AAV8-mediated gene transfer of microRNA-132 improves beta cell function in mice fed a high-fat diet

in Journal of Endocrinology
Correspondence should be addressed to J K Kruit: j.k.kruit@umcg.nl
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MicroRNAs have emerged as essential regulators of beta cell function and beta cell proliferation. One of these microRNAs, miR-132, is highly induced in several obesity models and increased expression of miR-132 in vitro modulates glucose-stimulated insulin secretion. The aim of this study was to investigate the therapeutic benefits of miR-132 overexpression on beta cell function in vivo. To overexpress miR-132 specifically in beta cells, we employed adeno-associated virus (AAV8)-mediated gene transfer using the rat insulin promoter in a double-stranded, self-complementary AAV vector to overexpress miR-132. Treatment of mice with dsAAV8-RIP-mir132 increased miR-132 expression in beta cells without impacting expression of miR-212 or miR-375. Surprisingly, overexpression of miR-132 did not impact glucose homeostasis in chow-fed animals. Overexpression of miR-132 did improve insulin secretion and hence glucose homeostasis in high-fat diet-fed mice. Furthermore, miR-132 overexpression increased beta cell proliferation in mice fed a high-fat diet. In conclusion, our data show that AAV8-mediated gene transfer of miR-132 to beta cells improves beta cell function in mice in response to a high-fat diet. This suggests that increased miR-132 expression is beneficial for beta cell function during hyperglycemia and obesity.

 

      Society for Endocrinology

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    Hyperglycemia and obesity-induced miR-132 expression resulting in increased insulin secretion. (A) Several miRNAs were differently expressed in pancreatic islets of 12-week-old ob/ob mice (n = 6). (B) Increased miR-132 expression in islets isolated from ob/ob (n = 4), 10-week high-fat diet-fed C57Bl6 mice (n = 4) or 14-month-old C57Bl6 mice (n = 7–8). (C) Culture of primary islet for 24 h in high glucose media or media containing 2 mM palmitate-induced miR-132 expression (n = 4). (D) Overexpression of miR-132 in INS1E cells resulted in increased glucose-stimulated insulin secretion (n = 4).

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    AAV8-mediated gene transfer resulted in miR-132 overexpression in beta cells. (A) Representative image of immunofluorescent staining against GFP (green) and insulin (red) in control AAV-RIP-GFP or AAV8-RIP-miR132-treated mice. (B) Increased expression of miR-132 in islets isolated from AAV8-RIP-miR132-treated mice compared to islets isolated from control mice (n = 4–6). (C) Expression of miR-375, miR-184 and miR-212 in islets was comparable between AAV8-RIP-miR132-treated and control mice (n = 4–6). (D) Representative image of islet morphology based immunofluorescent staining against insulin (green) and glucagon (red).

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    Impact of miR-132 overexpression in beta cells on glucose homeostasis. (A) Body weights of AAV8-RIP-GFP control and AAV8-RIP-miR132-treated mice (n = 6) during chow and high-fat diet (HFD) feeding. (B) AAV8-RIP-miR132-treated mice showed similar glucose levels during fasting or after oral glucose bolus as control mice fed a chow diet (n = 6). (C) Insulin levels at 0 and 15 min after oral glucose bolus were similar between the two groups (n = 6). (D) Oral glucose tolerance testing showed improved glucose tolerance in AAV8-RIP-miR-132-treated mice after 4 weeks of high-fat diet (n = 6). (E) Analysis of insulin levels at 0 and 15 min after oral glucose bolus showed increased insulin secretion in mice overexpressing miR-132 after the glucose bolus (n = 6). (F) Ex vivo analysis of glucose-stimulated insulin secretion showed increased insulin secretion at 16.7 mM glucose in miR-132-overexpressing islets (n = 6). (G) Isolated islets of AAV8-RIP-miR132-treated mice showed normal gene expression of beta cell-related genes (n = 4–6). (H) Increased expression of miR-132 coincided with reduced expression of CACT mRNA and protein levels (n = 4–6).

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    Signs of increased proliferation in beta cells of AAV8-RIP-miR132-treated mice. (A) Pancreatic sections of control or AAV8-RIP-miR-132-treated mice stained using immunofluorescence for insulin (green) and BrdU (red). (B) Percentage of BrdU-positive beta cells in pancreata of control and AAV8-RIP-miR-132 mice (n = 4). (C) Isolated islets of AAV8-RIP-miR132-treated mice showed increased ki67 gene expression (n = 4–6). (D) Decreased RB protein levels in isolated islets of AAV8-RIP-miR132-treated mice, of which the quantification is shown in (E) (n = 6).

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