The type 2 diabetes-associated HMG20A gene is mandatory for islet beta cell functional maturity

Abstract

HMG20A (also known as iBRAF) is a chromatin factor involved in neuronal differentiation and maturation. Recently small nucleotide polymorphisms (SNPs) in the HMG20A gene have been linked to type 2 diabetes mellitus (T2DM) yet neither expression nor function of this T2DM candidate gene in islets is known. Herein we demonstrate that HMG20A is expressed in both human and mouse islets and that levels are decreased in islets of T2DM donors as compared to islets from non-diabetic donors. In vitro studies in mouse and human islets demonstrated that glucose transiently increased HMG20A transcript levels, a result also observed in islets of gestating mice. In contrast, HMG20A expression was not altered in islets from diet-induced obese and pre-diabetic mice. The T2DM-associated rs7119 SNP, located in the 3' UTR of the HMG20A transcript reduced the luciferase activity of a reporter construct in the human beta 1.1E7 cell line. Depletion of Hmg20a in the rat INS-1E cell line resulted in decreased expression levels of its neuronal target gene NeuroD whereas Rest and Pax4 were increased. Chromatin immunoprecipitation confirmed the interaction of HMG20A with the Pax4 gene promoter. Expression levels of Mafa, Glucokinase, and Insulin were also inhibited. Furthermore, glucose-induced insulin secretion was blunted in HMG20A-depleted islets. In summary, our data demonstrate that HMG20A expression in islet is essential for metabolism-insulin secretion coupling via the coordinated regulation of key islet-enriched genes such as NeuroD and Mafa and that depletion induces expression of genes such as Pax4 and Rest implicated in beta cell de-differentiation. More importantly we assign to the T2DM-linked rs7119 SNP the functional consequence of reducing HMG20A expression likely translating to impaired beta cell mature function.

Fig. 1. HMG20A is expressed in both mouse and human islets and is decreased in islets from T2DM donors.

a HMG20A transcript levels were assessed by qPCR in islets, brain, liver, muscle, white adipose tissue (WAT), and brown adipose tissue (BAT) from mice (n = 6). Representative images of b mouse and c human islets co-stained for HMG20A (green) along with INSULIN (INS), GLUCAGON (GLUC) or SOMATOSTATIN (SMT) (red). Nuclei are stained using DAPI (blue). Magnification 40×. White boxes define areas enlarged in panels bellow. d HMG20A mRNA levels were measured by qPCR in human islets isolated from normoglycemic (control) or type 2 diabetic (T2DM) organ donors (n = 7). Data are depicted as dot plots with means ± S.E.M. p values were determined using unpaired two-tailed Student's t-test. **p < 0.01

Fig. 2. HMG20A protein expression is not altered in islets of high fat diet (HFD)-induced obesity and pre-diabetic mice.

a Insulin tolerance test (ITT) and area under the curve (AUC) analysis in both control (CT) and HFD mice (CT, n = 10; HFD, n = 15). b Fasting blood glucose levels in both control and HFD mice (CT, n = 10; HFD, n = 15). c Representative images of pancreatic sections from HFD and CT mice stained and quantified for HMG20A (n = 4). d Hmg20a mRNA levels were assessed by qPCR in INS-1E cells cultured with 0.05 mM palmitate for 24, 48, 72 or 96 h (n = 3 independent experiments performed in triplicates). Data are represented as means + S.E.M. p values were determined by ordinary one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.0001

Fig. 3. HMG20A is regulated by glucose.

a–c HMG20A and d–f NEUROD mRNA levels were assessed by qPCR in human (n = 3 donors performed in triplicates) and mouse islets (three independent preparations of pooled islets from three different isolation) as well as INS-1E cells cultured in 24 mM glucose for 24, 48, 72, or 96 h (n = 3 independent experiments of all time points executed in triplicates). Data are represented as means + S.E.M. p values were determined by ordinary one-way ANOVA. *p < 0.05, compared to the control 0 time point

Fig. 4. HMG20A is transiently increased in mouse islets during pregnancy.

a Representative images of pancreatic sections from pregnant mice co-stained for HMG20A (green) and INSULIN (INS) (red). Nuclei were stained using DAPI (blue). Magnification 40×. White boxes define areas enlarged in panels below. p.c. post coitum. b Quantification of integrated fluorescence intensity for HMG20A in beta cells of islets from pregnant mice (n = 3 mice per time point with 35-71 islets counted per time point). Data are depicted as dot plots with means ± S.E.M. c HMG20A mRNA levels in islets from pregnant mice (n = 5 mice) at 12.5 and 14.5 days of pregnancy. d Oral glucose tolerance test (OGTT) and area under the curve (AUC) analysis of non-pregnant (NP) and pregnant (day 14.5 of pregnancy; D14.5) mice (NP, n = 8; D14.5, n = 6). Data are represented as means ± S.E.M. p values were determined by the unpaired two-tailed Student´s t-test (d for AUC and c) or ordinary one-way ANOVA with Dunnet’s multiple comparison test (b). *p < 0.05, **p < 0.01, ****p < 0.0001, compared to the control 0 time point

Fig. 5. The rs7119 SNP within the HMG20A 3′ UTR decreases protein levels.

Expression profile of putative miRNAs binding to the “mut” allele of the HMG20A transcript in a human 1.1E7 cells and b mouse islets. Data are presented respect to miR375 expression levels. c Schematic representation of the HMG20A gene depicting the promoter region (HMG20A promoter), transcription starting site (tss) and 11 exons. Gaussia luciferase (GLuc) reporter constructs harboring the 3′ UTR corresponding to either wild type (wt) or rs7119 mutant (mut) allele under pSV40 promoter are shown below. d HMG20A mRNA levels assessed by qPCR in the human pancreatic beta cell line 1.1E7 cultured in 24 mM glucose for 24, 48, 72, or 96 h (n = 5 independent experiments performed in triplicates). e Human pancreatic beta 1.1E7 and f RPE1 cell lines were transfected with the reporter construct bearing either wt or rs7119 mut variant of the HMG20A 3′ UTR. Both the Gaussia Luciferase activity and SEAP activity were determined 24 h post transfection (n = 3 independent experiments). Data are represented as means + S.E.M. p values were determined by ordinary one-way ANOVA (d) or unpaired Student's t-test (e, f). *p < 0.05

Fig. 6. HMG20A regulates metabolism-secretion coupling genes in INS-1E cells.

a Hmg20a was silenced by specific siRNA in INS-1E cells (n = 6 independent experiments, each performed in triplicates). b Representative images of INS-1E cells immuno-stained for HMG20A (green) and DAPI (blue) confirming decreased protein level after treatment with siHMG20A. Magnification 40×. c Beta cell development and maturity genes, as Neurod, Pax4, Mafa, Pdx1, and Rest transcripts levels after silencing of Hmg20a in INS-1E cells (n = 4 independent experiments performed in triplicates). d H3K4m2 and e HMG20A occupancy of promoter, exon 1 and ATG regions of the Pax4 gene after chromatin immunoprecipitation using anti-H3K4m2 or anti-HMG20A antibodies, with IgG as a control for non-specific interactions (n = 4 independent experiments performed in triplicates). f Beta cell metabolism-secretion coupling genes transcripts levels after silencing of Hmg20a in INS-1E cells (n = 4 independent experiments, performed in triplicates). g Alteration in the expression of key genes was confirmed in INS-1E cells using a second siHMG20A (n = 4 independent experiments performed in triplicates). Data are represented as means + S.E.M. p values were determined by one-way ANOVA with Tukey’s multiple comparisons test (a, c, f, g). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 7. Hmg20a silencing impairs insulin secretion in INS-E1 cells as well as in mouse islets without affecting cell death or proliferation.

a Cell death (n = 3), b proliferation (n = 3), and c glucose-induced insulin secretion (GSIS, n = 4) were assessed subsequent to siRNA-mediated HMG20A depletion in INS-1E cells. d Effect of siRNA-mediated HMG20A depletion on e cell proliferation (n = 6 islet preparations) and f GSIS in mouse islets (n = 6 islet preparations). Data are represented as mean + S.E.M. p values were determined by unpaired two-tailed Student's t-test. *p < 0.05, **p < 0.01