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Type 2 diabetes (T2D) is a metabolic disorder characterized by hyperglycemia, hyperinsulinemia, and insulin resistance (IR). During the early phase of T2D, insulin synthesis and secretion by pancreatic β cells is enhanced, which can lead to proinsulin misfolding that aggravates endoplasmic reticulum (ER) protein homeostasis in β cells. Moreover, increased circulating insulin may contribute to fatty liver disease. Medical interventions aimed at alleviating ER stress in β cells while maintaining optimal insulin secretion are therefore an attractive therapeutic strategy for T2D. Previously, we demonstrated that germline Chop gene deletion preserved β cells in high-fat diet (HFD)-fed mice and in leptin receptor-deficient db/db mice. In the current study, we further investigated whether targeting Chop/Ddit3 specifically in murine β cells conferred therapeutic benefits. First, we showed that Chop deletion in β cells alleviated β cell ER stress and delayed glucose-stimulated insulin secretion (GSIS) in HFD-fed mice. Second, β cell-specific Chop deletion prevented liver steatosis and hepatomegaly in aged HFD-fed mice without affecting basal glucose homeostasis. Third, we provide mechanistic evidence that Chop depletion reduces ER Ca2+ buffering capacity and modulates glucose-induced islet Ca2+ oscillations, leading to transcriptional changes of ER chaperone profile ("ER remodeling"). Last, we demonstrated that a GLP1-conjugated Chop antisense oligonucleotide strategy recapitulated the reduction in liver triglycerides and pancreatic insulin content. In summary, our results demonstrate that Chop depletion in β cells provides a therapeutic strategy to alleviate dysregulated insulin secretion and consequent fatty liver disease in T2D.

Cell replication is a fundamental attribute of growth and repair in multicellular organisms. Pancreatic beta-cells in adults rarely enter cell cycle, hindering the capacity for regeneration in diabetes. Efforts to drive beta-cells into cell cycle have so far largely focused on regulatory molecules such as cyclins and cyclin-dependent kinases (CDKs). Investigations in cancer biology have uncovered that adaptive changes in metabolism, the mitochondrial network, and cellular Ca2+ are critical for permitting cells to progress through the cell cycle. Here, we investigated these parameters in the replication-competent beta-cell line INS 832/13. Cell cycle synchronization of this line permitted evaluation of cell metabolism, mitochondrial network, and cellular Ca2+ compartmentalization at key cell cycle stages. The mitochondrial network is interconnected and filamentous at G1/S but fragments during the S and G2/M phases, presumably to permit sorting to daughter cells. Pyruvate anaplerosis peaks at G1/S, consistent with generation of biomass for daughter cells, whereas mitochondrial Ca2+ and respiration increase during S and G2/M, consistent with increased energy requirements for DNA and lipid synthesis. This synchronization approach may be of value to investigators performing live cell imaging of Ca2+ or mitochondrial dynamics commonly undertaken in INS cell lines because without synchrony widely disparate data from cell to cell would be expected depending on position within cell cycle. Our findings also offer insight into why replicating beta-cells are relatively nonfunctional secreting insulin in response to glucose. They also provide guidance on metabolic requirements of beta-cells for the transition through the cell cycle that may complement the efforts currently restricted to manipulating cell cycle to drive beta-cells through cell cycle.

Type 2 diabetes (T2DM) results when increases in beta cell function and/or mass cannot compensate for rising insulin resistance. Numerous studies have documented the longitudinal changes in metabolism that occur during the development of glucose intolerance and lead to T2DM. However, the role of changes in insulin secretion, both amount and temporal pattern, has been understudied. Most of the insulin secreted from pancreatic beta cells of the pancreas is released in a pulsatile pattern, which is disrupted in T2DM. Here we review the evidence that changes in beta cell pulsatility occur during the progression from glucose intolerance to T2DM in humans, and contribute significantly to the etiology of the disease. We review the evidence that insulin pulsatility improves the efficacy of secreted insulin on its targets, particularly hepatic glucose production, but also examine evidence that pulsatility alters or is altered by changes in peripheral glucose uptake. Finally, we summarize our current understanding of the biophysical mechanisms responsible for oscillatory insulin secretion. Understanding how insulin pulsatility contributes to normal glucose homeostasis and is altered in metabolic disease states may help improve the treatment of T2DM.

Primary defects in folding of mutant proinsulin can cause dominant‐negative proinsulin accumulation in the endoplasmic reticulum (ER), impaired anterograde proinsulin trafficking, perturbed ER homeostasis, diminished insulin production, and β‐cell dysfunction. Conversely, if primary impairment of ER‐to‐Golgi trafficking (which also perturbs ER homeostasis) drives misfolding of nonmutant proinsulin—this might suggest bi‐directional entry into a common pathological phenotype (proinsulin misfolding, perturbed ER homeostasis, and deficient ER export of proinsulin) that can culminate in diminished insulin storage and diabetes. Here, we've challenged β‐cells with conditions that impair ER‐to‐Golgi trafficking, and devised an accurate means to assess the relative abundance of distinct folded/misfolded forms of proinsulin using a novel nonreducing SDS‐PAGE/immunoblotting protocol. We confirm abundant proinsulin misfolding upon introduction of a diabetogenic INS mutation, or in the islets of db/db mice. Whereas blockade of proinsulin trafficking in Golgi/post‐Golgi compartments results in intracellular accumulation of properly‐folded proinsulin (bearing native disulfide bonds), impairment of ER‐to‐Golgi trafficking (regardless whether such impairment is achieved by genetic or pharmacologic means) results in decreased native proinsulin with more misfolded proinsulin. Remarkably, reversible ER‐to‐Golgi transport defects (such as treatment with brefeldin A or cellular energy depletion) upon reversal quickly restore the ER folding environment, resulting in the disappearance of pre‐existing misfolded proinsulin while preserving proinsulin bearing native disulfide bonds. Thus, proper homeostatic balance of ER‐to‐Golgi trafficking is linked to a more favorable proinsulin folding (as well as trafficking) outcome. [ABSTRACT FROM AUTHOR]

Pancreatic beta cells secrete insulin in response to plasma glucose. The ATP‐sensitive potassium channel (KATP) links glucose metabolism to islet electrical activity in these cells by responding to increased cytosolic [ATP]/[ADP]. It was recently proposed that pyruvate kinase (PK) in close proximity to beta cell KATP locally produces the ATP that inhibits KATP activity. This proposal was largely based on the observation that applying phosphoenolpyruvate (PEP) and ADP to the cytoplasmic side of excised inside‐out patches inhibited KATP. To test the relative contributions of local vs. mitochondrial ATP production, we recorded KATP activity using mouse beta cells and INS‐1 832/13 cells. In contrast to prior reports, we could not replicate inhibition of KATP activity by PEP + ADP. However, when the pH of the PEP solutions was not corrected for the addition of PEP, strong channel inhibition was observed as a result of the well‐known action of protons to inhibit KATP. In cell‐attached recordings, perifusing either a PK activator or an inhibitor had little or no effect on KATP channel closure by glucose, further suggesting that PK is not an important regulator of KATP. In contrast, addition of mitochondrial inhibitors robustly increased KATP activity. Finally, by measuring the [ATP]/[ADP] responses to imposed calcium oscillations in mouse beta cells, we found that oxidative phosphorylation could raise [ATP]/[ADP] even when ADP was at its nadir during the burst silent phase, in agreement with our mathematical model. These results indicate that ATP produced by mitochondrial oxidative phosphorylation is the primary controller of KATP in pancreatic beta cells. Key points: Phosphoenolpyruvate (PEP) plus adenosine diphosphate does not inhibit KATP activity in excised patches. PEP solutions only inhibit KATP activity if the pH is unbalanced.Modulating pyruvate kinase has minimal effects on KATP activity.Mitochondrial inhibition, in contrast, robustly potentiates KATP activity in cell‐attached patches.Although the ADP level falls during the silent phase of calcium oscillations, mitochondria can still produce enough ATP via oxidative phosphorylation to close KATP.Mitochondrial oxidative phosphorylation is therefore the main source of the ATP that inhibits the KATP activity of pancreatic beta cells. [ABSTRACT FROM AUTHOR]

The standard model for Ca2+ oscillations in insulin-secreting pancreatic β cells centers on Ca2+ entry through voltage-activated Ca2+ channels. These work in combination with ATP-dependent K+ channels, which are the bridge between the metabolic state of the cells and plasma membrane potential. This partnership underlies the ability of the β cells to secrete insulin appropriately on a minute-to-minute time scale to control whole body plasma glucose. Though this model, developed over more than 40 years through many cycles of experimentation and mathematical modeling, has been very successful, it has been challenged by a hypothesis that calcium-induced calcium release from the endoplasmic reticulum through ryanodine or inositol trisphosphate (IP3) receptors is instead the key driver of islet oscillations. We show here that the alternative model is in fact incompatible with a large body of established experimental data and that the new observations offered in support of it can be better explained by the standard model. [ABSTRACT FROM AUTHOR]

A maternal diet that is low in protein increases the susceptibility of offspring to type 2 diabetes by inducing long-term alterations in β cell mass and function. Nutrients and growth factor signaling converge through mTOR, suggesting that this pathway participates in β cell programming during fetal development. Here, we revealed that newborns of dams exposed to low-protein diet (LP0.5) throughout pregnancy exhibited decreased insulin levels, a lower β cell fraction, and reduced mTOR signaling. Adult offspring of LP0.5-exposed mothers exhibited glucose intolerance as a result of an insulin secretory defect and not β cell mass reduction. The β cell insulin secretory defect was distal to glucose-dependent [Ca.sup.2+] influx and resulted from reduced proinsulin biosynthesis and insulin content. Islets from offspring of LP0.5-fed dams exhibited reduced mTOR and increased expression of a subset of microRNAs, and blockade of microRNA-199a-3p and-342 in these islets restored mTOR and insulin secretion to normal. Finally, transient β cell activation of mTORC1 signaling in offspring during the last week of pregnancy of mothers fed a LP0.5 rescued the defect in the neonatal β cell fraction and metabolic abnormalities in the adult. Together, these findings indicate that a maternal low-protein diet alters microRNA and mTOR expression in the offspring, influencing insulin secretion and glucose homeostasis., Introduction The pervasiveness of type 2 diabetes (T2D) is a major public health concern worldwide. The elevated prevalence of this disease results in part from an increased rate of obesity [...]