Insulin clamp studies have shown that the suppressive actions of insulin on endogenous glucose production (EGP) are markedly more sensitive than for stimulating glucose disposal (Rd). However, clamp conditions do not adequately mimic postprandial physiological responses. Here, using the variable infusion dual-tracer approach, we used a threefold range of ingested glucose doses (25, 50, and 75 g) to investigate how physiological changes in plasma insulin influence EGP in healthy subjects. Remarkably, the glucose responses were similar for all doses tested, yet there was a dose-dependent increase in insulin secretion and plasma insulin levels. Nonetheless, EGP was suppressed with the same rapidity and magnitude (∼55%) across all doses. The progressive hyperinsulinemia, however, caused a dose-dependent increase in the estimated rates of Rd, which likely accounts for the lack of a dose effect on plasma glucose excursions. This suggests that after glucose ingestion, the body preferentially permits a transient and optimal degree of postprandial hyperglycemia to efficiently enhance insulin-induced changes in glucose fluxes, thereby minimizing the demand for insulin secretion. This may represent an evolutionarily conserved mechanism that not only reduces the secretory burden on β-cells but also avoids the potential negative consequences of excessive insulin release into the systemic arterial circulation.

Epac is a cAMP-activated guanine nucleotide exchange factor that mediates cAMP signaling in various types of cells, including β-cells, where it is involved in the control of insulin secretion. Upon activation, the protein redistributes to the plasma membrane, but the underlying molecular mechanisms and functional consequences are unclear. Using quantitative high-resolution microscopy, we found that cAMP elevation caused rapid binding of Epac2A to the β-cell plasma membrane, where it accumulated specifically at secretory granules and rendered them more prone to undergo exocytosis. cAMP-dependent membrane binding required the high-affinity cyclic nucleotide-binding (CNB) and Ras association domains, but not the disheveled-Egl-10-pleckstrin domain. Although the N-terminal low-affinity CNB domain (CNB-A) was dispensable for the translocation to the membrane, it was critical for directing Epac2A to the granule sites. Epac1, which lacks the CNB-A domain, was recruited to the plasma membrane but did not accumulate at granules. We conclude that Epac2A controls secretory granule release by binding to the exocytosis machinery, an effect that is enhanced by prior cAMP-dependent accumulation of the protein at the plasma membrane.

The ventromedial hypothalamus (VMH) regulates glucose and energy metabolism in mammals. Optogenetic stimulation of VMH neurons that express steroidogenic factor 1 (SF1) induces hyperglycemia. However, leptin acting via the VMH stimulates whole-body glucose utilization and insulin sensitivity in some peripheral tissues, and this effect of leptin appears to be mediated by SF1 neurons. We examined the effects of activation of SF1 neurons with DREADD (designer receptors exclusively activated by designer drugs) technology. Activation of SF1 neurons by an intraperitoneal injection of clozapine-N-oxide (CNO), a specific hM3Dq ligand, reduced food intake and increased energy expenditure in mice expressing hM3Dq in SF1 neurons. It also increased whole-body glucose utilization and glucose uptake in red-type skeletal muscle, heart, and interscapular brown adipose tissue, as well as glucose production and glycogen phosphorylase a activity in the liver, thereby maintaining blood glucose levels. During hyperinsulinemic-euglycemic clamp, such activation of SF1 neurons increased insulin-induced glucose uptake in the same peripheral tissues and tended to enhance insulin-induced suppression of glucose production by suppressing gluconeogenic gene expression and glycogen phosphorylase a activity in the liver. DREADD technology is thus an important tool for studies of the role of the brain in the regulation of insulin sensitivity in peripheral tissues.

Induction of endoplasmic reticulum stress and activation of the intrinsic apoptotic pathway is widely believed to contribute to β-cell death in type 1 diabetes (T1D). MCL-1 is an antiapoptotic member of the BCL-2 protein family, whose depletion causes apoptosis in rodent β-cells in vitro. Importantly, decreased MCL-1 expression was observed in islets from patients with T1D. We report here that MCL-1 downregulation is associated with cytokine-mediated killing of human β-cells, a process partially prevented by MCL-1 overexpression. By generating a β-cell-specific Mcl-1 knockout mouse strain (βMcl-1KO), we observed that, surprisingly, MCL-1 ablation does not affect islet development and function. β-Cells from βMcl-1KO mice were, however, more susceptible to cytokine-induced apoptosis. Moreover, βMcl-1KO mice displayed higher hyperglycemia and lower pancreatic insulin content after multiple low-dose streptozotocin treatment. We found that the kinase GSK3β, the E3 ligases MULE and βTrCP, and the deubiquitinase USP9x regulate cytokine-mediated MCL-1 protein turnover in rodent β-cells. Our results identify MCL-1 as a critical prosurvival protein for preventing β-cell death and clarify the mechanisms behind its downregulation by proinflammatory cytokines. Development of strategies to prevent MCL-1 loss in the early stages of T1D may enhance β-cell survival and thereby delay or prevent disease progression.

A truncated form of human glucose-dependent insulinotropic polypeptide (GIP), GIP(3-30)NH2, was recently identified as an antagonist of the human GIP receptor. This study examined the ability of GIP(3-30)NH2 to antagonize the physiological actions of GIP in glucose metabolism, subcutaneous abdominal adipose tissue blood flow (ATBF), and lipid metabolism in humans. Eight lean subjects were studied by measuring arteriovenous concentrations of metabolites and ATBF on three different occasions during hyperglycemic-hyperinsulinemic clamps with concomitant infusions of GIP, GIP(3-30)NH2, or both GIP and GIP(3-30)NH2 During infusion of GIP(3-30)NH2 alone and in combination with GIP, insulin levels and the total glucose amount infused to maintain the clamp were lower than during GIP alone. In addition, ATBF remained constant during the antagonist and increased only slightly in combination with GIP, whereas it increased fivefold during GIP alone. Adipose tissue triacylglyceride (TAG) and glucose uptake decreased, and the free fatty acid/glycerol ratio increased during the antagonist alone and in combination with GIP. The changes in glucose infusion rates and plasma insulin levels demonstrate an inhibitory effect of the antagonist on the incretin effect of GIP. In addition, the antagonist inhibited GIP-induced increase in ATBF and decreased the adipose tissue TAG uptake, indicating that GIP also plays a crucial role in lipid metabolism.

Macrophages are critical for the initiation and resolution of the inflammatory phase of wound repair. In diabetes, macrophages display a prolonged inflammatory phenotype in late wound healing. Mixed-lineage leukemia-1 (MLL1) has been shown to direct gene expression by regulating nuclear factor-κB (NF-κB)-mediated inflammatory gene transcription. Thus, we hypothesized that MLL1 influences macrophage-mediated inflammation in wound repair. We used a myeloid-specific Mll1 knockout (Mll1f/fLyz2Cre+ ) to determine the function of MLL1 in wound healing. Mll1f/fLyz2Cre+ mice display delayed wound healing and decreased wound macrophage inflammatory cytokine production compared with control animals. Furthermore, wound macrophages from Mll1f/fLyz2Cre+ mice demonstrated decreased histone H3 lysine 4 trimethylation (H3K4me3) (activation mark) at NF-κB binding sites on inflammatory gene promoters. Of note, early wound macrophages from prediabetic mice displayed similarly decreased MLL1, H3K4me3 at inflammatory gene promoters, and inflammatory cytokines compared with controls. Late wound macrophages from prediabetic mice demonstrated an increase in MLL1, H3K4me3 at inflammatory gene promoters, and inflammatory cytokines. Prediabetic macrophages treated with an MLL1 inhibitor demonstrated reduced inflammation. Finally, monocytes from patients with type 2 diabetes had increased Mll1 compared with control subjects without diabetes. These results define an important role for MLL1 in regulating macrophage-mediated inflammation in wound repair and identify a potential target for the treatment of chronic inflammation in diabetic wounds.

This cross-sectional study evaluated the relationship between 1) functional and structural measurements of neurodegeneration in the initial stages of diabetic retinopathy (DR) and 2) the presence of neurodegeneration and early microvascular impairment. We analyzed baseline data of 449 patients with type 2 diabetes enrolled in the European Consortium for the Early Treatment of Diabetic Retinopathy (EUROCONDOR) study (NCT01726075). Functional studies by multifocal electroretinography (mfERG) evaluated neurodysfunction, and structural measurements using spectral domain optical coherence tomography (SD-OCT) evaluated neurodegeneration. The mfERG P1 amplitude was more sensitive than the P1 implicit time and was lower in patients with Early Treatment of Diabetic Retinopathy Study (ETDRS) level 20-35 than in patients with ETDRS level <20 (P = 0.005). In 58% of patients, mfERG abnormalities were present in the absence of visible retinopathy. Correspondence between SD-OCT thinning and mfERG abnormalities was shown in 67% of the eyes with ETDRS <20 and in 83% of the eyes with ETDRS level 20-35. Notably, 32% of patients with ETDRS 20-35 presented no abnormalities in mfERG or SD-OCT. We conclude that there is a link between mfERG and SD-OCT measurements that increases with the presence of microvascular impairment. However, a significant proportion of patients in our particular study population (ETDRS ≤35) had normal ganglion cell-inner plexiform layer thickness and normal mfERG findings. We raise the hypothesis that neurodegeneration may play a role in the pathogenesis of DR in many but not in all patients with type 2 diabetes.

Salivary and pancreatic amylases (encoded by AMY1 and AMY2 genes, respectively) are responsible for digesting starchy foods. AMY1 and AMY2 show copy number variations that affect differences in amylase amount and activity, and AMY1 copies have been associated with adiposity. We investigated whether genetic variants determining amylase gene copies are associated with 2-year changes in adiposity among 692 overweight and obese individuals who were randomly assigned to diets varying in macronutrient content. We found that changes in body weight (BW) and waist circumference (WC) were significantly different according to the AMY1-AMY2 rs11185098 genotype. Individuals carrying the A allele (indicating higher amylase amount and activity) showed a greater reduction in BW and WC at 6, 12, 18, and 24 months than those without the A allele (P < 0.05 for all). The association was stronger for long-term changes compared with short-term changes of these outcomes. The genetic effects on these outcomes did not significantly differ across diet groups. In conclusion, the genetic variant determining starch metabolism influences the response to weight-loss dietary intervention. Overweight and obese individuals carrying the AMY1-AMY2 rs11185098 genotype associated with higher amylase activity may have greater loss of adiposity during low-calorie diet interventions.

Neuropilin 1 (Nrp1), a coreceptor for class 3 semaphorins and growth factors, is highly expressed in vascular cells and myeloid cells, including macrophages. Unlike well-characterized proangiogenic functions of endothelial cell Nrp1, the contributions of macrophage Nrp1 within the context of metabolic dysfunction remain to be established. The aim of this study was to determine the contributions of macrophage Nrp1 in high-fat diet (HFD)-instigated insulin resistance in vivo. Insulin sensitivity and Nlrp3 inflammasome activation were monitored in wild-type (WT) and myeloid cell-specific Nrp1 knockout (Nrp1myel-KO) mice fed an HFD (60% kcal) for 16 weeks. HFD-fed mice exhibited insulin resistance with reduced levels of Nrp1 in macrophages compared with chow-fed mice. Further, HFD-fed Nrp1myel-KO mice displayed accentuated insulin resistance, enhanced systemic inflammation, and dramatically increased Nlrp3 inflammasome priming and activation. Importantly, knockout of Nlrp3 ablated HFD-induced insulin resistance and inflammation in Nrp1myel-KO mice, indicating that Nrp1 reduction in macrophages instigates insulin resistance by increasing macrophage Nlrp3 inflammasome activation. Mechanistically, Nrp1 deletion activates the nuclear factor-κB pathway, which in turn accentuates the priming of Nlrp3, promotes Nlrp3-ASC inflammasome assembly, and results in the activation of Nlrp3. We conclude that the HFD-instigated Nrp1 reduction in macrophages exacerbates insulin resistance by promoting Nlrp3 inflammasome priming and activation.

A logical cure for type 1 diabetes (T1D) involves replacing the lost insulin-producing cells with new ones, preferably cells from a well-characterized and unlimited source of human insulin-producing cells. This straightforward and simple solution to provide a cure for T1D is immensely attractive but entails at least two inherent and thus far unresolved hurdles: 1) provision of an unlimited source of functional human insulin-producing cells and 2) prevention of rejection without the side effects of systemic immunosuppression. Generation of transplantable insulin-producing cells from human embryonic stem cells or induced pluripotent stem cells is at present close to reality, and we are currently awaiting the first clinical studies. Focus is now directed to foster development of novel means to control the immune system to enable large-scale clinical application. Encapsulation introduces a physical barrier that prevents access of immune cells to the transplanted cells but also hinders blood vessel ingrowth. Therefore, oxygen, nutrient, and hormonal passage over the encapsulation membrane is solely dependent on diffusion over the immune barrier, contributing to delays in glucose sensing and insulin secretion kinetics. This Perspective focuses on the physiological possibilities and limitations of an encapsulation strategy to establish near-normoglycemia in subjects with T1D, assuming that glucose-responsive insulin-producing cells are available for transplantation.

On the basis of studies that investigated the intraportal versus systemic insulin infusion and transendothelial transport of insulin, we proposed the "single gateway hypothesis," which supposes an indirect regulation of hepatic glucose production by insulin; the rate-limiting transport of insulin across the adipose tissue capillaries is responsible for the slow suppression of free fatty acids (FFAs), which in turn is responsible for delayed suppression of hepatic endogenous glucose production (EGP) during insulin infusion. Preventing the fall in plasma FFAs during insulin infusion either by administering intralipids or by inhibiting adipose tissue lipolysis led to failure in EGP suppression, thus supporting our hypothesis. More recently, mice lacking hepatic Foxo1 in addition to Akt1 and Akt2 (L-AktFoxo1TKO), all required for insulin signaling, surprisingly showed normal glycemia. Inhibiting the fall of plasma FFAs in these mice prevented the suppression of EGP during a clamp, reaffirming that the site of insulin action to control EGP is extrahepatic. Measuring whole-body turnover rates of glucose and FFAs in L-AktFoxo1TKO mice also confirmed that hepatic EGP was regulated by insulin-mediated control of FFAs. The knockout mouse model in combination with sophisticated molecular techniques confirmed our physiological findings and the single gateway hypothesis.

The Edwin Bierman Award Lecture is presented in honor of the memory of Edwin L. Bierman, MD, an exemplary scientist, mentor, and leader in the field of diabetes, obesity, hyperlipidemia, and atherosclerosis. The award and lecture recognizes a leading scientist in the field of macrovascular complications and contributing risk factors in diabetes. Clay F. Semenkovich, MD, the Irene E. and Michael M. Karl Professor and Chief of the Division of Endocrinology, Metabolism and Lipid Research at Washington University School of Medicine in St. Louis, St. Louis, MO, received the prestigious award at the American Diabetes Association's 76th Scientific Sessions, 10-14 June 2016, in New Orleans, LA. He presented the Edwin Bierman Award Lecture, "We Know More Than We Can Tell About Diabetes and Vascular Disease," on Sunday, 12 June 2016.Diabetes is a disorder of abnormal lipid metabolism, a notion strongly supported by the work of Edwin Bierman, for whom this eponymous lecture is named. This abnormal lipid environment continues to be associated with devastating vascular complications in diabetes despite current therapies, suggesting that our understanding of the pathophysiology of blood vessel disease in diabetes is limited. In this review, potential new insights into the nature of diabetic vasculopathy will be discussed. Recent observations suggest that while the concept of distinct macrovascular and microvascular complications of diabetes has been useful, vascular diseases in diabetes may be more interrelated than previously appreciated. Moreover, the intermediary metabolic pathway of de novo lipogenesis, which synthesizes lipids from simple precursors, is robustly sensitive to insulin and may contribute to these complications. De novo lipogenesis requires fatty acid synthase, and recent studies of this enzyme suggest that endogenously produced lipids are channeled to specific intracellular sites to affect physiology. These findings raise the possibility that novel approaches to treating diabetes and its complications could be based on altering the intracellular lipid milieu.

Adipose tissues considerably influence metabolic homeostasis, and both white (WAT) and brown (BAT) adipose tissue play significant roles in lipid and glucose metabolism. O-linked N-acetylglucosamine (O-GlcNAc) modification is characterized by the addition of N-acetylglucosamine to various proteins by O-GlcNAc transferase (Ogt), subsequently modulating various cellular processes. However, little is known about the role of O-GlcNAc modification in adipose tissues. Here, we report the critical role of O-GlcNAc modification in cold-induced thermogenesis. Deletion of Ogt in WAT and BAT using adiponectin promoter-driven Cre recombinase resulted in severe cold intolerance with decreased uncoupling protein 1 (Ucp1) expression. Furthermore, Ogt deletion led to decreased mitochondrial protein expression in conjunction with decreased peroxisome proliferator-activated receptor γ coactivator 1-α protein expression. This phenotype was further confirmed by deletion of Ogt in BAT using Ucp1 promoter-driven Cre recombinase, suggesting that O-GlcNAc modification in BAT is responsible for cold-induced thermogenesis. Hypothermia was significant under fasting conditions. This effect was mitigated after normal diet consumption but not after consumption of a fatty acid-rich ketogenic diet lacking carbohydrates, suggesting impaired diet-induced thermogenesis, particularly by fat. In conclusion, O-GlcNAc modification is essential for cold-induced thermogenesis and mitochondrial biogenesis in BAT. Glucose flux into BAT may be a signal to maintain BAT physiological responses.

Intensive glycemic control (IGC) targeting HbA1c fails to show an unequivocal reduction of macrovascular complications in type 2 diabetes (T2D); however, the underlying mechanisms remain elusive. Epigenetic changes are emerging as important mediators of cardiovascular damage and may play a role in this setting. This study investigated whether epigenetic regulation of the adaptor protein p66Shc, a key driver of mitochondrial oxidative stress, contributes to persistent vascular dysfunction in patients with T2D despite IGC. Thirty-nine patients with uncontrolled T2D (HbA1c >7.5%) and 24 age- and sex-matched healthy control subjects were consecutively enrolled. IGC was implemented for 6 months in patients with T2D to achieve a target HbA1c of ≤7.0%. Brachial artery flow-mediated dilation (FMD), urinary 8-isoprostaglandin F2α (8-isoPGF2α), and epigenetic regulation of p66Shc were assessed at baseline and follow-up. Continuous glucose monitoring was performed to determine the mean amplitude of glycemic excursion (MAGE) and postprandial incremental area under the curve (AUCpp). At baseline, patients with T2D showed impaired FMD, increased urinary 8-isoPGF2α, and p66Shc upregulation in circulating monocytes compared with control subjects. FMD, 8-isoPGF2α, and p66Shc expression were not affected by IGC. DNA hypomethylation and histone 3 acetylation were found on the p66Shc promoter of patients with T2D, and IGC did not change such adverse epigenetic remodeling. Persistent downregulation of methyltransferase DNMT3b and deacetylase SIRT1 may explain the observed p66Shc-related epigenetic changes. MAGE and AUCpp but not HbA1c were independently associated with the altered epigenetic profile on the p66Shc promoter. Hence, glucose fluctuations contribute to chromatin remodeling and may explain persistent vascular dysfunction in patients with T2D with target HbA1c levels.

Growing attention has been focused on the roles of the proximal tubules (PTs) of the kidney in glucose metabolism, including the mechanism of regulation of gluconeogenesis. In this study, we found that PT-specific insulin receptor substrate 1/2 double-knockout mice, established by using the newly generated sodium-glucose cotransporter 2 (SGLT2)-Cre transgenic mice, exhibited impaired insulin signaling and upregulated gluconeogenic gene expression and renal gluconeogenesis, resulting in systemic insulin resistance. In contrast, in streptozotocin-treated mice, although insulin action was impaired in the PTs, the gluconeogenic gene expression was unexpectedly downregulated in the renal cortex, which was restored by administration of an SGLT1/2 inhibitor. In the HK-2 cells, the gluconeogenic gene expression was suppressed by insulin, accompanied by phosphorylation and inactivation of forkhead box transcription factor 1 (FoxO1). In contrast, glucose deacetylated peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1α), a coactivator of FoxO1, via sirtuin 1, suppressing the gluconeogenic gene expression, which was reversed by inhibition of glucose reabsorption. These data suggest that both insulin signaling and glucose reabsorption suppress the gluconeogenic gene expression by inactivation of FoxO1 and PGC1α, respectively, providing insight into novel mechanisms underlying the regulation of gluconeogenesis in the PTs.

The objective of this study was to examine the effect of renal sodium-glucose cotransporter inhibition with empagliflozin on the fasting plasma glucose (FPG) concentration and β-cell function in subjects with impaired fasting glucose (IFG). Eight subjects with normal fasting glucose (NFG) and eight subjects with IFG received empagliflozin (25 mg/day) for 2 weeks. FPG concentration and β-cell function was measured with a nine-step hyperglycemic clamp before and 48 h and 14 days after the start of empagliflozin. Empagliflozin caused 50 ± 4 and 45 ± 4 g glucosuria on day 2 in subjects with IFG and NFG, respectively, and the glucosuria was maintained for 2 weeks in both groups. The FPG concentration decreased only in subjects with IFG from 110 ± 2 to 103 ± 3 mg/dL (P < 0.01) after 14 days. The FPG concentration remained unchanged (95 ± 2 to 94 ± 2 mg/dL) in subjects with NFG. Empagliflozin enhanced β-cell function only in subjects with IFG. The incremental area under the plasma C-peptide concentration curve during the hyperglycemic clamp increased by 22 ± 4 and 23 ± 4% after 48 h and 14 days, respectively (P < 0.01); the plasma C-peptide response remained unchanged in subjects with NFG. Insulin sensitivity during the hyperglycemic clamp was not affected by empagliflozin in either IFG or NFG. Thus, β-cell function measured with the insulin secretion/insulin sensitivity (disposition) index increased significantly in IFG, but not in subjects with normal glucose tolerance. Inhibition of renal sodium-glucose cotransport with empagliflozin in subjects with IFG and NFG produces comparable glucosuria but lowers the plasma glucose concentration and improves β-cell function only in subjects with IFG.

Insulin exocytosis is regulated by ion channels that control excitability and Ca2+ influx. Channels also play an increasingly appreciated role in microdomain structure. In this study, we examine the mechanism by which the voltage-dependent K+ (Kv) channel Kv2.1 (KCNB1) facilitates depolarization-induced exocytosis in INS 832/13 cells and β-cells from human donors with and without type 2 diabetes (T2D). We find that Kv2.1, but not Kv2.2 (KCNB2), forms clusters of 6-12 tetrameric channels at the plasma membrane and facilitates insulin exocytosis. Knockdown of Kv2.1 expression reduces secretory granule targeting to the plasma membrane. Expression of the full-length channel (Kv2.1-wild-type) supports the glucose-dependent recruitment of secretory granules. However, a truncated channel (Kv2.1-ΔC318) that retains electrical function and syntaxin 1A binding, but lacks the ability to form clusters, does not enhance granule recruitment or exocytosis. Expression of KCNB1 appears reduced in T2D islets, and further knockdown of KCNB1 does not inhibit Kv current in T2D β-cells. Upregulation of Kv2.1-wild-type, but not Kv2.1-ΔC318, rescues the exocytotic phenotype in T2D β-cells and increases insulin secretion from T2D islets. Thus, the ability of Kv2.1 to directly facilitate insulin exocytosis depends on channel clustering. Loss of this structural role for the channel might contribute to impaired insulin secretion in diabetes.