Mouse islets were isolated by Histopaque gradient following collagenase P shot towards the pancreatic duct, as described63 previously. of ChREBP is essential for adaptive -cell extension in response to metabolic issues. Conversely, chronic excessive -cell-specific overexpression of ChREBP results in loss of -cell identity, apoptosis, loss of -cell mass, and diabetes. Furthermore, -cell glucolipotoxicity can be prevented by deletion of ChREBP. Moreover, ChREBP-mediated cell death is usually mitigated by overexpression of the alternate CHREBP gene product, ChREBP, or by activation of the antioxidant Nrf2 pathway in rodent and human -cells. We conclude that ChREBP, whether adaptive or maladaptive, is an important determinant of -cell fate and a potential target for the preservation of -cell M2I-1 mass in diabetes. (h), or (ChREBP) (i) gene promoters. The data are the means??SEM of the percent input after subtraction of the IgG control. promoter as a target because it contains a well-studied ChoRE5,12,20,21. The recruitment over time of ChREBP to both the ChoRE region and the ChREBP (mice (Fig.?3a). In addition, human islets labeled with a RIP-ZsGreen-expressing adenovirus were sorted to obtain pure -cells22. The ratio of ChREBP to ChREBP mRNA expression was significantly higher in subjects with T2D compared to non-diabetic control donors (Fig.?3b). Furthermore, we found that treatment of mice with adipsin, which preserves -cell mass in diabetic mice23, decreased ChREBP abundance in a manner proportionate to the improved glycemia and plasma insulin levels, concordant with the idea that ChREBP expression contributes to the glucose toxicity seen in the mouse model of T2D (Fig.?3cCf). Open in a separate window Fig. 3 ChREBP expression and nuclear localization correlates with adaptive expansion of -cells and with glucose toxicity in diabetes.a The N-terminal or the C-terminal antibodies recognizing ChREBP were used to stain pancreatic tissue slices from C57Bl/6 mice fed on a standard chow diet, or fed a high-fat diet for 1 week, or from diabetic mice. All micrographs represent at least 3 independent experiments. b Ratio of expression of ChREBP to ChREBP FPKM M2I-1 from RNAseq performed from FACS-sorted human -cells isolated from non-diabetic (mice treated with control AAV (GFP) or with AAV expressing adipsin for 24 weeks. Correlation between percentages of nuclear ChREBP in insulin-positive -cells and blood glucose levels (e) or FCGR2A blood insulin levels (f), where each data point represents an individual mouse. All micrographs represent at least 3 M2I-1 independent experiments. We next M2I-1 explored nuclear localization of ChREBP in human -cells in vivo using a minimal human islet transplant model in immunosuppressed mice24. In this experiment, human islets were transduced with adenoviruses and then transplanted under the kidney capsules of streptozotocin-induced diabetic immunocompromised mice (Fig.?4a). 1500 islet equivalents (IEQs) were sufficient to normalize glucose levels (Fig.?4b). 500 IEQs transduced with a control Cre-expressing adenovirus was a minimal mass of -cells sufficient to keep the animals alive, but hyperglycemic (around 400?mg/dL). When 500 IEQs were transduced with an adenovirus expressing ChREBP, which activates the Nrf2 antioxidant pathway8, blood glucose, plasma insulin, and glucose tolerance all approached normal levels (Fig.?4bCe). At the end of the experiment, a uninephrectomy was performed and glucose levels rose to diabetic levels, confirming that this transplanted human -cells provided the only insulin in the recipient mice (Fig.?4b). Kidneys made up of islet grafts were fixed and immunolabeled for insulin and ChREBP using the C-terminal antibody (Fig.?4f). This revealed abundant nuclear ChREBP in -cells transduced with control, Cre-expressing adenovirus, but nuclear labeling was nearly absent in the ChREBP–treated -cells. In addition, there was a strong correlation between glucose levels prior to the harvesting of the graft and nuclear ChREBP abundance (Fig.?4g). Thus, nuclear ChREBP expression is usually proportionate to glucose levels in human -cells in vivo. Open in a.
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