Improvement in type 2 diabetes after Roux-en-Y gastric bypass (RYGB) has been attributed partly to weight loss, but mechanisms beyond weight loss remain unclear. We performed an ancillary study to the Diabetes Surgery Study to assess changes in incretins, insulin sensitivity, and secretion 1 year after randomization to lifestyle modification and intensive medical management (LS/IMM) alone (n = 34) or in conjunction with RYGB (n = 34). The RYGB group lost more weight and had greater improvement in HbA1c. Fasting glucose was lower after RYGB than after LS/IMM, although the glucose area under the curve decreased comparably for both groups. Insulin sensitivity increased in both groups. Insulin secretion was unchanged after LS/IMM but decreased after RYGB, except for a rapid increase during the first 30 min after meal ingestion. Glucagon-like peptide 1 (GLP-1) was substantially increased after RYGB, while gastric inhibitory polypeptide and glucagon decreased. Lower HbA1c was most strongly correlated with the percentage of weight loss for both groups. At baseline, a greater C-peptide index and 90-min postprandial C-peptide level were predictive of lower HbA1c at 1 year after RYGB. β-Cell glucose sensitivity, which improved only after RYGB, and improved disposition index were associated with lower HbA1c in both groups, independent of weight loss. Weight loss and preserved β-cell function both predominantly determine the greatest glycemic benefit after RYGB.

During pancreas development, endocrine precursors and their progeny differentiate, migrate, and cluster to form nascent islets. The transcription factor Neurogenin 3 (Neurog3) is required for islet development in mice, but its role in these dynamic morphogenetic steps has been inferred from fixed tissues. Moreover, little is known about the molecular genetic functions of NEUROG3 in human islet development. We developed methods for gene transduction by viral microinjection in the epithelium of cultured Neurog3-null mutant fetal pancreas, permitting genetic complementation in a developmentally relevant context. In addition, we developed methods for quantitative assessment of live-cell phenotypes in single developing islet cells. Delivery of wild-type NEUROG3 rescued islet differentiation, morphogenesis, and live cell deformation, whereas the patient-derived NEUROG3(R107S) allele partially restored indicators of islet development. NEUROG3(P39X), a previously unreported patient allele, failed to restore islet differentiation or morphogenesis and was indistinguishable from negative controls, suggesting that it is a null mutation. Our systems also permitted genetic suppression analysis and revealed that targets of NEUROG3, including NEUROD1 and RFX6, can partially restore islet development in Neurog3-null mutant mouse pancreata. Thus, advances described here permitted unprecedented assessment of gene functions in regulating crucial dynamic aspects of islet development in the fetal pancreas.

The aim of this study was to determine whether antecedent stimulation of γ-aminobutyric acid (GABA) A receptors with the benzodiazepine alprazolam can blunt physiologic responses during next-day moderate (90 min) exercise in healthy man. Thirty-one healthy individuals (16 male/15 female aged 28 ± 1 year, BMI 23 ± 3 kg/m(2)) were studied during separate, 2-day protocols. Day 1 consisted of morning and afternoon 2-h hyperinsulinemic-euglycemic or hypoglycemic clamps with or without 1 mg alprazolam given 30 min before a clamp. Day 2 consisted of 90-min euglycemic cycling exercise at 50% VO2max. Despite similar euglycemia (5.3 ± 0.1 mmol/L) and insulinemia (46 ± 6 pmol/L) during day 2 exercise studies, GABA A activation with alprazolam during day 1 euglycemia resulted in significant blunting of plasma epinephrine, norepinephrine, glucagon, cortisol, and growth hormone responses. Lipolysis (glycerol, nonesterified fatty acids) and endogenous glucose production during exercise were also reduced, and glucose infusion rates were increased following prior euglycemia with alprazolam. Prior hypoglycemia with alprazolam resulted in further reduction of glucagon and cortisol responses during exercise. We conclude that prior activation of GABA A pathways can play a significant role in blunting key autonomous nervous system, neuroendocrine, and metabolic physiologic responses during next-day exercise in healthy man.

Staphylococcus aureus is an opportunistic bacterium capable of causing a wide range of severe diseases when it gains access to underlying tissues. Paradoxically, S. aureus is a common inhabitant of the skin microflora and colonizes the nares and other human mucosa. The purpose of this study was to determine the genetic basis for the differences in the pathogenic versus colonizing potential of S. aureus isolated from diabetic foot ulcers (DFUs). By performing optical map comparisons of a collection of S. aureus strains isolated from DFUs, we brought to light a prophage present in noninfecting bacteria. The phage, namely ROSA-like, was localized in a hotspot region ΦNM2 near the locus isd, the iron surface determinant system. The integrated phage significantly reduces the virulence of the strain and increases the biofilm formation. DFUs seem to be a specific niche of this colonizing strain. The ROSA-like phage represents the first description of a mobile element present mainly in S. aureus isolated from DFUs, which modulates the relationship of the bacteria with its human host. This phage appears to attenuate bacterial virulence and promote colonization.

Aberrant Wnt signaling appears to play an important role in the onset of diabetes. Moreover, the insulin signaling pathway is defective in the nucleus tractus solitarii (NTS) of spontaneously hypertensive rats (SHRs) and fructose-fed rats. Nevertheless, the relationships between Wnt signaling and the insulin pathway and the related modulation of blood pressure (BP) in the central nervous system have yet to be established. The aim of this study was to investigate the potential signaling pathways involved in Wnt-mediated BP regulation in the NTS. Pretreatment with the LDL receptor-related protein (LRP) antagonist Dickkopf-1 (DKK1) significantly attenuated the Wnt3a-induced depressor effect and nitric oxide production. Additionally, the inhibition of LRP6 activity using DKK1 significantly abolished Wnt3a-induced glycogen synthase kinase 3β (GSK-3β)(S9), extracellular signal-regulated kinases 1/2(T202/Y204), ribosomal protein S6 kinase(T359/S363), and Akt(S473) phosphorylation; and increased insulin receptor substrate 1 (IRS1)(S332) phosphorylation. GSK-3β was also found to bind directly to IRS1 and to induce the phosphorylation of IRS1 at serine 332 in the NTS. By contrast, administration of the GSK-3β inhibitor TWS119 into the brain decreased the BP of hypertensive rats by enhancing IRS1 activity. Taken together, these results suggest that the GSK-3β-IRS1 pathway may play a significant role in Wnt-mediated central BP regulation.

The aim of this study was 1) to clarify β-cell regenerative capacity in the face of glucocorticoid (GC)-induced insulin resistance and 2) to clarify the change in β- and α-cell mass in GC-induced diabetes in humans. We obtained the pancreases from 100 Japanese autopsy case subjects. The case subjects were classified according to whether or not they had received GC therapy before death and the presence or absence of diabetes. Fractional β-cell area (%BCA) and α-cell area (%ACA) were quantified, and the relationship with GC therapy was evaluated. As a result, in case subjects without diabetes, there was no significant difference in %BCA between case subjects with and without GC therapy (1.66 ± 1.05% vs. 1.21 ± 0.59%, P = 0.13). %ACA was also not significantly different between the two groups. In case subjects with type 2 diabetes, %BCA and %ACA were both significantly reduced compared with control subjects without diabetes; however, neither %BCA nor %ACA was significantly decreased in case subjects with GC-induced diabetes. There was a significant negative correlation between %BCA and HbA1c measured before death; however, this relationship was attenuated in case subjects with GC therapy. In conclusion, the current study suggests that β- and α-cell mass remain largely unchanged in the face of GC-induced insulin resistance in Japanese individuals, implying limited capacity of β-cell regeneration in adult humans. The absence of apparent β-cell deficit in case subjects with GC-induced diabetes suggests that GC-induced diabetes is mainly caused by insulin resistance and/or β-cell dysfunction, but not necessarily a deficit of β-cell mass.

Glucagon-like peptide-1 (GLP-1) and cholecystokinin (CCK) exert important complementary beneficial metabolic effects. This study assessed the biological actions and therapeutic utility of a novel (pGlu-Gln)-CCK-8/exendin-4 hybrid peptide compared with the stable GLP-1 and CCK mimetics exendin-4 and (pGlu-Gln)-CCK-8, respectively. All peptides significantly enhanced in vitro insulin secretion. Administration of the peptides, except (pGlu-Gln)-CCK-8 alone, in combination with glucose significantly lowered plasma glucose and increased plasma insulin in mice. All treatments elicited appetite-suppressive effects. Twice-daily administration of the novel (pGlu-Gln)-CCK-8/exendin-4 hybrid, (pGlu-Gln)-CCK-8 alone, or (pGlu-Gln)-CCK-8 in combination with exendin-4 for 21 days to high-fat-fed mice significantly decreased energy intake, body weight, and circulating plasma glucose. HbA1c was reduced in the (pGlu-Gln)-CCK-8/exendin-4 hybrid and combined parent peptide treatment groups. Glucose tolerance and insulin sensitivity also were improved by all treatment modalities. Interestingly, locomotor activity was decreased in the hybrid peptide group, and these mice also exhibited reductions in circulating triglyceride and cholesterol levels. Pancreatic islet number and area, as well β-cell area and insulinotropic responsiveness, were dramatically improved by all treatments. These studies highlight the clear potential of dual activation of GLP-1 and CCK1 receptors for the treatment of type 2 diabetes.

Insulin stimulates the translocation fatty acid transport protein 1 (FATP1) to plasma membrane, and thus greater free fatty acid (FFA) uptake, in adipocyte cell models. Whether insulin stimulates greater FFA clearance into adipose tissue in vivo is unknown. We tested this hypothesis by comparing direct FFA storage in subcutaneous adipose tissue during insulin versus niacin-medicated suppression of lipolysis. We measured direct FFA storage in abdominal and femoral subcutaneous fat in 10 and 11 adults, respectively, during euglycemic hyperinsulinemia or after oral niacin to suppress FFA compared with 11 saline control experiments. Direct palmitate storage was assessed using a [U-(13)C]palmitate infusion to measure palmitate kinetics and an intravenous palmitate radiotracer bolus/timed biopsy. Plasma palmitate concentrations and flux were suppressed to 23 ± 3 and 26 ± 5 µmol ⋅ L(-1) (P = 0.91) and 44 ± 4 and 39 ± 5 µmol ⋅ min(-1) (P = 0.41) in the insulin and niacin groups, respectively, much less (P < 0.001) than the saline control group (102 ± 8 and 104 ± 12 µmol ⋅ min(-1), respectively). In the insulin, niacin, and saline groups, abdominal palmitate storage rates were 0.25 ± 0.05 vs. 0.25 ± 0.07 vs. 0.32 ± 0.05 µmol ⋅ kg adipose lipid(-1) ⋅ min(-1), respectively (P = NS), and femoral adipose storage rates were 0.19 ± 0.06 vs. 0.20 ± 0.05 vs. 0.31 ± 0.05 µmol ⋅ kg adipose lipid(-1) ⋅ min(-1), respectively (P = NS). In conclusion, insulin does not increase FFA storage in adipose tissue compared with niacin, which suppresses lipolysis via a different pathway.

26RFa is a hypothalamic neuropeptide that promotes food intake. 26RFa is upregulated in obese animal models, and its orexigenic activity is accentuated in rodents fed a high-fat diet, suggesting that this neuropeptide might play a role in the development and maintenance of the obese status. As obesity is frequently associated with type 2 diabetes, we investigated whether 26RFa may be involved in the regulation of glucose homeostasis. In the current study, we show a moderate positive correlation between plasma 26RFa levels and plasma insulin in patients with diabetes. Plasma 26RFa concentration also increases in response to an oral glucose tolerance test. In addition, we found that 26RFa and its receptor GPR103 are present in human pancreatic β-cells as well as in the gut. In mice, 26RFa attenuates the hyperglycemia induced by a glucose load, potentiates insulin sensitivity, and increases plasma insulin concentrations. Consistent with these data, 26RFa stimulates insulin production by MIN6 insulinoma cells. Finally, we show, using in vivo and in vitro approaches, that a glucose load induces a massive secretion of 26RFa by the small intestine. Altogether, the present data indicate that 26RFa acts as an incretin to regulate glucose homeostasis.

The prevalence of obesity-related diabetes is increasing worldwide. Here we report the identification of a pentapeptide, GLP-1(32-36)amide (LVKGRamide), derived from the glucoincretin hormone GLP-1, that increases basal energy expenditure and curtails the development of obesity, insulin resistance, diabetes, and hepatic steatosis in diet-induced obese mice. The pentapeptide inhibited weight gain, reduced fat mass without change in energy intake, and increased basal energy expenditure independent of physical activity. Analyses of tissues from peptide-treated mice reveal increased expression of UCP-1 and UCP-3 in brown adipose tissue and increased UCP-3 and inhibition of acetyl-CoA carboxylase in skeletal muscle, findings consistent with increased fatty acid oxidation and thermogenesis. In palmitate-treated C2C12 skeletal myotubes, GLP-1(32-36)amide activated AMPK and inhibited acetyl-CoA carboxylase, suggesting activation of fat metabolism in response to energy depletion. By mass spectroscopy, the pentapeptide is rapidly formed from GLP-1(9-36)amide, the major form of GLP-1 in the circulation of mice. These findings suggest that the reported insulin-like actions of GLP-1 receptor agonists that occur independently of the GLP-1 receptor might be mediated by the pentapeptide, and the previously reported nonapeptide (FIAWLVKGRamide). We propose that by increasing basal energy expenditure, GLP-1(32-36)amide might be a useful treatment for human obesity and associated metabolic disorders.

Elevated cardiac triacylglycerol (TAG) content is traditionally equated with cardiolipotoxicity and suggested to be a culprit in cardiac dysfunction. However, previous work demonstrated that myosin heavy-chain-mediated cardiac-specific overexpression of diacylglycerol transferase 1 (MHC-DGAT1), the primary enzyme for TAG synthesis, preserved cardiac function in two lipotoxic mouse models despite maintaining high TAG content. Therefore, we examined whether increased cardiomyocyte TAG levels due to DGAT1 overexpression led to changes in cardiac TAG turnover rates under normoxia and ischemia-reperfusion conditions. MHC-DGAT1 mice had elevated TAG content and synthesis rates, which did not alter cardiac function, substrate oxidation, or myocardial energetics. MHC-DGAT1 hearts had ischemia-induced lipolysis; however, when a physiologic mixture of long-chain fatty acids was provided, enhanced TAG turnover rates were associated with improved functional recovery from low-flow ischemia. Conversely, exogenous supply of palmitate during reperfusion suppressed elevated TAG turnover rates and impaired recovery from ischemia in MHC-DGAT1 hearts. Collectively, this study shows that elevated TAG content, accompanied by enhanced turnover, does not adversely affect cardiac function and, in fact, provides cardioprotection from ischemic stress. In addition, the results highlight the importance of exogenous supply of fatty acids when assessing cardiac lipid metabolism and its relationship with cardiac function.

Activation of AMPK suppresses inflammation, but the underlying mechanisms remain poorly understood. This study was designed to characterize the molecular mechanisms by which AMPK suppresses vascular inflammation. In cultured human aortic smooth muscle cells, pharmacologic or genetic activation of AMPK inhibited the signal transducer and activator of transcription-1 (STAT1), while inhibition of AMPK had opposite effects. Deletion of AMPKα1 or AMPKα2 resulted in activation of STAT1 and in increases in proinflammatory mediators, both of which were attenuated by administration of STAT1 small interfering RNA or fludarabine, a selective STAT1 inhibitor. Moreover, AMPK activation attenuated the proinflammatory actions induced by STAT1 activators such as interferon-γ and angiotensin II (AngII). Mechanistically, we found that AMPK activation increased, whereas AMPK inhibition decreased, the levels of mitogen-activated protein kinase phosphatase-1 (MKP-1), an inducible nuclear phosphatase, by regulating proteasome-dependent degradation of MKP-1. Gene silencing of MKP-1 increased STAT1 phosphorylation and prevented 5-aminoimidazole-4-carboxyamide ribonucleoside-reduced STAT1 phosphorylation. Finally, we found that infusion of AngII caused a more severe inflammatory response in AMPKα2 knockout mouse aortas, all of which were suppressed by chronic administration of fludarabine. We conclude that AMPK activation suppresses STAT1 signaling and inhibits vascular inflammation through the upregulation of MKP-1.

Understanding the molecular networks controlling ectopic lipid deposition, glucose tolerance, and insulin sensitivity is essential to identifying new pharmacological approaches to treat type 2 diabetes. We recently identified serine/threonine protein kinase 25 (STK25) as a negative regulator of glucose and insulin homeostasis based on observations in myoblasts with acute depletion of STK25 and in STK25-overexpressing transgenic mice. Here, we challenged Stk25 knockout mice and wild-type littermates with a high-fat diet and showed that STK25 deficiency suppressed development of hyperglycemia and hyperinsulinemia, improved systemic glucose tolerance, reduced hepatic gluconeogenesis, and increased insulin sensitivity. Stk25(-/-) mice were protected from diet-induced liver steatosis accompanied by decreased protein levels of acetyl-CoA carboxylase, a key regulator of both lipid oxidation and synthesis. Lipid accumulation in Stk25(-/-) skeletal muscle was reduced, and expression of enzymes controlling the muscle oxidative capacity (Cpt1, Acox1, Cs, Cycs, Ucp3) and glucose metabolism (Glut1, Glut4, Hk2) was increased. These data are consistent with our previous study of STK25 knockdown in myoblasts and reciprocal to the metabolic phenotype of Stk25 transgenic mice, reinforcing the validity of the results. The findings suggest that STK25 deficiency protects against the metabolic consequences of chronic exposure to dietary lipids and highlight the potential of STK25 antagonists for the treatment of type 2 diabetes.