Doc2b Protects β-Cells Against Inflammatory Damage and Enhances Function

Loss of functional β-cell mass is an early feature of type 1 diabetes. To release insulin, β-cells require soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, as well as SNARE complex regulatory proteins like double C2 domain-containing protein β (Doc2b). We hypothesized that Doc2b deficiency or overabundance may confer susceptibility or protection, respectively, to the functional β-cell mass. Indeed, Doc2b+/- knockout mice show an unusually severe response to multiple-low-dose streptozotocin (MLD-STZ), resulting in more apoptotic β-cells and a smaller β-cell mass. In addition, inducible β-cell-specific Doc2b-overexpressing transgenic (βDoc2b-dTg) mice show improved glucose tolerance and resist MLD-STZ-induced disruption of glucose tolerance, fasting hyperglycemia, β-cell apoptosis, and loss of β-cell mass. Mechanistically, Doc2b enrichment enhances glucose-stimulated insulin secretion (GSIS) and SNARE activation and prevents the appearance of apoptotic markers in response to cytokine stress and thapsigargin. Furthermore, expression of a peptide containing the Doc2b tandem C2A and C2B domains is sufficient to confer the beneficial effects of Doc2b enrichment on GSIS, SNARE activation, and apoptosis. These studies demonstrate that Doc2b enrichment in the β-cell protects against diabetogenic and proapoptotic stress. Furthermore, they identify a Doc2b peptide that confers the beneficial effects of Doc2b and may be a therapeutic candidate for protecting functional β-cell mass

The actin-related p41ARC subunit contributes to p21-activated kinase-1 (PAK1)-mediated glucose uptake into skeletal muscle cells

Defects in translocation of the glucose transporter GLUT4 are associated with peripheral insulin resistance, preclinical diabetes, and progression to type 2 diabetes. GLUT4 recruitment to the plasma membrane of skeletal muscle cells requires F-actin remodeling. Insulin signaling in muscle requires p21-activated kinase-1 (PAK1), whose downstream signaling triggers actin remodeling, which promotes GLUT4 vesicle translocation and glucose uptake into skeletal muscle cells. Actin remodeling is a cyclic process, and although PAK1 is known to initiate changes to the cortical actin-binding protein cofilin to stimulate the depolymerizing arm of the cycle, how PAK1 might trigger the polymerizing arm of the cycle remains unresolved. Toward this, we investigated whether PAK1 contributes to the mechanisms involving the actin-binding and -polymerizing proteins neural Wiskott-Aldrich syndrome protein (N-WASP), cortactin, and ARP2/3 subunits. We found that the actin-polymerizing ARP2/3 subunit p41ARC is a PAK1 substrate in skeletal muscle cells. Moreover, co-immunoprecipitation experiments revealed that insulin stimulates p41ARC phosphorylation and increases its association with N-WASP coordinately with the associations of N-WASP with cortactin and actin. Importantly, all of these associations were ablated by the PAK inhibitor IPA3, suggesting that PAK1 activation lies upstream of these actin-polymerizing complexes. Using the N-WASP inhibitor wiskostatin, we further demonstrated that N-WASP is required for localized F-actin polymerization, GLUT4 vesicle translocation, and glucose uptake. These results expand the model of insulin-stimulated glucose uptake in skeletal muscle cells by implicating p41ARC as a new component of the insulin-signaling cascade and connecting PAK1 signaling to N-WASP-cortactin-mediated actin polymerization and GLUT4 vesicle translocation

DOC2B promotes insulin sensitivity in mice via a novel KLC1-dependent mechanism in skeletal muscle

Aims/hypothesis: Skeletal muscle accounts for >80% of insulin-stimulated glucose uptake; dysfunction of this process underlies insulin resistance and type 2 diabetes. Insulin sensitivity is impaired in mice deficient in the double C2 domain β (DOC2B) protein, while whole-body overexpression of DOC2B enhances insulin sensitivity. Whether insulin sensitivity in the skeletal muscle is affected directly by DOC2B or is secondary to an effect on other tissues is unknown; the underlying molecular mechanisms also remain unclear. Methods: Human skeletal muscle samples from non-diabetic or type 2 diabetic donors were evaluated for loss of DOC2B during diabetes development. For in vivo analysis, new doxycycline-inducible skeletal-muscle-specific Doc2b-overexpressing mice fed standard or high-fat diets were evaluated for insulin and glucose tolerance, and insulin-stimulated GLUT4 accumulation at the plasma membrane (PM). For in vitro analyses, a DOC2B-overexpressing L6-GLUT4-myc myoblast/myotube culture system was coupled with an insulin resistance paradigm. Biochemical and molecular biology methods such as site-directed mutagenesis, co-immunoprecipitation and mass spectrometry were used to identify the molecular mechanisms linking insulin stimulation to DOC2B. Results: We identified loss of DOC2B (55% reduction in RNA and 40% reduction in protein) in the skeletal muscle of human donors with type 2 diabetes. Furthermore, inducible enrichment of DOC2B in skeletal muscle of transgenic mice enhanced whole-body glucose tolerance (AUC decreased by 25% for female mice) and peripheral insulin sensitivity (area over the curve increased by 20% and 26% for female and male mice, respectively) in vivo, underpinned by enhanced insulin-stimulated GLUT4 accumulation at the PM. Moreover, DOC2B enrichment in skeletal muscle protected mice from high-fat-diet-induced peripheral insulin resistance, despite the persistence of obesity. In L6-GLUT4-myc myoblasts, DOC2B enrichment was sufficient to preserve normal insulin-stimulated GLUT4 accumulation at the PM in cells exposed to diabetogenic stimuli. We further identified that DOC2B is phosphorylated on insulin stimulation, enhancing its interaction with a microtubule motor protein, kinesin light chain 1 (KLC1). Mutation of Y301 in DOC2B blocked the insulin-stimulated phosphorylation of DOC2B and interaction with KLC1, and it blunted the ability of DOC2B to enhance insulin-stimulated GLUT4 accumulation at the PM. Conclusions/interpretation: These results suggest that DOC2B collaborates with KLC1 to regulate insulin-stimulated GLUT4 accumulation at the PM and regulates insulin sensitivity. Our observation provides a basis for pursuing DOC2B as a novel drug target in the muscle to prevent/treat type 2 diabetes

Rosuvastatin treatment affects both basal and glucose-induced insulin secretion in INS-1 832/13 cells

Rosuvastatin is a member of the statin family. Like the other statins it is prescribed to lower cholesterol levels and thereby reduce the risk of cardiovascular events. Rosuvastatin lowers the cholesterol levels by inhibiting the key enzyme 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase) in the cholesterol producing mevalonate pathway. It has been recognized that apart from their beneficial lipid lowering effects, statins also exhibit diabetogenic properties. The molecular mechanisms behind these remain unresolved. To investigate the effects of rosuvastatin on insulin secretion, we treated INS-1 832/13 cells with varying doses (20 nM to 20 μM) of rosuvastatin for 48 h. At concentrations of 2 μM and above basal insulin secretion was significantly increased. Using diazoxide we could determine that rosuvastatin did not increase basal insulin secretion by corrupting the KATP channels. Glucose-induced insulin secretion on the other hand seemed to be affected differently at different rosuvastatin concentrations. Rosuvastatin treatment (20 μM) for 24-48 h inhibited voltage-gated Ca2+ channels, which lead to reduced depolarization-induced exocytosis of insulin-containing granules. At lower concentrations of rosuvastatin (≤ 2 μM) the stimulus-secretion coupling pathway was intact downstream of the KATP channels as assessed by the patch clamp technique. However, a reduction in glucose-induced insulin secretion could be observed with rosuvastatin concentrations as low as 200 nM. The inhibitory effects of rosuvastatin on glucose-induced insulin secretion could be reversed with mevalonate, but not squalene, indicating that rosuvastatin affects insulin secretion through its effects on the mevalonate pathway, but not through the reduction of cholesterol biosynthesis. Taken together, these data suggest that rosuvastatin has the potential to increase basal insulin secretion and reduce glucose-induced insulin secretion. The latter is possibly an unavoidable side effect of rosuvastatin treatment as it occurs through the same mechanisms as the lipid-lowering effects of the drug

Effects of 48 h of rosuvastatin treatment on insulin secretion in INS-1 832/13 cells.

<p>(A) Insulin secretion at 2.8 mM glucose measured in the presence of rosuvastatin (Ros) at concentrations ranging from 20 nM-20 μM as indicated in the figure. Statistical significance is calculated compared to the control (DMSO). (B) Same as in (A) but insulin secretion is measured at 16.7 mM glucose instead. (C) Insulin secretion at 2.8 mM glucose from cells treated with 20μM rosuvastatin, 200 μM diazoxide (Dzx) or a combination of the two. (D) Insulin secretion at 2.8 mM glucose and 50 mM K⁺ with and without 20μM rosuvastatin. (E) Insulin secretion at 16.7 mM glucose from cells treated with 100 nM GLP-1 with and without 20 μM rosuvastatin. Data are given as mean ± SEM from 3 experiments with 3 technical replicates in each experiment. * p≤ 0.05; ** p≤ 0.01; *** p≤ 0.001.</p

Effect of mevalonate and squalene on insulin secretion from rosuvastatin-treated INS-1 832/13 cells.

<p>(A) Insulin secretion at basal (2.8 mM) concentrations of glucose from cells treated with 20 μM rosuvastatin (Ros), 50 μM mevalonate (MVA), and 100 μM squalene (Sq) for 48 h as indicated in the figure. (B) Same as in (A) but with stimulatory (16.7 mM) concentrations of glucose instead. (C) Same as in (A) but the cells are stimulated with 50 mM K⁺ as well. Data are given as mean ± SEM from 3 experiments with 3 technical replicates in each experiment. * p≤ 0.05.</p

Effects of 24–48 h of rosuvastatin treatment on exocytosis in INS-1 832/13 cells.

<p>(A) Example traces of depolarization-induced exocytosis measured as changes in cell membrane capacitance, from rosuvastatin-treated cells (20 μM; black trace) and control cells (grey trace). Exocytosis was evoked by a train of ten 500 ms depolarizing pulses from -70 mV to 0 mV. (B) Summary of the total capacitance change during the train in control cells (white bars) and rosuvastatin-treated cells (Ros; black bars). The concentration of rosuvastatin in these experiments ranged from 20 nM-20 μM as marked in the figure. (C) A graph describing the exocytotic response to all 10 pulses (∑_all) to the first 2 pulses (∑_1–2) or to the latter 8 pulses (∑_3–10) in cells incubated with 20 μM rosuvastatin (black bars) and their controls (white bars). (D) Calcium sensitivity of cells incubated with 20 μM rosuvastatin (Ros; black bars) or their controls (white bars). Calcium sensitivity is calculated by dividing the exocytotic response to the first pulse with the calcium charge measured during the same pulse. Data are given as mean ± SEM of 21–31 cells. * p≤ 0.05; ** p≤ 0.01.</p

Electrophysiological characterization of voltage-gated ion channels in rosuvastatin treated INS-1 832/13.

<p>Cells were treated with 20 μM rosuvastatin for 24-48h. (A) Example traces of currents evoked by a depolarization to 0 mV in a single rosuvastatin-treated (Ros; black trace) and control (grey trace) cell. I_sus and I_p measured in (B) and (D) are marked. (B) Sustained current (I_sus)-voltage (V) relationship (C) charge (Q)-voltage (V) relationship. Charge is measured as the area enclosed by the curve in (A). (D) peak current (I_p)-voltage (V) relationship in INS-1 832/13 cells treated with 20 μM rosuvastatin (Ros; black dots) or control cells (Control; white squares). Data are given as mean ± SEM of 28–38 cells.</p

DOC2B promotes insulin sensitivity in mice via a novel KLC1-dependent mechanism in skeletal muscle

Aims/hypothesis: Skeletal muscle accounts for >80% of insulin-stimulated glucose uptake; dysfunction of this process underlies insulin resistance and type 2 diabetes. Insulin sensitivity is impaired in mice deficient in the double C2 domain β (DOC2B) protein, while whole-body overexpression of DOC2B enhances insulin sensitivity. Whether insulin sensitivity in the skeletal muscle is affected directly by DOC2B or is secondary to an effect on other tissues is unknown; the underlying molecular mechanisms also remain unclear. Methods: Human skeletal muscle samples from non-diabetic or type 2 diabetic donors were evaluated for loss of DOC2B during diabetes development. For in vivo analysis, new doxycycline-inducible skeletal-muscle-specific Doc2b-overexpressing mice fed standard or high-fat diets were evaluated for insulin and glucose tolerance, and insulin-stimulated GLUT4 accumulation at the plasma membrane (PM). For in vitro analyses, a DOC2B-overexpressing L6-GLUT4-myc myoblast/myotube culture system was coupled with an insulin resistance paradigm. Biochemical and molecular biology methods such as site-directed mutagenesis, co-immunoprecipitation and mass spectrometry were used to identify the molecular mechanisms linking insulin stimulation to DOC2B. Results: We identified loss of DOC2B (55% reduction in RNA and 40% reduction in protein) in the skeletal muscle of human donors with type 2 diabetes. Furthermore, inducible enrichment of DOC2B in skeletal muscle of transgenic mice enhanced whole-body glucose tolerance (AUC decreased by 25% for female mice) and peripheral insulin sensitivity (area over the curve increased by 20% and 26% for female and male mice, respectively) in vivo, underpinned by enhanced insulin-stimulated GLUT4 accumulation at the PM. Moreover, DOC2B enrichment in skeletal muscle protected mice from high-fat-diet-induced peripheral insulin resistance, despite the persistence of obesity. In L6-GLUT4-myc myoblasts, DOC2B enrichment was sufficient to preserve normal insulin-stimulated GLUT4 accumulation at the PM in cells exposed to diabetogenic stimuli. We further identified that DOC2B is phosphorylated on insulin stimulation, enhancing its interaction with a microtubule motor protein, kinesin light chain 1 (KLC1). Mutation of Y301 in DOC2B blocked the insulin-stimulated phosphorylation of DOC2B and interaction with KLC1, and it blunted the ability of DOC2B to enhance insulin-stimulated GLUT4 accumulation at the PM. Conclusions/interpretation: These results suggest that DOC2B collaborates with KLC1 to regulate insulin-stimulated GLUT4 accumulation at the PM and regulates insulin sensitivity. Our observation provides a basis for pursuing DOC2B as a novel drug target in the muscle to prevent/treat type 2 diabetes

The highly expressed calcium-insensitive synaptotagmin-11 and synaptotagmin-13 modulate insulin secretion

AIM: SYT11 and SYT13, two calcium-insensitive synaptotagmins, are downregulated in islets from type-2 diabetic donors, but their function in insulin secretion is unknown. To address this, we investigated the physiological role of these two synaptotagmins in insulin secreting cells.METHODS: Correlations between gene expression levels were performed using previously described RNA-seq data on islets from 188 human donors. SiRNA knockdown was performed in EndoC-βH1 and INS-1 832/13 cells. Insulin secretion was measured with ELISA. Patch clamp was used for single cell electrophysiology. Confocal microscopy was used to determine intra-cellular localization.RESULTS: Human islet expression of the transcription factor PDX-1 was positively correlated with SYT11 (p = 2.4e -10 ) and SYT13 (p<2.2 e -16 ). Syt11 and Syt13 both co-localized with insulin, indicating their localization in insulin granules. Downregulation of Syt11 in INS-1 832/13 cells (siSYT11) resulted in increased basal and glucose-induced insulin secretion. Downregulation of Syt13 (siSYT13) decreased insulin secretion induced by glucose and K + .Interestingly, the cAMP raising agent forskolin was unable to enhance insulin secretion in siSYT13 cells. There was no difference in insulin content, exocytosis, or voltage-gated Ca 2+ currents in the two models. Double knockdown of Syt11 and Syt13 (DKD) resembled the results in siSYT13 cells. CONCLUSION: SYT11 and SYT13 have similar localization and transcriptional regulation but they regulate insulin secretion differentially. While downregulation of SYT11 might be a compensatory mechanism in type-2 diabetes, downregulation of SYT13 reduces the insulin secretory response and overrules the compensatory regulation of SYT11 in a way that could aggravate the disease