Insulin Signaling Regulates Mitochondrial Function in Pancreatic β-Cells

Insulin/IGF-I signaling regulates the metabolism of most mammalian tissues including pancreatic islets. To dissect the mechanisms linking insulin signaling with mitochondrial function, we first identified a mitochondria-tethering complex in β-cells that included glucokinase (GK), and the pro-apoptotic protein, BAD_S. Mitochondria isolated from β-cells derived from β-cell specific insulin receptor knockout (βIRKO) mice exhibited reduced BAD_S, GK and protein kinase A in the complex, and attenuated function. Similar alterations were evident in islets from patients with type 2 diabetes. Decreased mitochondrial GK activity in βIRKOs could be explained, in part, by reduced expression and altered phosphorylation of BAD_S. The elevated phosphorylation of p70S6K and JNK1 was likely due to compensatory increase in IGF-1 receptor expression. Re-expression of insulin receptors in βIRKO cells partially restored the stoichiometry of the complex and mitochondrial function. These data indicate that insulin signaling regulates mitochondrial function and have implications for β-cell dysfunction in type 2 diabetes.     

The Role of IRE1α in the Degradation of Insulin mRNA in Pancreatic β-Cells

Background

The endoplasmic reticulum (ER) is a cellular compartment for the biosynthesis and folding of newly synthesized secretory proteins such as insulin. Perturbations to ER homeostasis cause ER stress and subsequently activate cell signaling pathways, collectively known as the Unfolded Protein Response (UPR). IRE1α is a central component of the UPR. In pancreatic β-cells, IRE1α also functions in the regulation of insulin biosynthesis.

Principal Findings

Here we report that hyperactivation of IRE1α caused by chronic high glucose treatment or IRE1α overexpression leads to insulin mRNA degradation in pancreatic β-cells. Inhibition of IRE1α signaling using its dominant negative form prevents insulin mRNA degradation. Islets from mice heterozygous for IRE1α retain expression of more insulin mRNA after chronic high glucose treatment than do their wild-type littermates.

Conclusions/Significance

These results reveal a role of IRE1α in insulin mRNA expression under ER stress conditions caused by chronic high glucose. The rapid degradation of insulin mRNA could provide immediate relief for the ER and free up the translocation machinery. Thus, this mechanism would preserve ER homeostasis and help ensure that the insulin already inside the ER can be properly folded and secreted. This adaptation may be crucial for the maintenance of β-cell homeostasis and may explain why the β-cells of type 2 diabetic patients with chronic hyperglycemia stop producing insulin in the absence of apoptosis. This mechanism may also be involved in suppression of the autoimmune type 1 diabetes by reducing the amount of misfolded insulin, which could be a source of “neo-autoantigens.”     

Studies of the Mechanism of Activation of the Volume-Regulated Anion Channel in Rat Pancreatic β-Cells

There is evidence that depolarization of the pancreatic β cell by glucose involves cell swelling and activation of the volume-regulated anion channel (VRAC). However, it is unclear whether cell swelling per se or accompanying changes in intracellular osmolality and/or ionic strength are responsible for VRAC activation. VRAC activity was measured in rat β cells by conventional or perforated patch whole-cell recording. Cell volume was measured by video imaging. In conventional whole-cell recordings, VRAC activation was achieved by exposure of the cells to a hyposmotic bath solution, by application of positive pressure to the pipette, or by use of a hyperosmotic pipette solution. Increased concentrations of intracellular CsCl also caused channel activation, but with delayed kinetics. In perforated patch recordings, VRAC activation was induced by isosmotic addition of the permeable osmolytes urea, 3-Ο-methyl glucose, arginine, and NH₄Cl. These effects were all accompanied by β-cell swelling. It is concluded that increased cell volume, whether accompanied by raised intracellular osmolality or ionic strength, is a major determinant of VRAC activation in the β cell. However, increased intracellular ionic strength markedly reduced the rate of VRAC activation. These findings are consistent with the hypothesis that the accumulation of glucose metabolites in the β cell, and the resultant increase in cell volume, provides a signal coupling glucose metabolism with VRAC activation.     

Alteration of Endoplasmic Reticulum Lipid Rafts Contributes to Lipotoxicity in Pancreatic β-Cells

Chronic saturated fatty acid exposure causes β-cell apoptosis and, thus, contributes to type 2 diabetes. Although endoplasmic reticulum (ER) stress and reduced ER-to-Golgi protein trafficking have been implicated, the exact mechanisms whereby saturated fatty acids trigger β-cell death remain elusive. Using mass spectroscopic lipidomics and subcellular fractionation, we demonstrate that palmitate pretreatment of MIN6 β-cells promoted ER remodeling of both phospholipids and sphingolipids, but only the latter was causally linked to lipotoxic ER stress. Thus, overexpression of glucosylceramide synthase, previously shown to protect against defective protein trafficking and ER stress, partially reversed lipotoxic reductions in ER sphingomyelin (SM) content and aggregation of ER lipid rafts, as visualized using Erlin1-GFP. Using both lipidomics and a sterol response element reporter assay, we confirmed that free cholesterol in the ER was also reciprocally modulated by chronic palmitate and glucosylceramide synthase overexpression. This is consistent with the known coregulation and association of SM and free cholesterol in lipid rafts. Inhibition of SM hydrolysis partially protected against ATF4/C/EBP homology protein induction because of palmitate. Our results suggest that loss of SM in the ER is a key event for initiating β-cell lipotoxicity, which leads to disruption of ER lipid rafts, perturbation of protein trafficking, and initiation of ER stress.     

Temporal Proteomic Analysis of Pancreatic β-Cells in Response to Lipotoxicity and Glucolipotoxicity

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Highlights

• •Temporal proteome profiling of lipotoxicity and glucolipotoxicity in β-cells
• •Palmitate induced cholesterol metabolism earlier than fatty acid metabolism
• •Setd8 promotes palmitate + glucose-stimulated INS-1 cell proliferation
• •PA induced apoptosis partially via upregulation of Rhob in INS-1 cells

     

Chronic Exposure to Excess Nutrients Left-shifts the Concentration Dependence of Glucose-stimulated Insulin Secretion in Pancreatic β-Cells

Hyperinsulinemia (HI) is elevated plasma insulin at basal glucose. Impaired glucose tolerance is associated with HI, although the exact cause and effect relationship remains poorly defined. We tested the hypothesis that HI can result from an intrinsic response of the β-cell to chronic exposure to excess nutrients, involving a shift in the concentration dependence of glucose-stimulated insulin secretion. INS-1 (832/13) cells were cultured in either a physiological (4 mm) or high (11 mm) glucose concentration with or without concomitant exposure to oleate. Isolated rat islets were also cultured with or without oleate. A clear hypersensitivity to submaximal glucose concentrations was evident in INS-1 cells cultured in excess nutrients such that the 25% of maximal (S_0.25) glucose-stimulated insulin secretion was significantly reduced in cells cultured in 11 mm glucose (S_0.25 = 3.5 mm) and 4 mm glucose with oleate (S_0.25 = 4.5 mm) compared with 4 mm glucose alone (S_0.25 = 5.7 mm). The magnitude of the left shift was linearly correlated with intracellular lipid stores in INS-1 cells (r² = 0.97). We observed no significant differences in the dose responses for glucose stimulation of respiration, NAD(P)H autofluorescence, or Ca²⁺ responses between left- and right-shifted β-cells. However, a left shift in the sensitivity of exocytosis to Ca²⁺ was documented in permeabilized INS-1 cells cultured in 11 versus 4 mm glucose (S_0.25 = 1.1 and 1.7 μm, respectively). Our results suggest that the sensitivity of exocytosis to triggering is modulated by a lipid component, the levels of which are influenced by the culture nutrient environment.     

The Inactivation of Arx in Pancreatic α-Cells Triggers Their Neogenesis and Conversion into Functional β-Like Cells

Recently, it was demonstrated that pancreatic new-born glucagon-producing cells can regenerate and convert into insulin-producing β-like cells through the ectopic expression of a single gene, Pax4. Here, combining conditional loss-of-function and lineage tracing approaches, we show that the selective inhibition of the Arx gene in α-cells is sufficient to promote the conversion of adult α-cells into β-like cells at any age. Interestingly, this conversion induces the continuous mobilization of duct-lining precursor cells to adopt an endocrine cell fate, the glucagon⁺ cells thereby generated being subsequently converted into β-like cells upon Arx inhibition. Of interest, through the generation and analysis of Arx and Pax4 conditional double-mutants, we provide evidence that Pax4 is dispensable for these regeneration processes, indicating that Arx represents the main trigger of α-cell-mediated β-like cell neogenesis. Importantly, the loss of Arx in α-cells is sufficient to regenerate a functional β-cell mass and thereby reverse diabetes following toxin-induced β-cell depletion. Our data therefore suggest that strategies aiming at inhibiting the expression of Arx, or its molecular targets/co-factors, may pave new avenues for the treatment of diabetes.     

Selective Expression of Huntingtin-associated Protein 1 in ��-Cells of the Rat Pancreatic Islets

Huntingtin-associated protein-1 (HAP1) was initially identified as a binding partner of huntingtin, the Huntington''s disease protein. Based on its preferred distribution among neurons and endocrine cells, HAP1 has been suggested to play roles in vesicular transportation in neurons and hormonal secretion of endocrine cells. Given that HAP1 is selectively expressed in the islets of rat pancreas, in this study, we analyzed the expression pattern of HAP1 in the islets. In rats injected intraperitoneally with streptozotocin, which can selectively destroy β-cells of the pancreatic islets, the number of HAP1 immunoreactive cells was dramatically decreased and was accompanied by a parallel decrease in the number of insulin-immunoreactive cells. Immunofluorescent double staining of pancreas sections showed that, in rat islets, HAP1 is selectively expressed in the insulin-immunoreactive β-cells but not in the glucagon-immunoreactive α-cells and somatostatin immunoreactive δ-cells. In isolated rat pancreatic islets, ∼80% of cells expressed both HAP1 and insulin. Expression of HAP1 in the INS-1 rat insulinoma cell line was also demonstrated by immunofluorescent staining. Western blotting further revealed that HAP1 in both the isolated rat pancreatic islets and the INS-1 cells also has two isoforms, HAP1A and HAP1B, which are the same as those in the hypothalamus. These results demonstrated that HAP1 is selectively expressed in β-cells of rat pancreatic islets, suggesting the involvement of HAP1 in the regulation of cellular trafficking and secretion of insulin. (J Histochem Cytochem 58:255–263, 2010)     

Liganded Thyroid Hormone Receptor-α Enhances Proliferation of Pancreatic β-Cells

Failure of the functional pancreatic β-cell mass to expand in response to increased metabolic demand is a hallmark of type 2 diabetes. Lineage tracing studies indicate that replication of existing β-cells is important for β-cell proliferation in adult animals. In rat pancreatic β-cell lines (RIN5F), treatment with 100 nm thyroid hormone (triiodothyronine, T₃) enhances cell proliferation. This result suggests that T₃ is required for β-cell proliferation or replication. To identify the role of thyroid hormone receptor α (TRα) in the processes of β-cell growth and cell cycle regulation, we constructed a recombinant adenovirus vector, AdTRα. Infection with AdTRα to RIN5F cells increased the expression of cyclin D1 mRNA and protein. Overexpression of the cyclin D1 protein in AdTRα-infected cells led to activation of the cyclin D1/cyclin-dependent kinase/retinoblastoma protein/E2F pathway, along with cell cycle progression and cell proliferation following treatment with 100 nm T₃. Conversely, lowering cellular cyclin D1 by small interfering RNA knockdown in AdTRα-infected cells led to down-regulation of the cyclin D1/CDK/Rb/E2F pathway and inhibited cell proliferation. Furthermore, in immunodeficient mice with streptozotocin-induced diabetes, intrapancreatic injection of AdTRα led to the restoration of islet function and to an increase in the β-cell mass. These results support the hypothesis that liganded TRα plays a critical role in β-cell replication and in expansion of the β-cell mass during postnatal development. Thus, liganded TRα may be a target for therapeutic strategies that can induce the expansion and regeneration of β-cells.     

Evidence That Calcium Release-activated Current Mediates the Biphasic Electrical Activity of Mouse Pancreatic β-Cells

The electrical response of pancreatic β-cells to step increases in glucose concentration is biphasic, consisting of a prolonged depolarization with action potentials (Phase 1) followed by membrane potential oscillations known as bursts. We have proposed that the Phase 1 response results from the combined depolarizing influences of potassium channel closure and an inward, nonselective cation current (I _CRAN) that activates as intracellular calcium stores empty during exposure to basal glucose (Bertram et al., 1995). The stores refill during Phase 1, deactivating I _CRAN and allowing steady-state bursting to commence. We support this hypothesis with additional simulations and experimental results indicating that Phase 1 duration is sensitive to the filling state of intracellular calcium stores. First, the duration of the Phase 1 transient increases with duration of prior exposure to basal (2.8 mm) glucose, reflecting the increased time required to fill calcium stores that have been emptying for longer periods. Second, Phase 1 duration is reduced when islets are exposed to elevated K⁺ to refill calcium stores in the presence of basal glucose. Third, when extracellular calcium is removed during the basal glucose exposure to reduce calcium influx into the stores, Phase 1 duration increases. Finally, no Phase 1 is observed following hyperpolarization of the β-cell membrane with diazoxide in the continued presence of 11 mm glucose, a condition in which intracellular calcium stores remain full. Application of carbachol to empty calcium stores during basal glucose exposure did not increase Phase 1 duration as the model predicts. Despite this discrepancy, the good agreement between most of the experimental results and the model predictions provides evidence that a calcium release-activated current mediates the Phase 1 electrical response of the pancreatic β-cell. Received: 5 June 1996/Revised: 15 August 1996     

Epoxypukalide Induces Proliferation and Protects against Cytokine-Mediated Apoptosis in Primary Cultures of Pancreatic β-Cells

There is an urgency to find new treatments for the devastating epidemic of diabetes. Pancreatic β-cells viability and function are impaired in the two most common forms of diabetes, type 1 and type 2. Regeneration of pancreatic β-cells has been proposed as a potential therapy for diabetes. In a preliminary study, we screened a collection of marine products for β-cell proliferation. One unique compound (epoxypukalide) showed capability to induce β-cell replication in the cell line INS1 832/13 and in primary rat cell cultures. Epoxypukalide was used to study β-cell proliferation by [³H]thymidine incorporation and BrdU incorporation followed by BrdU/insulin staining in primary cultures of rat islets. AKT and ERK1/2 signalling pathways were analyzed. Cell cycle activators, cyclin D2 and cyclin E, were detected by western-blot. Apoptosis was studied by TUNEL and cleaved caspase 3. β-cell function was measured by glucose-stimulated insulin secretion. Epoxypukalide induced 2.5-fold increase in β-cell proliferation; this effect was mediated by activation of ERK1/2 signalling pathway and upregulation of the cell cycle activators, cyclin D2 and cyclin E. Interestingly, epoxypukalide showed protection from basal (40% lower versus control) and cytokine-induced apoptosis (80% lower versus control). Finally, epoxypukalide did not impair β-cell function when measured by glucose-stimulated insulin secretion. In conclusion, epoxypukalide induces β-cell proliferation and protects against basal and cytokine-mediated β-cell death in primary cultures of rat islets. These findings may be translated into new treatments for diabetes.     

Methylglyoxal Causes Swelling and Activation of a Volume-Sensitive Anion Conductance in Rat Pancreatic β-Cells

Membrane potential and whole-cell current were studied in rat pancreatic β-cells using the `perforated patch' technique and cell volume measured by a video-imaging method. Exposure of β-cells to the α-ketoaldehyde methylglyoxal (1 mm) resulted in depolarization and electrical activity. In cells voltage-clamped at −70 mV, this effect was accompanied by the development of inward current noise. In voltage-pulse experiments, methylglyoxal activated an outwardly rectifying conductance which was virtually identical to the volume-sensitive anion conductance previously described in these cells. Two inhibitors of this conductance, 4,4′-dithiocyanatostilbene-2,2′-disulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), also inhibited the depolarization and inward current evoked by methylglyoxal. Methylglyoxal increased β-cell volume to a relative value of 1.33 after 10 min with a gradual return towards basal levels following withdrawal of the α-ketoaldehyde. None of the effects of methylglyoxal was observed in response to t-butylglyoxal which, unlike methylglyoxal, is a poor substrate for the glyoxalase pathway. Methylglyoxal had no apparent effect on β-cell K⁺ channel activity. It is suggested that the metabolism of methylglyoxal to d-lactate causes β-cell swelling and activation of the volume-sensitive anion channel, leading to depolarization. These findings could be relevant to the stimulatory action of d-glucose, the metabolism of which generates significant quantities of l-lactate. Received: 15 May 1998/Revised: 25 September 1998     

Brain-selective Kinase 2 (BRSK2) Phosphorylation on PCTAIRE1 Negatively Regulates Glucose-stimulated Insulin Secretion in Pancreatic β-Cells

Brain-selective kinase 2 (BRSK2) has been shown to play an essential role in neuronal polarization. In the present study, we show that BRSK2 is also abundantly expressed in pancreatic islets and MIN6 β-cell line. Yeast two-hybrid screening, GST fusion protein pull-down, and co-immunoprecipitation assays reveal that BRSK2 interacts with CDK-related protein kinase PCTAIRE1, a kinase involved in neurite outgrowth and neurotransmitter release. In MIN6 cells, BRSK2 co-localizes with PCTAIRE1 in the cytoplasm and phosphorylates one of its serine residues, Ser-12. Phosphorylation of PCTAIRE1 by BRSK2 reduces glucose-stimulated insulin secretion (GSIS) in MIN6 cells. Conversely, knockdown of BRSK2 by siRNA increases serum insulin levels in mice. Our results reveal a novel function of BRSK2 in the regulation of GSIS in β-cells via a PCTAIRE1-dependent mechanism and suggest that BRSK2 is an attractive target for developing novel diabetic drugs.