leprdb-mouse-model-type-2-diabetes-pancreatic-islet-isolation-live

Lepr<sup>db</sup> Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

The University of Queensland

Pancreatic acinar cells secrete fluid and digestive enzymes. Both types of secretion are activated by a rise in intracellular calcium but how the stimulus-secretion cascade actually regulates secretory output is not well understood. It has long been known that the calcium response of acinar cells to physiological stimulation is complex. Dependent on the type and concentration of agonist, it consists of either local or global calcium increases as well as spreading waves of calcium across the cell. In the past it has been speculated that these different calcium signals drive different secretory responses. Now, recent employment of two-photon microscopy has enabled the simultaneous recording of both enzyme secretion and calcium signals and is beginning to resolve this issue. The data shows that local calcium responses exclusively drive fluid secretion. Where-as, global calcium responses drive both fluid and enzyme secretion. This differential control of secretory output is likely central to controlling the physiological responses of pancreatic acinar cells.

Pancreatic acinar cell: new insights into the control of secretion.

The relationship between the cellular Ca2+ signal and secretory vesicle fusion (exocytosis) is a key determinant of the regulation of the kinetics and magnitude of the secretory response. Here, we have investigated secretion in cells where the exocytic response is controlled by Ca2+ release from intracellular Ca2+ stores. Using live-cell two-photon microscopy that simultaneously records Ca2+signals and exocytic responses, we provide evidence that secretion is controlled by changes in Ca2+ concentration [Ca2+] in relatively large-volume microdomains. Our evidence includes: (1) long latencies (>2 seconds) between the rise in [Ca2+] and exocytosis, (2) observation of exocytosis all along the lumen and not clustered around Ca2+ release hot-spots, (3) high affinity (Kd=1.75 microM) Ca2+dependence of exocytosis, (4) significant reduction in exocytosis in the presence of cytosolic EGTA, (5) spatial exclusion of secretory granules from the cell membrane by the endoplasmic reticulum, and (6) inability of local Ca2+ responses to trigger exocytosis. These results strongly indicate that the control of exocytosis, triggered by Ca2+ release from stores, is through the regulation of cytosolic[Ca2+] within a microdomain.

Exocytosis, dependent on Ca2+ release from Ca2+ stores, is regulated by Ca2+ microdomains.

In dispersed single beta cells the response of each cell to glucose is heterogeneous. In contrast, within an islet, cell-to-cell communication leads to glucose inducing a more homogeneous response. For example, increases in NAD(P)H and calcium are relatively uniform across the cells of the islet. These data suggest that secretion of insulin from single beta cells within an islet should also be relatively homogeneous. The aim of this study was to test this hypothesis by determining the glucose dependence of single-cell insulin responses within an islet.

Glucose principally regulates insulin secretion in mouse islets by controlling the numbers of granule fusion events per cell.

We used the db/db mouse to determine the nature of the secretory defect in intact islets.

The secretory deficit in islets from db/db mice is mainly due to a loss of responding beta cells.

We set out to test the hypothesis that insulin secretion from beta cells is targeted towards the vasculature.

Jiun T. Low

School of Biomedical Sciences

The University of Queensland

Lepr^db Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Jiun T. Low

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The University of Queensland

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

School of Biomedical Sciences

Lepr^db Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets