Peroxisome proliferator-activated receptor γ coactivator 1 (PPARGCA1, PGC-1) transcriptional coactivators control gene programs important for nutrient metabolism. Islets of type 2 diabetic subjects have reduced PGC-1α expression and this is associated with decreased insulin secretion, yet little is known about why this occurs or what role it plays in the development of diabetes. Our goal was to delineate the role and importance of PGC-1 proteins to β-cell function and energy homeostasis.

The physiological role of serotonin, or 5-hydroxytryptamine (5-HT), in pancreatic β-cell function was previously elucidated using a pregnant mouse model. During pregnancy, 5-HT increases β-cell proliferation and glucose-stimulated insulin secretion (GSIS) through the Gαq-coupled 5-HT2b receptor (Htr2b) and the 5-HT3 receptor (Htr3), a ligand-gated cation channel, respectively. However, the role of 5-HT in β-cell function in an insulin-resistant state has yet to be elucidated. Here, we characterized the metabolic phenotypes of β-cell-specific Htr2b(-/-) (Htr2b βKO), Htr3a(-/-) (Htr3a knock-out [KO]), and β-cell-specific tryptophan hydroxylase 1 (Tph1)(-/-) (Tph1 βKO) mice on a high-fat diet (HFD). Htr2b βKO, Htr3a KO, and Tph1 βKO mice exhibited normal glucose tolerance on a standard chow diet. After 6 weeks on an HFD, beginning at 4 weeks of age, both Htr3a KO and Tph1 βKO mice developed glucose intolerance, but Htr2b βKO mice remained normoglycemic. Pancreas perfusion assays revealed defective first-phase insulin secretion in Htr3a KO mice. GSIS was impaired in islets isolated from HFD-fed Htr3a KO and Tph1 βKO mice, and 5-HT treatment improved insulin secretion from Tph1 βKO islets but not from Htr3a KO islets. Tph1 and Htr3a gene expression in pancreatic islets was not affected by an HFD, and immunostaining could not detect 5-HT in pancreatic islets from mice fed an HFD. Taken together, these results demonstrate that basal 5-HT levels in β-cells play a role in GSIS through Htr3, which becomes more evident in a diet-induced insulin-resistant state.

Glucagon, a peptide hormone secreted from the α-cells of the pancreatic islets, is critical for blood glucose homeostasis. We reviewed the literature and employed a computational systems analysis of intracellular metabolic and electrical regulation of glucagon secretion to better understand these processes. The mathematical model of α-cell metabolic parameters is based on our previous model for pancreatic β-cells. We also formulated an ionic model for action potentials that incorporates Ca ( 2+) , K (+) , Na (+) and Cl (-) currents. Metabolic and ionic models are coupled to the equations describing Ca ( 2+) homeostasis and glucagon secretion that depends on activation of specific voltage-gated Ca ( 2+) channels. Paracrine and endocrine regulations were analyzed with an emphasis on their effects on a hyperpolarization of membrane potential. This general model simulates and gives insight into the mechanisms of regulation of glucagon secretion under a wide range of experimental conditions. We also reviewed and analyzed dysfunctional mechanisms in α-cells to determine key pharmacological targets for modulating glucagon secretion in type 1 and 2 diabetes.

Glucose-induced β-cell action potential (AP) repolarization is regulated by potassium efflux through voltage gated (Kv) and calcium activated (K(Ca)) potassium channels. Thus, ablation of the primary Kv channel of the β-cell, Kv2.1, causes increased AP duration. However, Kv2.1(-/-) islet electrical activity still remains sensitive to the potassium channel inhibitor tetraethylammonium. Therefore, we utilized Kv2.1(-/-) islets to characterize Kv and K(Ca) channels and their respective roles in modulating the β-cell AP. The remaining Kv current present in Kv2.1(-/-) β-cells is inhibited with 5 μM CP 339818. Inhibition of the remaining Kv current in Kv2.1(-/-) mouse β-cells increased AP firing frequency by 39.6% but did not significantly enhance glucose stimulated insulin secretion (GSIS). The modest regulation of islet AP frequency by CP 339818 implicates other K(+) channels, possibly K(Ca) channels, in regulating AP repolarization. Blockade of the K(Ca) channel BK with slotoxin increased β-cell AP amplitude by 28.2%, whereas activation of BK channels with isopimaric acid decreased β-cell AP amplitude by 30.6%. Interestingly, the K(Ca) channel SK significantly contributes to Kv2.1(-/-) mouse islet AP repolarization. Inhibition of SK channels decreased AP firing frequency by 66% and increased AP duration by 67% only when Kv2.1 is ablated or inhibited and enhanced GSIS by 2.7-fold. Human islets also express SK3 channels and their β-cell AP frequency is significantly accelerated by 4.8-fold with apamin. These results uncover important repolarizing roles for both Kv and K(Ca) channels and identify distinct roles for SK channel activity in regulating calcium- versus sodium-dependent AP firing.

Calcium (Ca(2+)) signaling regulates insulin secretion in pancreatic beta-cells. STIM1 has been proposed to function as an endoplasmic reticulum (ER) Ca(2+) sensor regulating store-operated Ca(2+) entry (SOCE). Here we studied the translocation of EYFP-STIM1 in response to ER calcium depletion in mouse insulinoma MIN6 cells by fluorescent microscopy. While in resting cells EYFP-STIM1 is co-localized with an ER marker, in thapsigargin (Tg)-stimulated cells it occupied highly defined areas of the peri-PM space in punctae adjacent to, but not entirely coincident with the ER. Co-staining with fluorescent phalloidin revealed that EYFP-STIM1 punctae was located in actin-poor areas. Use of the SOCE blocker in MIN6 cells, 2-aminoethoxy diphenylborate (2-APB), prevented store depletion-dependent translocation of EYFP-STIM1 to the PM in a concentration-dependent (3.75-100muM) and reversible manner. TIRF microscopy revealed that 2-APB treatment led to the reversible disappearance of peri-PM EYFP-STIM1 punctae, while the ER structure in this compartment remained grossly unaffected. We conclude from this data that in these cells EYFP-STIM1 is delivered to a peri-PM location from the ER upon store depletion and this trafficking is reversibly blocked by 2-APB.

Multiple genome-wide association studies have identified a strong genetic linkage between the SKAP2 locus and type 1 diabetes (T1D), but how this leads to disease remains obscure. Here, we characterized the functional consequence of a novel SKAP2 coding mutation in a patient with T1D to gain further insight into how this impacts immune tolerance.

Insulin secretion from pancreatic beta cells is dependent on maturation and acidification of the secretory granule, processes necessary for prohormone convertase cleavage of proinsulin. Previous studies in isolated beta cells revealed that acidification may be dependent on the granule membrane chloride channel ClC-3, in a step permissive for a regulated secretory response. In this study, immuno-EM of beta cells revealed colocalization of ClC-3 and insulin on secretory granules. Clcn3(-/-) mice as well as isolated islets demonstrate impaired insulin secretion; Clcn3(-/-) beta cells are defective in regulated insulin exocytosis and granular acidification. Increased amounts of proinsulin were found in the majority of secretory granules in the Clcn3(-/-) mice, while in Clcn3(+/+) cells, proinsulin was confined to the immature secretory granules. These results demonstrate that in pancreatic beta cells, chloride channels, specifically ClC-3, are localized on insulin granules and play a role in insulin processing as well as insulin secretion through regulation of granular acidification.

Drosophila melanogaster has been widely used as a model of human Mendelian disease, but its value in modeling complex disease has received little attention. Fly models of complex disease would enable high-resolution mapping of disease-modifying loci and the identification of novel targets for therapeutic intervention. Here, we describe a fly model of permanent neonatal diabetes mellitus and explore the complexity of this model. The approach involves the transgenic expression of a misfolded mutant of human preproinsulin, hINS(C96Y), which is a cause of permanent neonatal diabetes. When expressed in fly imaginal discs, hINS(C96Y) causes a reduction of adult structures, including the eye, wing, and notum. Eye imaginal discs exhibit defects in both the structure and the arrangement of ommatidia. In the wing, expression of hINS(C96Y) leads to ectopic expression of veins and mechano-sensory organs, indicating disruption of wild-type signaling processes regulating cell fates. These readily measurable "disease" phenotypes are sensitive to temperature, gene dose, and sex. Mutant (but not wild-type) proinsulin expression in the eye imaginal disc induces IRE1-mediated XBP1 alternative splicing, a signal for endoplasmic reticulum stress response activation, and produces global change in gene expression. Mutant hINS transgene tester strains, when crossed to stocks from the Drosophila Genetic Reference Panel, produce F1 adults with a continuous range of disease phenotypes and large broad-sense heritability. Surprisingly, the severity of mutant hINS-induced disease in the eye is not correlated with that in the notum in these crosses, nor with eye reduction phenotypes caused by the expression of two dominant eye mutants acting in two different eye development pathways, Drop (Dr) or Lobe (L), when crossed into the same genetic backgrounds. The tissue specificity of genetic variability for mutant hINS-induced disease has, therefore, its own distinct signature. The genetic dominance of disease-specific phenotypic variability in our model of misfolded human proinsulin makes this approach amenable to genome-wide association study in a simple F1 screen of natural variation.

Over the last decade our insight into the causes of neonatal diabetes has greatly expanded. Neonatal diabetes was once considered a variant of type 1 diabetes that presented early in life. Recent advances in our understanding of this disorder have established that neonatal diabetes is not an autoimmune disease, but rather is a monogenic form of diabetes resulting from mutations in a number of different genes encoding proteins that play a key role in the normal function of the pancreatic beta-cell. Moreover, a correct genetic diagnosis can affect treatment and clinical outcome. This is especially true for patients with mutations in the genes KCNJ11 or ABCC8 that encode the two protein subunits (Kir6.2 and SUR1, respectively) of the ATP-sensitive potassium channel. These patients can be treated with oral sulfonylurea drugs with better glycemic control and quality of life. Recently, mutations in the insulin gene (INS) itself have been identified as another cause of neonatal diabetes. In this article, we review the role of INS mutations in the pathophysiology of neonatal diabetes.

The aim of this study was to assess short-term and long-term results of the pancreatic islet transplantation using the Edmonton protocol at the University of Chicago.

Although insulin resistance consistently occurs with type 1 diabetes, its predominant driver is uncertain. We therefore determined the relative contributions of hyperglycemia and iatrogenic hyperinsulinemia to insulin resistance using hyperinsulinemic-euglycemic clamps in three participant groups (n = 10/group) with differing insulinemia and glycemia: healthy control subjects (euinsulinemia and euglycemia), glucokinase-maturity-onset diabetes of the young (GCK-MODY; euinsulinemia and hyperglycemia), and type 1 diabetes (hyperinsulinemia and hyperglycemia matching GCK-MODY). We assessed the contribution of hyperglycemia by comparing insulin sensitivity in control and GCK-MODY and the contribution of hyperinsulinemia by comparing GCK-MODY and type 1 diabetes. Hemoglobin A1c was normal in control subjects and similarly elevated for type 1 diabetes and GCK-MODY. Basal insulin levels in control subjects and GCK-MODY were nearly equal but were 2.5-fold higher in type 1 diabetes. Low-dose insulin infusion suppressed endogenous glucose production similarly in all groups and suppressed nonesterified fatty acids similarly between control subjects and GCK-MODY, but to a lesser extent for type 1 diabetes. High-dose insulin infusion stimulated glucose disposal similarly in control subjects and GCK-MODY but was 29% and 22% less effective in type 1 diabetes, respectively. Multivariable linear regression showed that insulinemia-but not glycemia-was significantly associated with muscle insulin sensitivity. These data suggest that iatrogenic hyperinsulinemia predominates in driving insulin resistance in type 1 diabetes.

Many patients with type 1 diabetes (T1D) have residual β cells producing small amounts of C-peptide long after disease onset but develop an inadequate glucagon response to hypoglycemia following T1D diagnosis. The features of these residual β cells and α cells in the islet endocrine compartment are largely unknown, due to the difficulty of comprehensive investigation. By studying the T1D pancreas and isolated islets, we show that remnant β cells appeared to maintain several aspects of regulated insulin secretion. However, the function of T1D α cells was markedly reduced, and these cells had alterations in transcription factors constituting α and β cell identity. In the native pancreas and after placing the T1D islets into a non-autoimmune, normoglycemic in vivo environment, there was no evidence of α-to-β cell conversion. These results suggest an explanation for the disordered T1D counterregulatory glucagon response to hypoglycemia.

Clinical and pathologic heterogeneity in type 1 diabetes is increasingly being recognized. Findings in the islets and pancreas of a 22-year-old male with 8 years of type 1 diabetes were discordant with expected results and clinical history (islet autoantibodies negative, hemoglobin A1c 11.9%) and led to comprehensive investigation to define the functional, molecular, genetic, and architectural features of the islets and pancreas to understand the cause of the donor's diabetes. Examination of the donor's pancreatic tissue found substantial but reduced β-cell mass with some islets devoid of β cells (29.3% of 311 islets) while other islets had many β cells. Surprisingly, isolated islets from the donor pancreas had substantial insulin secretion, which is uncommon for type 1 diabetes of this duration. Targeted and whole-genome sequencing and analysis did not uncover monogenic causes of diabetes but did identify high-risk human leukocyte antigen haplotypes and a genetic risk score suggestive of type 1 diabetes. Further review of pancreatic tissue found islet inflammation and some previously described α-cell molecular features seen in type 1 diabetes. By integrating analysis of isolated islets, histological evaluation of the pancreas, and genetic information, we concluded that the donor's clinical insulin deficiency was most likely the result autoimmune-mediated β-cell loss but that the constellation of findings was not typical for type 1 diabetes. This report highlights the pathologic and functional heterogeneity that can be present in type 1 diabetes.

This multicenter prospective cohort study compared pancreas volume as assessed by MRI, metabolic scores derived from oral glucose tolerance testing (OGTT), and a combination of pancreas volume and metabolic scores for predicting progression to stage 3 type 1 diabetes (T1D) in individuals with multiple diabetes-related autoantibodies.

Using an integrated approach to characterize the pancreatic tissue and isolated islets from a 33-year-old with 17 years of type 1 diabetes (T1D), we found that donor islets contained β cells without insulitis and lacked glucose-stimulated insulin secretion despite a normal insulin response to cAMP-evoked stimulation. With these unexpected findings for T1D, we sequenced the donor DNA and found a pathogenic heterozygous variant in the gene encoding hepatocyte nuclear factor-1α (HNF1A). In one of the first studies of human pancreatic islets with a disease-causing HNF1A variant associated with the most common form of monogenic diabetes, we found that HNF1A dysfunction leads to insulin-insufficient diabetes reminiscent of T1D by impacting the regulatory processes critical for glucose-stimulated insulin secretion and suggest a rationale for a therapeutic alternative to current treatment.

The brain influences glucose homeostasis, partly by supplemental control over insulin and glucagon secretion. Without this central regulation, diabetes and its complications can ensue. Yet, the neuronal network linking to pancreatic islets has never been fully mapped. Here, we refine this map using pseudorabies virus (PRV) retrograde tracing, indicating that the pancreatic islets are innervated by efferent circuits that emanate from the hypothalamus. We found that the hypothalamic arcuate nucleus (ARC), ventromedial nucleus (VMN), and lateral hypothalamic area (LHA) significantly overlap PRV and the physiological glucose-sensing enzyme glucokinase. Then, experimentally lowering glucose sensing, specifically in the ARC, resulted in glucose intolerance due to deficient insulin secretion and no significant effect in the VMN, but in the LHA it resulted in a lowering of the glucose threshold that improved glucose tolerance and/or improved insulin sensitivity, with an exaggerated counter-regulatory response for glucagon secretion. No significant effect on insulin sensitivity or metabolic homeostasis was noted. Thus, these data reveal novel direct neuronal effects on pancreatic islets and also render a functional validation of the brain-to-islet neuronal map. They also demonstrate that distinct regions of the hypothalamus differentially control insulin and glucagon secretion, potentially in partnership to help maintain glucose homeostasis and guard against hypoglycemia.

Conditional gene targeting has been extensively used for in vivo analysis of gene function in β-cell biology. The objective of this study was to examine whether mouse transgenic Cre lines, used to mediate β-cell- or pancreas-specific recombination, also drive Cre expression in the brain.

Acute insulin secretion determines the efficiency of glucose clearance. Moreover, impaired acute insulin release is characteristic of reduced glucose control in the prediabetic state. Incretin hormones, which increase β-cell cAMP, restore acute-phase insulin secretion and improve glucose control. To determine the physiological role of the cAMP-dependent protein kinase (PKA), a mouse model was developed to increase PKA activity specifically in the pancreatic β-cells. In response to sustained hyperglycemia, PKA activity potentiated both acute and sustained insulin release. In contrast, a glucose bolus enhanced acute-phase insulin secretion alone. Acute-phase insulin secretion was increased 3.5-fold, reducing circulating glucose to 58% of levels in controls. Exendin-4 increased acute-phase insulin release to a similar degree as PKA activation. However, incretins did not augment the effects of PKA on acute-phase insulin secretion, consistent with incretins acting primarily via PKA to potentiate acute-phase insulin secretion. Intracellular calcium signaling was unaffected by PKA activation, suggesting that the effects of PKA on acute-phase insulin secretion are mediated by the phosphorylation of proteins involved in β-cell exocytosis. Thus, β-cell PKA activity transduces the cAMP signal to dramatically increase acute-phase insulin secretion, thereby enhancing the efficiency of insulin to control circulating glucose.

Pancreatic beta-cells respond to rising blood glucose by increasing oxidative metabolism, leading to an increased ATP/ADP ratio in the cytoplasm. This leads to a closure of KATP channels, depolarization of the plasma membrane, influx of calcium and the eventual secretion of insulin. Such mechanism suggests that beta-cell metabolism should have a functional regulation specific to secretion, as opposed to coupling to contraction. The goal of this work is to uncover contributions of the cytoplasmic and mitochondrial processes in this secretory coupling mechanism using mathematical modeling in a systems biology approach.

The gene KCNJ11 encodes Kir6.2 a major subunit of the ATP-sensitive potassium channel (KATP) expressed in both the pancreas and brain. Heterozygous gain of function mutations in KCNJ11 can cause neonatal diabetes mellitus (NDM). In addition, many patients exhibit neurological defects ranging from modest learning disorders to severe cognitive dysfunction and seizures. However, it remains unclear to what extent these neurological deficits are due to direct brain-specific activity of mutant KATP. We have generated cerebral organoids derived from human induced pluripotent stem cells (hiPSCs) possessing the KCNJ11 mutation p.Val59Met (V59M) and from non-pathogenic/normal hiPSCs (i.e., control/WT). Control cerebral organoids developed neural networks that could generate stable synchronized bursting neuronal activity whereas those derived from V59M cerebral organoids showed reduced synchronization. Histocytochemical studies revealed a marked reduction in neurons localized to upper cortical layer-like structures in V59M cerebral organoids suggesting dysfunction in the development of cortical neuronal network. Examination of temporal transcriptional profiles of neural stem cell markers revealed an extended window of SOX2 expression in V59M cerebral organoids. Continuous treatment of V59M cerebral organoids with the KATP blocker tolbutamide partially rescued the neurodevelopmental differences. Our study demonstrates the utility of human cerebral organoids as an investigative platform for studying the effects of KCNJ11 mutations on neurophysiological outcome.