secretion with significantly enhanced effects at 16.7 mmol/L compared with 5.6 mmol/L glucose. In obese diabetic (ob/ob) mice, GIP and (Sar(2))GIP significantly increased (1.4-fold to 1.5-fold; P <.05) plasma insulin concentrations, whereas (Abu(2))GIP exerted only minor effects. Changes in plasma glucose were small reflecting the severe insulin resistance of this mutant. The present data show that substitution of the penultimate N-terminal Ala(2) in GIP by Abu or Sar results in analogs with moderately reduced metabolic stability and biological activity in vitro, but with preserved biological activity in vivo. (C) 2003 Elsevier Inc. All rights reserved.

Effects of nateglinide on the secretion of glycated insulin and glucose tolerance in type 2 diabetes

Aims: Glycation of insulin has been demonstrated within pancreatic beta-cells and the resulting impaired bioactivity may contribute to insulin resistance in diabetes. We used a novel radioimmunoassay to evaluate the effect of nateglinide on plasma concentrations of glycated insulin and glucose tolerance in type 2 diabetes. Methods. Ten patients (5 M/5 F, age 57.8 +/- 1.9 years, HbA(1c), 7.6 +/- 0.5%,, fasting plasma glucose 9.4 +/- 1.2 mmol/l, creatinine 81.6 +/- 4.5 mumol/l) received oral nateglinide 120 mg or placebo, 10 min prior to 75 g oral glucose in a random, single blind, crossover design, 1 week apart. Blood samples were taken for glycated insulin, glucose, insulin and C-peptide over 225 min. Results: Plasma glucose and glycated insulin responses were reduced by 9% (P = 0.005) and 38% (P = 0.047), respectively, following nateglinide compared with placebo. Corresponding AUC measures for insulin and C-peptide were enhanced by 36% (P = 0.005) and 25% (P = 0.007) by nateglinide. Conclusions: Glycated insulin in type 2 diabetes is reduced in response to the insulin secretagogue nateglinide, resulting in preferential release of native insulin. Since glycated insulin exhibits impaired biological activity, reduced glycated insulin release may contribute to the anti hyperglycaemic action of nateglinide. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

Comparison of 5% versus 15% sucrose intakes as part of a eucaloric diet in overweight and obese subjects:effects on insulin sensitivity, glucose metabolism, vascular compliance, body composition and lipid profile. A randomised controlled trial

AIMS: The effect of dietary sucrose on insulin resistance and the pathogenesis of diabetes and vascular disease is unclear. We assessed the effect of 5% versus 15% sucrose intakes as part of a weight maintaining, eucaloric diet in overweight/obese subjects.

METHODS: Thirteen subjects took part in a randomised controlled crossover study (M:F 9:4, median age 46 years, range 37-56 years, BMI 31.7±0.9 kg/m(2)). Subjects completed two 6 week dietary periods separated by 4 week washout. Diets were designed to have identical macronutrient profile. Insulin action was assessed using a two-step hyperinsulinaemic euglycaemic clamp; glucose tolerance, vascular compliance, body composition and lipid profiles were also assessed.

RESULTS: There was no change in weight or body composition between diets. There was no difference in peripheral glucose utilization or suppression of endogenous glucose production. Fasting glucose was significantly lower after the 5% diet. There was no demonstrated effect on lipid profiles, blood pressure or vascular compliance.

CONCLUSION: A low-sucrose diet had no beneficial effect on insulin resistance as measured by the euglycaemic glucose clamp. However, reductions in fasting glucose, one hour insulin and insulin area under the curve with the low sucrose diet on glucose tolerance testing may indicate a beneficial effect and further work is required to determine if this is the case. Clinical Trial Registration number ISRCTN50808730.

Potential Role of Growth Hormone in Impairment of Insulin Signaling in Skeletal Muscle, Adipose Tissue, and Liver of Rats Chronically Treated with Arginine

We have shown that rats chronically treated with Arginine (Arg), although normoglycemic, exhibit hyperinsulinemia and decreased blood glucose disappearance rate after an insulin challenge. Attempting to investigate the processes underlying these alterations, male Wistar rats were treated with Arg (35 mg/d), in drinking water, for 4 wk. Rats were then acutely stimulated with insulin, and the soleus and extensorum digitalis longus muscles, white adipose tissue (WAT), and liver were excised for total and/or phosphorylated insulin receptor (IR), IR substrate 1/2, Akt, Janus kinase 2, signal transducer and activator of transcription (STAT) 1/3/5, and p85 alpha/55 alpha determination. Muscles and WAT were also used for plasma membrane (PM) and microsome evaluation of glucose transporter (GLUT) 4 content. Pituitary GH mRNA, GH, and liver IGF-I mRNA expression were estimated. It was shown that Arg treatment: 1) did not affect phosphotyrosine-IR, whereas it decreased phosphotyrosine-IR substrate 1/2 and phosphoserine-Akt content in all tissues studied, indicating that insulin signaling is impaired at post-receptor level; 2) decreased PM GLUT4 content in both muscles and WAT; 3) increased the pituitary GH mRNA, GH, and liver IGF-I mRNA expression, the levels of phosphotyrosine-STAT5 in both muscles, phosphotyrosine-Janus kinase 2 in extensorum digitalis longus, phosphotyrosine-STAT3 in liver, and WAT as well as total p85 alpha in soleus, indicating that GH signaling is enhanced in these tissues; and 4) increased p55 alpha total content in muscles, WAT, and liver. The present findings provide the molecular mechanisms by which insulin resistance and, by extension, reduced GLUT4 content in PM of muscles and WAT take place after chronic administration of Arg, and further suggest a putative role for GH in its genesis, considering its diabetogenic effect. (Endocrinology 150: 2080-2086, 2009)

Smad5 regulates Akt2 expression and insulin-induced glucose uptake in L6 myotubes

Insulin-induced glucose uptake by skeletal muscle results from Akt2 activation and is severely impaired during insulin resistance Recently, we and others have demonstrated that BMP9 improves glucose homeostasis in diabetic and non-diabetic rodents. However, the mechanism by which BMP9 modulates insulin action remains unknown. Here we demonstrate that Smad5. a transcription factor activated by BMP9, and Akt2. are upregulated in differentiated L6 myotubes. Smad5, rather than Smad1/8, is downregulated ""in vivo"" and ""in vitro"" by dexamethasone Smad5 knockdown decreased Akt2 expression and serine phosphorylation and insulin-induced glucose uptake, and increased the expression of the lipid phosphatase Ship2. Additionally, binding of Smad5 to Akt2 gene is decreased in dexamethasone-treated rats and Increased in L6 myotubes compared to myoblasts The present study indicates that Smad5 regulates glucose uptake in skeletal muscle by controlling Akt2 expression and phosphorylation These finding reveals Smad5 as a potential target for the therapeutic of type 2 diabetes. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Modulation of Bone Morphogenetic Protein-9 Expression and Processing by Insulin, Glucose, and Glucocorticoids: Possible Candidate for Hepatic Insulin-Sensitizing Substance

Bone morphogenetic protein 9 (BMP-9), a member of the TGF-beta superfamily predominantly expressed in nonparenchymal liver cells, has been demonstrated to improve glucose homeostasis in diabetic mice. Along with this therapeutic effect, BMP-9 was proposed as a candidate for the hepatic insulin-sensitizing substance ( HISS). Whether BMP-9 plays a physiological role in glucose homeostasis is still unknown. In the present study, we show that BMP-9 expression and processing is severely reduced in the liver of insulin-resistant rats. BMP-9 expression and processing was directly stimulated by in situ exposition of the liver to the combination of glucose and insulin and oral glucose in overnight fasted rats. Additionally, prolonged fasting ( 72 h) abrogated refeeding-induced BMP-9 expression and processing. Previous exposition to dexamethasone, a known inductor of insulin resistance, reduced BMP-9 processing stimulated by the combination of insulin and glucose. Finally, we show that neutralization of BMP-9 with an anti-BMP-9 antibody induces glucose intolerance and insulin resistance in 12-h fasted rats. Collectively, the present results demonstrate that BMP-9 plays an important role in the control of glucose homeostasis of the normal rat. Additionally, BMP-9 is expressed and processed in an HISS-like fashion, which is impaired in the presence of insulin resistance. BMP-9 regulation according to the feeding status and the presence of diabetogenic factors reinforces the hypothesis that BMP-9 might exert the role of HISS in glucose homeostasis physiology. ( Endocrinology 149: 6326-6335, 2008)

Glucocorticoids in Vivo Induce Both Insulin Hypersecretion and Enhanced Glucose Sensitivity of Stimulus-Secretion Coupling in Isolated Rat Islets

Although glucocorticoids are widely used as antiinflammatory agents in clinical therapies, they may cause serious side effects that include insulin resistance and hyperinsulinemia. To study the potential functional adaptations of the islet of Langerhans to in vivo glucocorticoid treatment, adult Wistar rats received dexamethasone (DEX) for 5 consecutive days, whereas controls (CTL) received only saline. The analysis of insulin release in freshly isolated islets showed an enhanced secretion in response to glucose in DEX-treated rats. The study of Ca(2+) signals by fluorescence microscopy also demonstrated a higher response to glucose in islets from DEX-treated animals. However, no differences in Ca(2+) signals were found between both groups with tolbutamide or KCl, indicating that the alterations were probably related to metabolism. Thus, mitochondrial function was explored by monitoring oxidation of nicotinamide dinucleotide phosphate autofluorescence and mitochondrial membrane potential. Both parameters revealed a higher response to glucose in islets from DEX-treated rats. The mRNA and protein content of glucose transporter-2, glucokinase, and pyruvate kinase was similar in both groups, indicating that changes in these proteins were probably not involved in the increased mitochondrial function. Additionally, we explored the status of Ca(2+)-dependent signaling kinases. Unlike calmodulin kinase II, we found an augmented phosphorylation level of protein kinase C alpha as well as an increased response of the phospholipase C/inositol 1,4,5-triphosphate pathway in DEX-treated rats. Finally, an increased number of docked secretory granules were observed in the beta-cells of DEX animals using transmission electron microscopy. Thus, these results demonstrate that islets from glucocorticoid-treated rats develop several adaptations that lead to an enhanced stimulus-secretion coupling and secretory capacity. (Endocrinology 151: 85-95, 2010)

Elevation in tanis expression alters glucose metabolism and insulin sensitivity in H4IIE cells

Increased hepatic glucose output and decreased glucose utilization are implicated in the development of type 2 diabetes. We previously reported that the expression of a novel gene, Tanis, was upregulated in the liver during fasting in the obese/diabetic animal model Psammomys obesus. Here, we have further studied the protein and its function. Cell fractionation indicated that Tanis was localized in the plasma membrane and microsomes but not in the nucleus, mitochondria, or soluble protein fraction. Consistent with previous gene expression data, hepatic Tanis protein levels increased more significantly in diabetic P. obesus than in nondiabetic controls after fasting. We used a recombinant adenovirus to increase Tanis expression in hepatoma H4IIE cells and investigated its role in metabolism. Tanis overexpression reduced glucose uptake, basal and insulin-stimulated glycogen synthesis, and glycogen content and attenuated the suppression of PEPCK gene expression by insulin, but it did not affect insulin-stimulated insulin receptor phosphorylation or triglyceride synthesis. These results suggest that Tanis may be involved in the regulation of glucose metabolism, and increased expression of Tanis could contribute to insulin resistance in the liver.

High glucose-induced impairment in insulin secretion is associated with reduction in islet glucokinase in a mouse model of susceptibility to islet dysfunction

Type 2 diabetes is characterized by islet dysfunction resulting in hyperglycemia, which can then lead to further deterioration in islet function. A possible mechanism for hyperglycemia-induced islet dysfunction is the accumulation of advanced glycation end products (AGE). The DBA/2 mouse develops pancreatic islet dysfunction when exposed to a high glucose environment and/or obesity-induced insulin resistance. To determine the biochemical cause of dysfunction, DBA/2 and C57BL/6 control islets were incubated in 11.1 mM or 40 mM glucose in the absence or presence of the AGE inhibitor aminoguanidine (AG) for 10 days. Basal (2.8 mM glucose) insulin release was increased in both DBA/2 and C57BL/6 islets incubated with 40 mM vs 11.1 mM glucose for 10 days. Chronic exposure to hyperglycemia decreased glucose (20 mM)-stimulated insulin secretion in DBA/2 but not in C57BL/6 islets. AG significantly increased fold-induced insulin release in high glucose cultured DBA/2 mouse islets, but did not affect C57BL/6 islet function. DBA/2 islet glucokinase was significantly reduced following 40 mM glucose culture, compared with 11.1 mM glucose cultured DBA/2 islets and 40 mM glucose cultured C57BL/6 islets. Incubation of islets with AG resulted in a normalization of DBA/2 islet glucokinase levels. In conclusion, chronic high glucose-induced increases in AGE can result in islet dysfunction and this is associated with reduced glucokinase levels in a mouse model with susceptibility to islet failure.

Postexercise muscle glycogen resynthesis in obese insulin-resistant Zucker rats

We determined the effect of an acute bout of swimming (8 × 30 min) followed by either carbohydrate administration (0.5 mg/g glucose ip and ad libitum access to chow; CHO) or fasting (Fast) on postexercise glycogen resynthesis in soleus muscle and liver from female lean (ZL) and obese insulin-resistant (ZO) Zucker rats. Resting soleus muscle glycogen concentration ([glycogen]) was similar between genotypes and was reduced by 73 (ZL) and 63% (ZO) after exercise (P < 0.05). Liver [glycogen] at rest was greater in ZO than ZL (334 ± 31 vs. 247 ± 16 μmol/g wet wt; P < 0.01) and fell by 44 and 94% after exercise (P < 0.05). The fractional activity of glycogen synthase (active/total) increased immediately after exercise (from 0.22 ± 0.05 and 0.32 ± 0.04 to 0.63 ± 0.08 vs. 0.57 ± 0.05; P < 0.01 for ZL and ZO rats, respectively) and remained elevated above resting values after 30 min of recovery. During this time, muscle [glycogen] in ZO increased 68% with CHO (P < 0.05) but did not change in Fast. Muscle [glycogen] was unchanged in ZL from postexercise values after both treatments. After 6 h recovery, GLUT-4 protein concentration was increased above resting levels by a similar extent for both genotypes in both fasted (∼45%) and CHO-supplemented (∼115%) rats. Accordingly, during this time CHO refeeding resulted in supercompensation in both genotypes (68% vs. 44% for ZL and ZO). With CHO, liver [glycogen] was restored to resting levels in ZL but remained at postexercise values for ZO after both treatments. We conclude that the increased glucose availability with carbohydrate refeeding after glycogen-depleting exercise resulted in glycogen supercompensation, even in the face of muscle insulin-resistance.