A new beta cell-specific mitophagy reporter mouse shows that metabolic stress leads to accumulation of dysfunctional mitochondria despite increased mitophagy.

AIMS/HYPOTHESIS: Mitophagy, the selective autophagy of mitochondria, is essential for maintenance of mitochondrial function. Recent studies suggested that defective mitophagy in beta cells caused diabetes. However, because of technical difficulties, the development of a convenient and reliable method to evaluate mitophagy in beta cells in vivo is needed. The aim of this study was to establish beta cell-specific mitophagy reporter mice and elucidate the role of mitophagy in beta cell function under metabolically stressed conditions induced by a high-fat diet (HFD). METHODS: Mitophagy was assessed using newly generated conditional mitochondrial matrix targeting mitophagy reporter (CMMR) mice, in which mitophagy can be visualised specifically in beta cells in vivo using a fluorescent probe sensitive to lysosomal pH and degradation. Metabolic stress was induced in mice by exposure to the HFD for 20 weeks. The accumulation of dysfunctional mitochondria was examined by staining for functional/total mitochondria and reactive oxygen species (ROS) using specific fluorescent dyes and antibodies. To investigate the molecular mechanism underlying mitophagy in beta cells, overexpression and knockdown experiments were performed. HFD-fed mice were examined to determine whether chronic insulin treatment for 6 weeks could ameliorate mitophagy, mitochondrial function and impaired insulin secretion. RESULTS: Exposure to the HFD increased the number of enlarged (HFD-G) islets with markedly elevated mitophagy. Mechanistically, HFD feeding induced severe hypoxia in HFD-G islets, which upregulated mitophagy through the hypoxia-inducible factor 1-ɑ (Hif-1ɑ)/BCL2 interacting protein 3 (BNIP3) axis in beta cells. However, HFD-G islets unexpectedly showed the accumulation of dysfunctional mitochondria due to excessive ROS production, suggesting an insufficient capacity of mitophagy for the degradation of dysfunctional mitochondria. Chronic administration of insulin ameliorated hypoxia and reduced ROS production and dysfunctional mitochondria, leading to decreased mitophagy and restored insulin secretion. CONCLUSIONS/INTERPRETATION: We demonstrated that CMMR mice enabled the evaluation of mitophagy in beta cells. Our results suggested that metabolic stress induced by the HFD caused the aberrant accumulation of dysfunctional mitochondria, which overwhelmed the mitophagic capacity and was associated with defective maintenance of mitochondrial function and impaired insulin secretion.

Serotonin regulates glucose-stimulated insulin secretion from pancreatic ß cells during pregnancy.

In preparation for the metabolic demands of pregnancy, ß cells in the maternal pancreatic islets increase both in number and in glucose-stimulated insulin secretion (GSIS) per cell. Mechanisms have been proposed for the increased ß cell mass, but not for the increased GSIS. Because serotonin production increases dramatically during pregnancy, we tested whether flux through the ionotropic 5-HT3 receptor (Htr3) affects GSIS during pregnancy. Pregnant Htr3a(-/-) mice exhibited impaired glucose tolerance despite normally increased ß cell mass, and their islets lacked the increase in GSIS seen in islets from pregnant wild-type mice. Electrophysiological studies showed that activation of Htr3 decreased the resting membrane potential in ß cells, which increased Ca(2+) uptake and insulin exocytosis in response to glucose. Thus, our data indicate that serotonin, acting in a paracrine/autocrine manner through Htr3, lowers the ß cell threshold for glucose and plays an essential role in the increased GSIS of pregnancy.

Association of worker's mental health with changes in exercise time, going-out time, and screen time (TV time, internet time, and game time) before and after the COVID-19 pandemic: A cross-sectional study.

The coronavirus disease 2019 (COVID-19) pandemic and government regulations have affected the daily lives and mental health of individuals worldwide. This study aimed to determine how much the change in time spent on exercise (exercise time), outdoor activities ("going-out" time), and screen usage (screen time) before and after the COVID-19 pandemic has affected mental health (depression, anxiety, and insomnia). In June 2021, during the third wave of the COVID-19 pandemic, a web-based, cross-sectional survey was conducted in Japan through an online research company. A total of 824 workers participated in this study. Depression, anxiety, and insomnia were assessed using the Patient Health Questionnaire-9, General Anxiety Disorder-7, and Insomnia Severity Index, respectively. The symptoms of depression were associated with age and decreased exercise time. Symptoms of anxiety were associated with not decreased going-out time. Symptoms of insomnia were associated with reduced exercise time. The results indicated that during the COVID-19 pandemic, an increase in exercise time could have prevented depression and insomnia. Similarly, a decrease in going-out time could have prevented anxiety. Furthermore, in the event of future outbreaks of unpredictable infections, such as COVID-19, decreased going out and increased exercise may help maintain mental health.

Imeglimin mitigates the accumulation of dysfunctional mitochondria to restore insulin secretion and suppress apoptosis of pancreatic ß-cells from db/db mice.

Mitochondrial dysfunction in pancreatic ß-cells leads to impaired glucose-stimulated insulin secretion (GSIS) and type 2 diabetes (T2D), highlighting the importance of autophagic elimination of dysfunctional mitochondria (mitophagy) in mitochondrial quality control (mQC). Imeglimin, a new oral anti-diabetic drug that improves hyperglycemia and GSIS, may enhance mitochondrial activity. However, chronic imeglimin treatment's effects on mQC in diabetic ß-cells are unknown. Here, we compared imeglimin, structurally similar anti-diabetic drug metformin, and insulin for their effects on clearance of dysfunctional mitochondria through mitophagy in pancreatic ß-cells from diabetic model db/db mice and mitophagy reporter (CMMR) mice. Pancreatic islets from db/db mice showed aberrant accumulation of dysfunctional mitochondria and excessive production of reactive oxygen species (ROS) along with markedly elevated mitophagy, suggesting that the generation of dysfunctional mitochondria overwhelmed the mitophagic capacity in db/db ß-cells. Treatment with imeglimin or insulin, but not metformin, reduced ROS production and the numbers of dysfunctional mitochondria, and normalized mitophagic activity in db/db ß-cells. Concomitantly, imeglimin and insulin, but not metformin, restored the secreted insulin level and reduced ß-cell apoptosis in db/db mice. In conclusion, imeglimin mitigated accumulation of dysfunctional mitochondria through mitophagy in diabetic mice, and may contribute to preserving ß-cell function and effective glycemic control in T2D.

Ten-year trends in long-term sickness absence among Japanese public servants: 2009-2018.

Sickness absences are a significant public health and economic problem worldwide. However, sickness absence diagnoses and trends have not been reported in much detail in Japan. This study was a retrospective cohort study. We examined data on certified diagnoses and the durations of sickness absence lasting over 90 days (long-term sickness absence) from 2009-2018 among city public servants in Japan. We found that 1) "Mental and behavioral disorders" (495.0-780.6 per 100,000 employees) was the most prevalent reason for long-term sickness absence, and "Mood disorders" (318.6-584.3 per 100,000 employees) was the most prevalent mental disorders diagnosis in each study year; 2) the prevalence of long-term sickness absence for mental disorders showed decreasing trends (781/100,000 in 2009 to 622/100,000 in 2018; [p=0.005, for the trend test]); 3) the trends differed by gender (p<0.05) and age (p<0.001); and 4) the duration of long-term sickness absence related to mental disorders (13.2 ± 9.0 months) was longer than long-term sickness absence resulting from all physical disorders except for diseases of the circulatory system (15.1 ± 11.6 months). Increased focus on significant depressive and neurotic disorders is needed when promoting mental health in the workplace.

Imaging analysis reveals mechanistic differences between first- and second-phase insulin exocytosis.

The mechanism of glucose-induced biphasic insulin release is unknown. We used total internal reflection fluorescence (TIRF) imaging analysis to reveal the process of first- and second-phase insulin exocytosis in pancreatic beta cells. This analysis showed that previously docked insulin granules fused at the site of syntaxin (Synt)1A clusters during the first phase; however, the newcomers fused during the second phase external to the Synt1A clusters. To reveal the function of Synt1A in phasic insulin exocytosis, we generated Synt1A-knockout (Synt1A(-/-)) mice. Synt1A(-/-) beta cells showed fewer previously docked granules with no fusion during the first phase; second-phase fusion from newcomers was preserved. Rescue experiments restoring Synt1A expression demonstrated restoration of granule docking status and fusion events. Inhibition of other syntaxins, Synt3 and Synt4, did not affect second-phase insulin exocytosis. We conclude that the first phase is Synt1A dependent but the second phase is not. This indicates that the two phases of insulin exocytosis differ spatially and mechanistically.

Relationships between Occupational Stress, Change in Work Environment during the COVID-19 Pandemic, and Depressive and Anxiety Symptoms among Non-Healthcare Workers in Japan: A Cross-Sectional Study.

This study aims to clarify the effect of occupational stress and changes in the work environment on non-healthcare workers' (HCWs) mental health during the third wave of the COVID-19 pandemic in Japan. A web-based, cross-sectional survey was conducted from 16 to 17 December 2020. Data from 807 non-HCWs were included. We evaluated occupational stress using the Generic Job Stress Questionnaire (GJSQ). Depressive and anxiety symptoms were assessed using the Japanese version of the Patient Health Questionnaire-9 and the Generalized Anxiety Disorder 7-item scale, respectively. We collected demographic variables, work-related variables, and the variables associated with COVID-19. The adjusted odds ratios for depressive and anxiety groups were estimated using multivariate logistic regression analyses, adjusted for all the demographic variables, work-related variables, COVID-19-related variables, and the six subdivided GJSQ subscales. The results confirm a relationship between variance in workload, job future ambiguity, social support from coworkers, having contact with COVID-19 patients, and depressive and anxiety symptoms. Paying attention to job future ambiguity, the variance in workload at the workplace and individual perspectives, promoting contact and support among coworkers using online communication tools, and reducing contact with COVID-19 patients, will be useful for decreasing the depressive and anxiety symptoms among non-HCWs.

DPP-4 inhibitor des-F-sitagliptin treatment increased insulin exocytosis from db/db mice ß cells.

Incretin promotes insulin secretion acutely. Recently, orally-administered DPP-4 inhibitors represent a new class of anti-hyperglycemic agents. Indeed, inhibitors of dipeptidyl peptidase-IV (DPP-4), sitagliptin, has just begun to be widely used as therapeutics for type 2 diabetes. However, the effects of sitagliptin-treatment on insulin exocytosis from single ß-cells are yet unknown. We therefore investigated how sitagliptin-treatment in db/db mice affects insulin exocytosis by treating db/db mice with des-F-sitagliptin for 2 weeks. Perfusion studies showed that 2 weeks-sitagliptin treatment potentiated insulin secretion. We then analyzed insulin granule motion and SNARE protein, syntaxin 1, by TIRF imaging system. TIRF imaging of insulin exocytosis showed the increased number of docked insulin granules and increased fusion events from them during first-phase release. In accord with insulin exocytosis data, des-F-sitagliptin-treatment increased the number of syntaxin 1 clusters on the plasma membrane. Thus, our data demonstrated that 2-weeks des-F-sitagliptin-treatment increased the fusion events of insulin granules, probably via increased number of docked insulin granules and that of syntaxin 1 clusters.

Insulin/phosphoinositide 3-kinase pathway accelerates the glucose-induced first-phase insulin secretion through TrpV2 recruitment in pancreatic ß-cells.

Functional insulin receptor and its downstream effector PI3K (phosphoinositide 3-kinase) have been identified in pancreatic ß-cells, but their involvement in the regulation of insulin secretion from ß-cells remains unclear. In the present study, we investigated the physiological role of insulin and PI3K in glucose-induced biphasic insulin exocytosis in primary cultured ß-cells and insulinoma Min6 cells using total internal reflection fluorescent microscopy. The pretreatment of ß-cells with insulin induced the rapid increase in intracellular Ca2+ levels and accelerated the exocytotic response without affecting the second-phase insulin secretion. The inhibition of PI3K not only abolished the insulin-induced rapid development of the exocytotic response, but also potentiated the second-phase insulin secretion. The rapid development of Ca2+ and accelerated exocytotic response induced by insulin were accompanied by the translocation of the Ca2+-permeable channel TrpV2 (transient receptor potential V2) in a PI3K-dependent manner. Inhibition of TrpV2 by the selective blocker tranilast, or the expression of shRNA (short-hairpin RNA) against TrpV2 suppressed the effect of insulin in the first phase, but the second phase was not affected. Thus our results demonstrate that insulin treatment induced the acceleration of the exocytotic response during the glucose-induced first-phase response by the insertion of TrpV2 into the plasma membrane in a PI3K-dependent manner.

Relationship between initial self-perceived depressive symptoms and disease severity in working patients with first-onset major depressive disorder.

The severity of major depressive disorder (MDD), which is related to the depressive symptoms, is a predictor of clinical outcomes and may be used to determine the appropriate treatment. However, there is a lack of systematic research on the relationship between early depressive symptoms and MDD severity. This study aimed to clarify the association between initial depressive symptoms and MDD severity in working patients. We assessed 118 patients aged over 20 years who visited the Neuropsychiatry Department of the Osaka City University Hospital following their first episode of MDD. Logistic regression analyses were performed to estimate the odds ratios (OR) with 95% confidence intervals (CI) for the associations between age, gender, marital status, working hours, and initial self-perceived depressive symptoms and MDD severity. Age and working hours were analyzed as continuous variables, and gender (man, woman), marital status (married, single) and severity (mild to moderate MDD, severe to very severe MDD) were analyzed as categorical variables. The most common initial self-perceived symptom was "depressed mood," followed by "fatigue or loss of energy nearly every day." The univariate analysis found no association between age, gender, marital status, or working hours and MDD severity. Initial self-perceived non-somatic symptoms were associated with increased odds of having severe MDD (odds ratio = 3.32, 95% confidence interval 1.46-7.58), and this association persisted in the adjusted model (odds ratio = 3.35, 95% confidence interval 1.47-7.60). Initial self-perceived non-somatic depressive symptoms are significantly associated with MDD severity at its first onset. Workplace support may lead to the early detection and treatment of working patients with non-somatic symptoms.

Pattern of rise in subplasma membrane Ca2+ concentration determines type of fusing insulin granules in pancreatic beta cells.

We simultaneously analyzed insulin granule fusion with insulin fused to green fluorescent protein and the subplasma membrane Ca2+ concentration ([Ca2+](PM)) with the Ca2+ indicator Fura Red in rat beta cells by dual-color total internal reflection fluorescence microscopy. We found that rapid and marked elevation in [Ca2+](PM) caused insulin granule fusion mostly from previously docked granules during the high KCl-evoked release and high glucose-evoked first phase release. In contrast, the slow and sustained elevation in [Ca2+](PM) induced fusion from newcomers translocated from the internal pool during the low KCl-evoked release and glucose-evoked second phase release. These data suggest that the pattern of the [Ca2+](PM) rise directly determines the types of fusing granules.

Imaging exocytosis of single glucagon-like peptide-1 containing granules in a murine enteroendocrine cell line with total internal reflection fluorescent microscopy.

To analyze the exocytosis of glucagon-like peptide-1 (GLP-1) granules, we imaged the motion of GLP-1 granules labeled with enhanced yellow fluorescent protein (Venus) fused to human growth hormone (hGH-Venus) in an enteroendocrine cell line, STC-1 cells, by total internal reflection fluorescent (TIRF) microscopy. We found glucose stimulation caused biphasic GLP-1 granule exocytosis: during the first phase, fusion events occurred from two types of granules (previously docked granules and newcomers), and thereafter continuous fusion was observed mostly from newcomers during the second phase. Closely similar to the insulin granule fusion from pancreatic beta cells, the regulated biphasic exocytosis from two types of granules may be a common mechanism in glucose-evoked hormone release from endocrine cells.

Insulin exocytosis in Goto-Kakizaki rat beta-cells subjected to long-term glinide or sulfonylurea treatment.

Sulfonylurea and glinide drugs display different effects on insulin granule motion in single beta-cells in vitro. We therefore investigated the different effects that these drugs manifest towards insulin release in an in vivo long-term treatment model. Diabetic GK (Goto-Kakizaki) rats were treated with nateglinide, glibenclamide or insulin for 6 weeks. Insulin granule motion in single beta-cells and the expression of SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) proteins were then analysed. Perifusion studies showed that decreased first-phase insulin release was partially recovered when GK rats were treated with nateglinide or insulin for 6 weeks, whereas no first-phase release occurred with glibenclamide treatment. In accord with the perifusion results, TIRF (total internal reflection fluorescence) imaging of insulin exocytosis showed restoration of the decreased number of docked insulin granules and the fusion events from them during first-phase release for nateglinide or insulin, but not glibenclamide, treatment; electron microscopy results confirmed the TIRF microscopy data. Relative to vehicle-treated GK beta-cells, an increased number of SNARE clusters were evident in nateglinide- or insulin-treated cells; a lesser increase was observed in glibenclamide-treated cells. Immunostaining for insulin showed that nateglinide treatment better preserved pancreatic islet morphology than did glibenclamide treatment. However, direct exposure of GK beta-cells to these drugs could not restore the decreased first-phase insulin release nor the reduced numbers of docked insulin granules. We conclude that treatment of GK rats with nateglinide and glibenclamide varies in long-term effects on beta-cell functions; nateglinide treatment appears overall to be more beneficial.

ELKS/Voltage-Dependent Ca2+ Channel-ß Subunit Module Regulates Polarized Ca2+ Influx in Pancreatic ß Cells.

Pancreatic ß cells secrete insulin by Ca2+-triggered exocytosis. However, there is no apparent secretory site similar to the neuronal active zones, and the cellular and molecular localization mechanism underlying polarized exocytosis remains elusive. Here, we report that ELKS, a vertebrate active zone protein, is used in ß cells to regulate Ca2+ influx for insulin secretion. ß cell-specific ELKS-knockout (KO) mice showed impaired glucose-stimulated first-phase insulin secretion and reduced L-type voltage-dependent Ca2+ channel (VDCC) current density. In situ Ca2+ imaging of ß cells within islets expressing a membrane-bound G-CaMP8b Ca2+ sensor demonstrated initial local Ca2+ signals at the ELKS-localized vascular side of the ß cell plasma membrane, which were markedly decreased in ELKS-KO ß cells. Mechanistically, ELKS directly interacted with the VDCC-ß subunit via the GK domain. These findings suggest that ELKS and VDCCs form a potent insulin secretion complex at the vascular side of the ß cell plasma membrane for polarized Ca2+ influx and first-phase insulin secretion from pancreatic islets.

TDP-43 regulates early-phase insulin secretion via CaV1.2-mediated exocytosis in islets.

TAR DNA-binding protein 43 kDa (TDP-43), encoded by TARDBP, is an RNA-binding protein, the nuclear depletion of which is the histopathological hallmark of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder affecting both upper and lower motor neurons. Besides motor symptoms, patients with ALS often develop nonneuronal signs including glucose intolerance, but the underlying pathomechanism is still controversial, i.e., whether it is impaired insulin secretion and/or insulin resistance. Here, we showed that ALS subjects reduced early-phase insulin secretion and that the nuclear localization of TDP-43 was lost in the islets of autopsied ALS pancreas. Loss of TDP-43 inhibited exocytosis by downregulating CaV1.2 calcium channels, thereby reducing early-phase insulin secretion in a cultured ß cell line (MIN6) and ß cell-specific Tardbp knockout mice. Overexpression of CaV1.2 restored early-phase insulin secretion in Tardbp knocked-down MIN6 cells. Our findings suggest that TDP-43 regulates cellular exocytosis mediated by L-type voltage-dependent calcium channels and thus plays an important role in the early phase of insulin secretion by pancreatic islets. Thus, nuclear loss of TDP-43 is implicated in not only the selective loss of motor neurons but also in glucose intolerance due to impaired insulin secretion at an early stage of ALS.

ELKS, a protein structurally related to the active zone-associated protein CAST, is expressed in pancreatic beta cells and functions in insulin exocytosis: interaction of ELKS with exocytotic machinery analyzed by total internal reflection fluorescence microscopy.

The cytomatrix at the active zone (CAZ) has been implicated in defining the site of Ca2+-dependent exocytosis of neurotransmitters. Here, we demonstrate the expression and function of ELKS, a protein structurally related to the CAZ protein CAST, in insulin exocytosis. The results of confocal and immunoelectron microscopic analysis showed that ELKS is present in pancreatic beta cells and is localized close to insulin granules docked on the plasma membrane-facing blood vessels. Total internal reflection fluorescence microscopy imaging in insulin-producing clonal cells revealed that the ELKS clusters are less dense and unevenly distributed than syntaxin 1 clusters, which are enriched in the plasma membrane. Most of the ELKS clusters were on the docking sites of insulin granules that were colocalized with syntaxin 1 clusters. Total internal reflection fluorescence images of single-granule motion showed that the fusion events of insulin granules mostly occurred on the ELKS cluster, where repeated fusion was sometimes observed. When the Bassoon-binding region of ELKS was introduced into the cells, the docking and fusion of insulin granules were markedly reduced. Moreover, attenuation of ELKS expression by small interfering RNA reduced the glucose-evoked insulin release. These data suggest that the CAZ-related protein ELKS functions in insulin exocytosis from pancreatic beta cells.

VAMP7 Regulates Autophagosome Formation by Supporting Atg9a Functions in Pancreatic ß-Cells From Male Mice.

Dysfunctional mitochondria are observed in ß-cells of diabetic patients, which are eventually removed by autophagy. Vesicle-associated membrane protein (VAMP)7, a vesicular SNARE protein, regulates autophagosome formation to maintain mitochondrial homeostasis and control insulin secretion in pancreatic ß-cells. However, its molecular mechanism is largely unknown. In this study, we investigated the molecular mechanism of VAMP7-dependent autophagosome formation using VAMP7-deficient ß-cells and ß-cell-derived Min6 cells. VAMP7 localized in autophagy-related (Atg)9a-resident vesicles of recycling endosomes (REs), which contributed to autophagosome formation, and it interacted with Hrb, Syntaxin16, and SNAP-47. Hrb recruited VAMP7 and Atg9a from the plasma membrane to REs. Syntaxin16 and SNAP-47 mediated autophagosome formation at a step later than the proper localization of VAMP7 to Atg9a-resident vesicles. Knockdown of Hrb, Syntaxin16, and SNAP-47 resulted in defective autophagosome formation, accumulation of dysfunctional mitochondria, and impairment of glucose-stimulated insulin secretion. Our data indicate that VAMP7 and Atg9a are initially recruited to REs to organize VAMP7 and Atg9a-resident vesicles in an Hrb-dependent manner. Additionally, VAMP7 forms a SNARE complex with Syntaxin16 and SNAP-47, which may cause fusions of Atg9a-resident vesicles during autophagosome formation. Thus, VAMP7 participates in autophagosome formation by supporting Atg9a functions that contribute to maintenance of mitochondrial quality.

Imaging docking and fusion of insulin granules induced by antidiabetes agents: sulfonylurea and glinide drugs preferentially mediate the fusion of newcomer, but not previously docked, insulin granules.

Sulfonylurea and glinide drugs, commonly used for antidiabetes therapies, are known to stimulate insulin release from pancreatic beta-cells by closing ATP-sensitive K+ channels. However, the specific actions of these drugs on insulin granule motion are largely unknown. Here, we used total internal reflection fluorescence (TIRF) microscopy to analyze the docking and fusion of single insulin granules in live beta-cells exposed to either the sulfonylurea drug glibenclamide or the glinide drug mitiglinide. TIRF images showed that both agents caused rapid fusion of newcomer insulin granules with the cell membrane in both control and diabetic Goto-Kakizaki (GK) rat pancreatic beta-cells. However, in the context of beta-cells from sulfonylurea receptor 1 (SUR1) knockout mice, TIRF images showed that only mitiglinide, but not glibenclamide, caused fusion of newcomer insulin granules. Compositely, our data indicate that 1) the mechanism by which both sulfonylurea and glinide drugs promote insulin release entails the preferential fusion of newcomer, rather than previously docked, insulin granules, and that 2) mitiglinide can induce insulin release by a mechanism independent of mitiglinide binding to SUR1.

VAMP7 Regulates Autophagy to Maintain Mitochondrial Homeostasis and to Control Insulin Secretion in Pancreatic ß-Cells.

VAMP7 is a SNARE protein that mediates specific membrane fusions in intracellular trafficking and was recently reported to regulate autophagosome formation. However, its function in pancreatic ß-cells is largely unknown. To elucidate the physiological role of VAMP7 in ß-cells, we generated pancreatic ß-cell-specific VAMP7 knockout (Vamp7(flox/Y);Cre) mice. VAMP7 deletion impaired glucose-stimulated ATP production and insulin secretion, though VAMP7 was not localized to insulin granules. VAMP7-deficient ß-cells showed defective autophagosome formation and reduced mitochondrial function. p62/SQSTM1, a marker protein for defective autophagy, was selectively accumulated on mitochondria in VAMP7-deficient ß-cells. These findings suggest that accumulation of dysfunctional mitochondria that are degraded by autophagy caused impairment of glucose-stimulated ATP production and insulin secretion in Vamp7(flox/Y);Cre ß-cells. Feeding a high-fat diet to Vamp7(flox/Y);Cre mice exacerbated mitochondrial dysfunction, further decreased ATP production and insulin secretion, and consequently induced glucose intolerance. Moreover, we found upregulated VAMP7 expression in wild-type mice fed a high-fat diet and in db/db mice, a model for diabetes. Thus our data indicate that VAMP7 regulates autophagy to maintain mitochondrial quality and insulin secretion in response to pathological stress in ß-cells.

Docking and fusion of insulin secretory granules in SUR1 knock out mouse beta-cells observed by total internal reflection fluorescence microscopy.

To explore how the sulfonylurea receptor (SUR1) is involved in docking and fusion of insulin granules, dynamic motion of single insulin secretory granules near the plasma membrane was examined in SUR1 knock-out (Sur1KO) beta-cells by total internal reflection fluorescence microscopy. Sur1KO beta-cells exhibited a marked reduction in the number of fusion events from previously docked granules. However, the number of docked granules declined during stimulation as a consequence of the release of docked granules into the cytoplasm vs. fusion with the plasma membrane. Thus, the impaired docking and fusion results in decreased insulin exocytosis from Sur1KO beta-cells.