Munc18c mediates exocytosis of pre-docked and newcomer insulin granules underlying biphasic glucose stimulated insulin secretion in human pancreatic beta-cells

Mitochondrial network functionality is vital for glucose-stimulated insulin secretion in pancreatic beta cells. Altered mitochondrial dynamics in pancreatic beta cells are thought to trigger the development of type 2 diabetes mellitus. Fission protein 1 (Fis1) might be a key player in this process. Thus, the aim of this study was to investigate mitochondrial morphology in dependence of beta cell function, after knockdown and overexpression of Fis1. We demonstrate that glucose-unresponsive cells with impaired glucose-stimulated insulin secretion (INS1-832/2) showed decreased mitochondrial dynamics compared with glucose-responsive cells (INS1-832/13). Accordingly, mitochondrial morphology visualised using MitoTracker staining differed between the two cell lines. INS1-832/2 cells formed elongated and clustered mitochondria, whereas INS1-832/13 cells showed a homogenous mitochondrial network. Fis1 overexpression using lentiviral transduction significantly improved glucose-stimulated insulin secretion and mitochondrial network homogeneity in glucose-unresponsive cells. Conversely, Fis1 downregulation by shRNA, both in primary mouse beta cells and glucose-responsive INS1-832/13 cells, caused unresponsiveness and significantly greater numbers of elongated mitochondria. Overexpression of FIS1 in primary mouse beta cells indicated an upper limit at which higher FIS1 expression reduced glucose-stimulated insulin secretion. Thus, FIS1 was overexpressed stepwise up to a high concentration in RINm5F cells using the RheoSwitch system. Moderate FIS1 expression improved glucose-stimulated insulin secretion, whereas high expression resulted in loss of glucose responsiveness and in mitochondrial artificial loop structures and clustering. Our data confirm that FIS1 is a key regulator in pancreatic beta cells, because both glucose-stimulated insulin secretion and mitochondrial dynamics were clearly adapted to precise expression levels of this fission protein.

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Microtubules Regulate Spatial Localization and Availability of Insulin Granules in Pancreatic Beta Cells

10.1101/581330 ◽

2019 ◽

Author(s):

Kai M. Bracey ◽

Kung-Hsien Ho ◽

Dmitry Yampolsky ◽

Guoqiang Gu ◽

Irina Kaverina ◽

...

Keyword(s):

Plasma Membrane ◽

Beta Cells ◽

Pancreatic Beta Cells ◽

Single Cells ◽

Critical Role ◽

Super Resolution ◽

Transport Dynamics ◽

Insulin Granules ◽

Resolution Imaging ◽

Glucose Stimulated Insulin Secretion

AbstractTwo key prerequisites for glucose stimulated insulin secretion (GSIS) in Beta cells are the proximity of insulin granules to the plasma membrane and their anchoring or docking to the plasma membrane (PM). While recent evidence has indicated that both of these factors are altered in the context of diabetes, it is unclear what regulates localization of insulin and its interactions with the PM within single cells. Here we demonstrate that microtubule (MT) motor mediated transport dynamics have a critical role in regulating both factors. Super-resolution imaging shows that while the MT cytoskeleton resembles a random meshwork in the cells’ interior, MTs near the cells surface are preferentially aligned with the PM. Computational modeling demonstrates two consequences of this alignment. First, this structured MT network preferentially withdraws granules from the PM. Second, the binding and transport of insulin granules by MT motors prevents their stable anchoring to the PM. The MT cytoskeleton thus negatively regulates GSIS by both limiting the amount of insulin proximal to the PM and preventing/breaking interactions between the PM and the remaining nearby insulin. These results predict that altering MT structure in beta cells can be used to tune GSIS. Thus, our study points to a potential of an alternative therapeutic strategy for diabetes by targeting specific MT regulators.

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