Conditional ablation of HDAC3 in islet beta cells results in glucose intolerance and enhanced susceptibility to STZ-induced diabetes

Abstract

Histone deacetylases (HDACs) are enzymes that regulate gene expression by modifying chromatin structure through removal of acetyl groups from target histones or non-histone proteins. Previous in vitro studies suggest that HDACs may be novel pharmacological targets in immune-mediated islet β-cell destruction. However, the role of specific HDAC in islet β-cell development and function remain unclear. Here, we generated a conditional islet β-cells specific HDAC3 deletion mouse model to determine the consequences of HDAC3 depletion on islet β-cell differentiation, maintenance and function. Islet morphology, insulin secretion, glucose tolerance, and multiple low-dose streptozotocin (STZ)-induced diabetes incidence were evaluated and compared between HDAC3 knockout and wild type littermate controls. Mice with β-cell-specific HDAC3 deletion displayed decreased pancreatic insulin content, disrupted glucose-stimulated insulin secretion, with intermittent spontaneous diabetes and dramatically enhanced susceptibility to STZ-induced diabetes. Furthermore, islet β-cell line, MIN6 cells with siRNA-mediated HDAC3 silence, showed decreased insulin gene transcription, which was mediated, at least partially, through the upregulation of suppressors of cytokine signaling 3 (SOCS3). These results indicate the critical role of HDAC3 in normal β-cell differentiation, maintenance and function.

Keywords: HDAC3; Pathology Section; autoimmune diabetes; glucose tolerance; insulin; knockout.

Figure 1. Generation of β-cell-specific HDAC3 knockout mice

A. The floxed and null alleles of HDAC3 as a result of recombination between LoxP sites. B. PCR results of genotyping from tail biopsies of RIP-Cre⁻ HDAC3^fl/fl (WT), RIP-Cre⁺ HDAC3^fl/+ (HET) and RIP-Cre⁺ HDAC3^fl/fl (KO) mice. C. Dispersed islet cells from WT and KO mice were immunostained for HDAC3 (red) and insulin (green). Yellow (merged) indicates coexpression of HDAC3 and insulin. (Scale bar, 20 μm).

Figure 2. β-cell-specific deletion of HDAC3 causes higher blood glucose level, glucose intolerance and impaired insulin secretion in mice

A. Random blood glucose levels were monitored in WT (left) and KO (right) mice, each line represents an individual mouse. n = 6-7 male mice/group. B. The summary of non-fasting blood glucose levels and C. body weight monitored from 6- until 25-week of age. n = 6-7 male mice per group. The overall difference between KO and WT group was analyzed by ANOVA. D. Fasting blood glucose level was measured in 8- to 12-week-old KO and WT male mice. n = 8-12 male mice/group. Glucose tolerance test, 1g/kg body weight E. or 2g/kg body weight F., was performed on 8- to 10-week-old KO and WT mice. n = 4-6 mice/group. G. Plasma insulin levels during glucose tolerance test at the dose of 2 g/kg body weight. n = 5-6 male mice/group. Data are presented as means ± SD. (*p < 0.05, ***p < 0.001).

Figure 3. RIP-Cre.HDAC3^fl/fl mice are more susceptible to multiple low-dose STZ-induced (MLD-STZ) diabetes

Blood glucose levels throughout the MLD-STZ experiment were monitored and compared between KO and WT for all mice A., male mice C. or female mice E., respectively. Diabetes incidence was monitored and compared between KO and WT mice in all mice B., male mice D. or female mice F., respectively. Data are presented as means ± SD. (*p < 0.05, **p < 0.01).

Figure 4. Histopathological analysis of islets of β-cell-specific HDAC3 KO mice

Pancreatic sections were stained with hematoxylin and eosin from WT and KO mice without STZ treatment A. and B. or 25 days after STZ injection C. and D., scale bar, 100 μm.). Islet area relative to total pancreas was evaluated and compared between KO and WT mice of untreated E., or 25 days after STZ treatment F.. n = 10-13 mice per group. Pancreatic insulin content was measure and compared between 8-12-week-old untreated KO and WT mice. n = 4-5 mice per group G.. The relative viability of Min6 cells transfected with non-targeting sequence siRNA or HDAC3-specific siRNA was compared using MTT assay H.. (*p < 0.05, **p < 0.01).

Figure 5. SOCS3 upregulation by HDAC3 depletion in islet β-cells contributes to insulin production defect

A. Real-time PCR analysis of insulin related gene mRNA changes in HDAC3 silenced Min6 cells. B. Real-time PCR analysis of changed SOCS3 mRNA levels in HDAC3 KO and WT control mice. C. CHIP assays were performed with Min6 cells. Chromatin was immunoprecipitated with anti-HDAC3 or rabbit IgG control antibody, and precipitated DNA was used as a template to detect the SOCS3 promoter. Nonimmunoprecipitated sample served as an input control. D. Flow cytometry analysis of Min6 cells with HDAC3 or SOCS3 silencing. E. Real-time PCR analysis of Insulin1 and Insulin2 mRNA levels in different siRNA transfected Min6 cells. Insulin content F. and glucose stimulated insulin secretion F. of Min6 cells with silenced HDAC3, SOCS3 or both. Data are presented as means ± SD. (*p < 0.05, **p < 0.01).