Protein profiling of pancreatic islets

The insulin-producing beta cell in the islet of Langerhans is central in glucose homeostasis. Its dysfunction is part of the pathogenesis of both Type 1 and 2 diabetes mellitus. In both forms of the disease, there is a cytotoxic component either induced by cytokines, as in Type 1 diabetes, or by elevated levels of glucose and fatty acids, as in Type 2 diabetes. To find the mechanisms responsible for the cytotoxic effects of these compounds proteomic approaches with 2D gel electrophoresis and surface-enhanced laser desorption/ionization time-of-flight mass spectrometry have been undertaken. In this article, we describe these methods, and other methodological aspects of protein profiling of pancreatic islets, and summarize the results obtained with these methods.     

How to make insulin-producing pancreatic β cells for diabetes treatment

Around 400 million people worldwide suffer from diabetes mellitus.The major pathological event for Type 1 diabetes and advanced Type 2 diabetes is loss or impairment of insulin-secreting β cells of the pancreas.For the past 100 years,daily insulin injection has served as a life-saving treatment for these patients.However,insulin injection often cannot achieve full glucose control,and over time poor glucose control leads to severe complications and mortality.As an alternative treatment,islet transplantation has been demonstrated to effectively maintain glucose homeostasis in diabetic patients,but its wide application is limited by the scarcity of donated islets.Therefore,it is important to define new strategies to obtain functional human β cells for transplantation therapies.Here,we summarize recent progress towards the production of β cells in vitro from pluripotent stem cells or somatic cell types including a cells,pancreatic exocrine cells,gastrointestinal stem cells,fibroblasts and hepatocytes.We also discuss novel methods for optimizing β cell transplantation and maintenance in vivo.From our perspective,the future of βcell replacement therapy is very promising although it is still challenging to control differentiation of β cells in vitro and to protect these cells from autoimmune attack in Type 1 diabetic patients.Overall,tremendous progress has been made in understanding βcell differentiation and producing functional β cells with different methods.In the coming years,we believe more clinical trials will be launched to move these technologies towards treatments to benefit diabetic patients.     

Estimates of in vivo insulin action in humans: comparison of the insulin clamp and the minimal model techniques

The insulin clamp technique, which is often assumed to measure the ability of insulin to stimulate glucose uptake, actually measures both insulin-independent and insulin-dependent glucose uptake. In contrast, the minimal model technique, recently introduced by Bergman, Philips and Cobelli (1981), attempts to directly estimate insulin sensitivity (insulin-dependent glucose uptake = S1) by measurement of plasma glucose and insulin values during a 3 hour intravenous glucose tolerance test (IVGTT). In the present study estimates of insulin action derived from the insulin clamp and the minimal model technique were compared in 20 humans with varying degrees of glucose tolerance. The insulin response during the IVGTT was too low to permit calculation of S1 in 5 subjects - 4 with Type II diabetes and 1 with normal glucose tolerance. Although the correlation coefficient between the two tests in the other 15 patients was statistically significant (r = 0.53, P less than 0.05), this statement is somewhat misleading. Thus, S1 in the 4/7 patients with Type II diabetes in whom it could be measured was zero, and the correlation between estimates of insulin action with the two techniques in the 11 non-diabetic patients was not statistically significant (r = 0.41, P = NS) when these 4 patients were removed from the analysis. In conclusion, these data indicate that there was only a weak correlation between estimates of insulin action assessed with the insulin clamp and the minimal model techniques. One explanation for this observation is that the insulin-independent component of total glucose disposal both varies widely among patients and contributes significantly to glucose uptake as assessed by the insulin clamp technique.(ABSTRACT TRUNCATED AT 250 WORDS)     

The progression of type 2 diabetes: Partly caused by deficiency of ASP-C5L2 pathway?

Type 2 diabetes is characterized by insulin resistance and β-cell dysfunction. The pathway of acylation-stimulating protein (ASP) and its specific receptor, C5a-like receptor 2 (C5L2), involves in the effective clearance of plasma glucose and free fat acid. Abnormal ASP-C5L2 pathway may induce insulin resistance, as well as cause hyperglycemia and elevated plasma free fat acid. High levels of plasma glucose and free fat acid induce β-cell apoptosis and dysfunction. We proposed that the abnormality of ASP-C5L2 pathway contributes to progression of type 2 diabetes.     

Computational Modeling of Glucose Transport in Pancreatic β-Cells Identifies Metabolic Thresholds and Therapeutic Targets in Diabetes

Pancreatic β-cell dysfunction is a diagnostic criterion of Type 2 diabetes and includes defects in glucose transport and insulin secretion. In healthy individuals, β-cells maintain plasma glucose concentrations within a narrow range in concert with insulin action among multiple tissues. Postprandial elevations in blood glucose facilitate glucose uptake into β-cells by diffusion through glucose transporters residing at the plasma membrane. Glucose transport is essential for glycolysis and glucose-stimulated insulin secretion. In human Type 2 diabetes and in the mouse model of obesity-associated diabetes, a marked deficiency of β-cell glucose transporters and glucose uptake occurs with the loss of glucose-stimulated insulin secretion. Recent studies have shown that the preservation of glucose transport in β-cells maintains normal insulin secretion and blocks the development of obesity-associated diabetes. To further elucidate the underlying mechanisms, we have constructed a computational model of human β-cell glucose transport in health and in Type 2 diabetes, and present a systems analysis based on experimental results from human and animal studies. Our findings identify a metabolic threshold or “tipping point” whereby diminished glucose transport across the plasma membrane of β-cells limits intracellular glucose-6-phosphate production by glucokinase. This metabolic threshold is crossed in Type 2 diabetes and results in β-cell dysfunction including the loss of glucose stimulated insulin secretion. Our model further discriminates among molecular control points in this pathway wherein maximal therapeutic intervention is achieved.     

Differential AMPK phosphorylation by glucagon and metformin regulates insulin signaling in human hepatic cells

Insulin and glucagon signaling in the liver are major contributors to glucose homeostasis. Patients with Type 1 and Type 2 diabetes have impaired glycemic control due, in part, to dysregulation of the opposing actions of these hormones. While hyperglucagonemia is a common feature in diabetes, its precise role in insulin resistance is not well understood. Recently, metformin, an AMPK activator, was shown to regulate hepatic glucose output via inhibition of glucagon-induced cAMP/PKA signaling; however, the mechanism for how PKA inhibition leads to AMPK activation in human hepatic cells is not known. Here we show that glucagon impairs insulin-mediated AKT phosphorylation in human hepatic cell line Huh7. This impairment of AKT activation by glucagon is due to PKA-mediated inhibition of AMPK via increased inhibitory phosphorylation of AMPK^Ser173 and reduced activating phosphorylation of AMPK^Thr172. In contrast, metformin decreases PKA activity, leading to decreased pAMPK^Ser173 and increased pAMPK^Thr172. These data support a novel mechanism involving PKA-dependent AMPK phosphorylation that provides new insight into how glucagon and metformin modulate hepatic insulin resistance.     

Abnormal glucose homeostasis and pancreatic islet function in mice with inactivation of the Fem1b gene

Type 2 diabetes mellitus is a disorder of glucose homeostasis involving complex gene and environmental interactions that are incompletely understood. Mammalian homologs of nematode sex determination genes have recently been implicated in glucose homeostasis and type 2 diabetes mellitus. These are the Hedgehog receptor Patched and Calpain-10, which have homology to the nematode tra-2 and tra-3 sex determination genes, respectively. Here, we have developed Fem1b knockout (Fem1b-KO) mice, with targeted inactivation of Fem1b, a homolog of the nematode fem-1 sex determination gene. We show that the Fem1b-KO mice display abnormal glucose tolerance and that this is due predominantly to defective glucose-stimulated insulin secretion. Arginine-stimulated insulin secretion is also affected. The Fem1b gene is expressed in pancreatic islets, within both beta cells and non-beta cells, and is highly expressed in INS-1E cells, a pancreatic beta-cell line. In conclusion, these data implicate Fem1b in pancreatic islet function and insulin secretion, strengthening evidence that a genetic pathway homologous to nematode sex determination may be involved in glucose homeostasis and suggesting novel genes and processes as potential candidates in the pathogenesis of diabetes mellitus.     

Type 2 Diabetes Mellitus: New Genetic Insights will Lead to New Therapeutics

Type 2 diabetes is a disorder of dysregulated glucose homeostasis. Normal glucose homeostasis is a complex process involving several interacting mechanisms, such as insulin secretion, insulin sensitivity, glucose production, and glucose uptake. The dysregulation of one or more of these mechanisms due to environmental and/or genetic factors, can lead to a defective glucose homeostasis. Hyperglycemia is managed by augmenting insulin secretion and/or interaction with hepatic glucose production, as well as by decreasing dietary caloric intake and raising glucose metabolism through exercise. Although these interventions can delay disease progression and correct blood glucose levels, they are not able to cure the disease or stop its progression entirely. Better management of type 2 diabetes is sorely needed. Advances in genotyping techniques and the availability of large patient cohorts have made it possible to identify common genetic variants associated with type 2 diabetes through genome-wide association studies (GWAS). So far, genetic variants on 19 loci have been identified. Most of these loci contain or lie close to genes that were not previously linked to diabetes and they may thus harbor targets for new drugs. It is also hoped that further genetic studies will pave the way for predictive genetic screening. The newly discovered type 2 diabetes genes can be classified based on their presumed molecular function, and we discuss the relation between these gene classes and current treatments. We go on to consider whether the new genes provide opportunities for developing alternative drug therapies.Key Words: Type 2 diabetes, drug targets, genetics, personalized medicine.     

Troglitazone can prevent development of type 1 diabetes induced by multiple low-dose streptozotocin in mice.

Recent investigations suggest that cytotoxic cytokines such as tumor necrosis factor (TNF)alpha and interleukin (IL)-1beta or free radicals play an essential role in destruction of pancreatic beta cells in Type 1 diabetes and that, therefore, anti-oxidant or anti-TNF alpha and IL-1beta therapy could prevent the development of Type I diabetes. Troglitazone belongs to a novel class of antidiabetic agent possessing the ability to enhance insulin action provably through activating PPAR gamma and to scavenge free radicals. In the present study, we examined whether troglitazone can prevent the development of Type 1 diabetes in multiple, low-dose streptozotocin (MLDSTZ)-injected mice. In addition, effects of troglitazone on cytokine-induced pancreatic beta cell damage were examined in vitro. Type 1 diabetes was induced by MLDSTZ injection to DBA/2 mice (40 mg/kg/day for 5 days). Troglitazone was administered as a 0.2% food admixture (240 mg/kg/day) for 4 weeks from the start of or immediately after STZ injection. MLDSTZ injection elevated plasma glucose to 615 +/- 8 mg/dl 4 weeks after final STZ injection and was accompanied by infiltration of leukocytes to pancreatic islets (insulitis). Troglitazone treatment with MLDSTZ injection prevented hyperglycemia (230 +/- 30 mg/dl) and, suppressed insulitis and TNF alpha production from intraperitoneal exudate cells. TNF alpha (10 pg/ml) and IL-1beta (1 pg/ml) addition to hamster insulinoma cell line HIT-T15 for 7 days in vitro decreased insulin secretion and cell viability. Simultaneous troglitazone addition (0.03 to approximately 3 microM) significantly improved cytokine-induced decrease in insulin secretion and in cell viability. These findings suggest that troglitazone prevents the development of Type 1 diabetes in the MLDSTZ model by suppressing insulitis associated with decreasing TNF alpha production from intraperitoneal exudate cells and the subsequent TNF alpha and IL-1beta-induced beta cell damage.     

Effect of experimental diabetes on the activity of hexokinase isoenzymes in tissues of the rat

The effect of experimental diabetes on the activity of hexokinase isoenzymes was studied in a wide range of tissues of the rat. In the tissues known to require insulin for glucose phosphorylation, the activity of hexokinase was markedly decreased; the fall being mainly in the Type IV (Glucokinase) in liver and Type II in other tissues, these tissues also exhibit glucose underutilization in diabetes. In the tissues which are commonly known not to require insulin, the activity of Type I hexokinase was significantly increased, these tissues exhibit aspects of glucose overutilization in diabetes in particular kidney and lens. These changes are discussed in relation to Spiro's hypothesis of glucose under and overutilization in tissues in diabetes.     

Role of changes in cardiac metabolism in development of diabetic cardiomyopathy

In patients with diabetes, an increased risk of symptomatic heart failure usually develops in the presence of hypertension or ischemic heart disease. However, a predisposition to heart failure might also reflect the effects of underlying abnormalities in diastolic function that can occur in asymptomatic patients with diabetes alone (termed diabetic cardiomyopathy). Evidence of cardiomyopathy has also been demonstrated in animal models of both Type 1 (streptozotocin-induced diabetes) and Type 2 diabetes (Zucker diabetic fatty rats and ob/ob or db/db mice). During insulin resistance or diabetes, the heart rapidly modifies its energy metabolism, resulting in augmented fatty acid and decreased glucose consumption. Accumulating evidence suggests that this alteration of cardiac metabolism plays an important role in the development of cardiomyopathy. Hence, a better understanding of this dysregulation in cardiac substrate utilization during insulin resistance and diabetes could provide information as to potential targets for the treatment of cardiomyopathy. This review is focused on evaluating the acute and chronic regulation and dysregulation of cardiac metabolism in normal and insulin-resistant/diabetic hearts and how these changes could contribute toward the development of cardiomyopathy.     

3-Dimensional histological reconstruction and imaging of the murine pancreas

Visualization of important disease-driving tissues in their native morphological state, such as the pancreas, given its importance in glucose homeostasis and diabetes, provides critical insight into the etiology and progression of disease and our understanding of how cellular changes impact disease severity. Numerous challenges to maintaining tissue morphology exist when one attempts to preserve or to recreate such tissues for histological evaluation. We have overcome many of these challenges and have developed new methods for visualizing the whole murine pancreas and single islets of Langerhans in an effort to gain a better understanding of how islet cell volume, spatial distribution, and vascularization are altered as diabetes progresses. These methods are readily adaptable without requirement for costly specialized equipment, such as magnetic resonance imaging, positron emission tomography, or computed tomography, and can be used to provide additional robust analysis of diabetes susceptibility in mouse models of Type 1 and Type II diabetes.     

Rat maternal diabetes impairs pancreatic beta-cell function in the offspring

It has been shown that maternal diabetes increases the risk for obesity, glucose intolerance, and Type 2 diabetes mellitus in the adult life of the offspring. Mechanisms for these effects on the offspring are not well understood, and little information is available to reveal the mechanisms. We studied the effect of maternal diabetes on beta-cell function in the offspring of streptozotocin (STZ)-induced diabetic rat mothers (STZ-offspring). STZ-offspring did not become glucose intolerant up to 15 wk of age. At this age, however, insulin secretion was significantly impaired, as measured by in vivo and in vitro studies. Consistent with these changes, islet glucose metabolism and some important glucose metabolic enzyme activities were reduced. No significant changes were found in islet morphological analysis. These data indicate that beta-cell function is impaired in adult STZ-offspring; these changes may contribute to the development of type 2 diabetes mellitus in adulthood.     

Health-promoting effects of bovine colostrum in Type 2 diabetic patients can reduce blood glucose, cholesterol, triglyceride and ketones

Bovine colostrum (BC) has been reported to enhance immune function, reduce fat accumulation and facilitate the movement of glucose to the muscle. However, very few attempts have been made to examine its anti-diabetic effects in diabetes patients. The aim of this study was to evaluate whether BC decreases blood glucose, as well as cholesterol, triglyceride (TG) and ketones levels, which can be elevated by obesity and stress in Type 2 diabetic patients. Sixteen patients (men=8, women=8) with Type 2 diabetes were randomized into the study. Each ingested 5 g of BC on an empty stomach every morning and night for 4 weeks. Blood glucose, ketones (beta-hydroxybutyric acid), total cholesterol and TGs were measured every week. In both the men and women, blood glucose levels at 2 and 8 h postprandial decreased continually during the experimental period. The rate of decrease in blood glucose at 8 h postprandial was not different between the men and women, but was higher in the women (14.25+/-2.66) than in the men (10.96+/-1.82%) at 2 h postprandial. Total cholesterol and TG levels decreased significantly in both the men and women after 4 weeks. Also, beta-hydroxybutyric acid level decreased with BC ingestion, but this was not significant. These results suggest that BC can decrease levels of blood glucose and ketones, as well as reduce cholesterol and TGs, all of which may cause complications in Type 2 diabetic patients.     

Characterization of Obesity Phenotypes in Psammomys Obesus (Israeli Sand Rats)

Psammomys obesus (the Israeli sand rat) has been well studied as an animal model of Type 2 diabetes. However, obesity phenotypes in these animals have not been fully characterized. We analyzed phenotypic data including body weight, percentage body fat, blood glucose and plasma insulin concentration for over 600 animals from the Psammomys obesus colony at Deakin University to investigate the relationships between body fat, body weight and Type 2 diabetes using regression analysis and general linear modelling. The body weight distribution in Psammomys obesus approximates a normal distribution and closely resembles that observed in human populations. Animals above the 75th percentile for body weight had increased body fat content and a greater risk of developing diabetes. Increased visceral fat content .was also associated with elevated blood glucose and plasma insulin concentrations in these animals. A familial effect was also demonstrated in Psammomys obesus, and accounted for 51% of the variation in body weight, and 23–26% of the variation in blood glucose and plasma insulin concentrations in these animals. Psammomys obesus represents an excellent animal model of.obesity and Type 2 diabetes that exhibits a phenotypic pattern closely resembling that observed in human population studies. The obesity described in these animals was familial in nature and was significantly associated with Type 2 diabetes.     

Molecular target structures in alloxan-induced diabetes in mice

Type 1 diabetes results from irreversible damage of insulin-producing beta-cells. In laboratory animals, diabetes can be induced with alloxan (ALX), a 2,4,5,6-tetraoxopyrimidine. ALX is a potent generator of reactive oxygen species (ROS), which can mediate beta-cell toxicity. However, the initial lesions on essential beta-cell structures are not known. In this study, we report that the glucose transporter 2 (GLUT2) and glucokinase (GK) are target molecules for ALX. Ex vivo, a gradual decrement of both GLUT2 and GK mRNA expression was found in islets isolated from ALX-treated C57BL/6 mice. This reduction was more pronounced for GLUT2 than for GK. The mRNA expression of beta-actin was also slightly affected with time after ALX exposure, the proinsulin mRNA, however, remained unaffected as well as the pancreatic total insulin content. Pretreatment with D-glucose (D-G) protected the mRNA expression of GLUT2 and GK against ALX toxicity and prevented diabetes. Yet, in these euglycemic mice, an impaired oral glucose tolerance persisted. Pretreatment with 5-thio-D-glucose (5-T-G) failed to prevent ALX diabetes, administration of zinc sulfate (Zn(2+))-enriched drinking water, however, reduced ALX-induced hyperglycemia. In conclusion, ALX exerted differential toxicity on beta-cell structures similar to in vitro results reported from this laboratory. Furthermore, the present results differ from those reported for the diabetogen streptozotocin (STZ). Injections of multiple low doses (MLD) of STZ reduced GLUT2 expression only, but failed to affect expression of GK and proinsulin as well as beta-actin as internal control. MLD-STZ diabetes was prevented by pretreatment with both D-G and 5-T-G and administration of Zn(2+)-enriched drinking water. Apparently, ALX and MLD-STZ exert diabetogenicity by different pathways requiring different interventional schedules for prevention.     

Experimental diabetes treated with trigonelline: effect on key enzymes related to diabetes and hypertension, β-cell and liver function

Type 2 diabetes is quite diverse, including the improvement of insulin sensitivity by dipeptidylpeptidase-4 (DPP-4) inhibitor, α-glucosidase inhibitors, and the protection of β-cells islet. The aim of this study was to search the effect of trigonelline (Trig) on DPP-4, α-glucosidase and angiotensin converting enzyme (ACE) activities as well as β-cells architecture, and starch and glucose tolerance test. In surviving diabetic rats, the supplement of Trig potentially inhibited DPP-4 and α-glucosidase activities in both plasma and small intestine. The pancreas islet and less β-cells damage were observed after the administration of trig to diabetic rats. The increase of GLP-1 in surviving diabetic rats suppressed the increase of blood glucose level and improved results in the oral glucose and starch tolerance test. Trig also normalized key enzyme related to hypertension as ACE and improved the hemoglobin A1c and lipid profiles (plasma triglyceride, HDL-cholesterol, LDL-cholesterol, and total cholesterol), and liver indices toxicity. Therefore, these results revealed that Trig was successful in improving glycemic control, metabolic parameters, and liver function in diabetic rats. It is therefore suggested that Trig may be a potential agent for the treatment of type 2 diabetes.     

Invited review: Effects of acute exercise and exercise training on insulin resistance.

Insulin resistance of skeletal muscle glucose transport is a key defect in the development of impaired glucose tolerance and Type 2 diabetes. It is well established that both an acute bout of exercise and chronic endurance exercise training can have beneficial effects on insulin action in insulin-resistant states. This review summarizes the present state of knowledge regarding these effects in the obese Zucker rat, a widely used rodent model of obesity-associated insulin resistance, and in insulin-resistant humans with impaired glucose tolerance or Type 2 diabetes. A single bout of prolonged aerobic exercise (30-60 min at approximately 60-70% of maximal oxygen consumption) can significantly lower plasma glucose levels, owing to normal contraction-induced stimulation of GLUT-4 glucose transporter translocation and glucose transport activity in insulin-resistant skeletal muscle. However, little is currently known about the effects of acute exercise on muscle insulin signaling in the postexercise state in insulin-resistant individuals. A well-established adaptive response to exercise training in conditions of insulin resistance is improved glucose tolerance and enhanced skeletal muscle insulin sensitivity of glucose transport. This training-induced enhancement of insulin action is associated with upregulation of specific components of the glucose transport system in insulin-resistant muscle and includes increased protein expression of GLUT-4 and insulin receptor substrate-1. It is clear that further investigations are needed to further elucidate the specific molecular mechanisms underlying the beneficial effects of acute exercise and exercise training on the glucose transport system in insulin-resistant mammalian skeletal muscle.     

Effects of stabilized rice bran, its soluble and fiber fractions on blood glucose levels and serum lipid parameters in humans with diabetes mellitus Types I and II

Stabilized rice bran (SRB), a source of complex carbohydrates, tocols, gamma-oryzanols, and polyphenols, was treated with carbohydrases and heat to yield two fractions, rice bran water solubles (RBWS), and rice bran fiber concentrates (RBFC). Stabilized rice bran and its fractions were fed for 60 days to insulin-dependent and noninsulin-dependent diabetes mellitus (IDDM = Type I and NIDDM = Type II) subjects to determine possible effects on serum hemoglobin, carbohydrate and lipid parameters. The Type I subjects (n = 22, 26, and 20) fed Stabilized rice bran, rice bran water solubles, and rice bran fiber concentrates plus AHA Step-1 diet reduced glycosylated hemoglobin 1%, 11%, and 10%, respectively. The fasting serum glucose levels were also reduced significantly (P < 0.01) with stabilized rice bran (9%), rice bran water solubles (29%), and rice bran fiber concentrates (19%).The Type II subjects (n = 31, and 26) fed rice bran water solubles and rice bran fiber concentrates plus AHA Step-1 diet had decreased levels of glycosylated hemoglobin (15% and 11%) and fasting glucose (33% and 22%; P < 0.001), respectively. Serum insulin levels were increased (4%) with rice bran water solubles in both types of diabetes. The reduction of glycosylated hemoglobin and a slight increase in insulin levels indicate that consumption of rice bran water solubles can control blood glucose levels in human diabetes. Serum total cholesterol, LDL-cholesterol, apolipoprotein B, and triglycerides levels were reduced with rice bran fiber concentrates in the Type I (10, 16, 10, 7%) and Type II groups (12, 15, 10, 8%), respectively. These results indicate that rice bran water solubles significantly reduces hyperglycemia (P < 0.01), whereas rice bran fiber concentrates reduces hyperlipidemia (P < 0.05) in both types of diabetes. Therefore, these natural products can be used as nutritional supplements for the control of both types of diabetes mellitus in humans.     

Contraction signaling to glucose transport in skeletal muscle.

Contracting skeletal muscles acutely increases glucose transport in both healthy individuals and in people with Type 2 diabetes, and regular physical exercise is a cornerstone in the treatment of the disease. Glucose transport in skeletal muscle is dependent on the translocation of GLUT4 glucose transporters to the cell surface. It has long been believed that there are two major signaling mechanisms leading to GLUT4 translocation. One mechanism is insulin-activated signaling through insulin receptor substrate-1 and phosphatidylinositol 3-kinase. The other is an insulin-independent signaling mechanism that is activated by contractions, but the mediators of this signal are still unknown. Accumulating evidence suggests that the energy-sensing enzyme AMP-activated protein kinase plays an important role in contraction-stimulated glucose transport. However, more recent studies in transgenic and knockout animals show that AMP-activated protein kinase is not the sole mediator of the signal to GLUT4 translocation and suggest that there may be redundant signaling pathways leading to contraction-stimulated glucose transport. The search for other possible signal intermediates is ongoing, and calcium, nitric oxide, bradykinin, and the Akt substrate AS160 have been suggested as possible candidates. Further research is needed because full elucidation of an insulin-independent signal leading to glucose transport would be a promising pharmacological target for the treatment of Type 2 diabetes.