Haploid insufficiency of suppressor enhancer Lin12 1-like (SEL1L) protein predisposes mice to high fat diet-induced hyperglycemia

Abstract

Increasing evidence suggests that endoplasmic reticulum (ER) stress plays an important role in the pathogenesis of type 2 diabetes mellitus. SEL1L is an ER membrane protein that is highly expressed in the pancreatic islet and acinar cells. We have recently reported that a deficiency of SEL1L causes systemic ER stress and leads to embryonic lethality in mice. Here we show that mice with one functional allele of Sel1l (Sel1l(+/-)) are more susceptible to high fat diet (HFD)-induced hyperglycemia. Sel1l(+/-) mice have a markedly reduced β-cell mass as a result of decreased β-cell proliferation. Consequently, Sel1l(+/-) mice are severely glucose-intolerant and exhibit significantly retarded glucose-stimulated insulin secretion. Pancreatic islets from Sel1l(+/-) mice stimulated with a high concentration of glucose in vitro express significantly higher levels of unfolded protein response genes than those from wild-type control mice. Furthermore, dominant-negative interference of SEL1L function in insulinoma cell lines severely impairs, whereas overexpression of SEL1L efficiently improves protein secretion. Taken together, our results indicate that haploid insufficiency of SEL1L predispose mice to high fat diet-induced hyperglycemia. Our findings highlight a critical and previously unknown function for SEL1L in regulating adult β-cell function and growth.

FIGURE 1.

Sel1l^+/− mice are susceptible to HFD-induced hyperglycemia. A, shown is a schematic representation of mouse groups and diets. 10-Week-old male Sel1l^+/− and Sel1l^+/+ mice were randomly divided and fed with NC or a HFD, respectively, for 20 weeks. B, shown are body weight gains of Sel1l^+/− and Sel1l^+/+ mice on a HFD or NC. *, p < 0.05; **, p < 0.01, Sel1l^+/− HFD versus NC; #, p < 0.05; ##, p < 0.01, Sel1l^+/+ HFD versus Sel1l^+/+ NC. C, shown are fasting blood glucose levels of Sel1l^+/− and Sel1l^+/+ mice on HFD or NC. **, p < 0.01, Sel1l^+/− HFD versus Sel1l^+/+ HFD. D, shown is insulin tolerance of Sel1l^+/− and Sel1l^+/+ mice fed with HFD for 24 weeks. E, shown is glucose tolerance of Sel1l^+/− and Sel1l^+/+ mice on HFD for 20 weeks. **, p < 0.01, Sel1l^+/− HFD (n = 10) versus Sel1l^+/+ HFD (n = 11). F, shown are fasting blood glucose levels of Sel1l^+/− and Sel1l^+/+ mice on HFD for 20 weeks and treated with either PBS or 4-PBA for 1 week; *, p < 0.05 4-PBA versus PBS (n = 5 per genotype). All values are expressed as the mean ± S.E.

FIGURE 2.

Sel1l^+/− mice fed with a high fat diet exhibit impaired GSIS and reduced β-cell mass. A, shown are plasma insulin levels of Sel1l^+/− and Sel1l^+/+ mice on NC or HFD for 20 weeks. **, p < 0.01 HFD-fed Sel1l^+/− versus Sel1l^+/+ mice (n = 7). B, shown is fold change of GSIS of Sel1l^+/− and Sel1l^+/+ mice. **, p < 0.01, Sel1l^+/− versus Sel1l^+/+ mice (n = 5 per genotype). C, shown are β-cell masses of Sel1l^+/− and Sel1l^+/+ mice on HFD for 8 weeks (n = 7 per genotype). D–E, shown are representative images of insulin immunostained pancreatic sections from Sel1l^+/+ (D) and Sel1l^+/− (E) mice on HFD for 20 weeks. F, shown is quantification of β-cell mass in Sel1l^+/+ and Sel1l^+/− mice on HFD for 20 weeks. **, p < 0.01 Sel1l^+/+ versus Sel1l^+/− mice (n = 5 per genotype). β-Cell mass was determined by timing the pancreatic weight with percentage of the β-cell area. G and H, representative images are shown of Ki67 immunofluorescence of pancreatic sections from Sel1l^+/+ (G) and Sel1l^+/− (H) mice on HFD for 20 weeks. Nuclei were countered stained with DAPI. I, shown is quantification of Ki67-positive cells in Sel1l^+/+ and Sel1l^+/− mice on HFD for 20 weeks. The rate of β-cell proliferation was calculated by dividing the number of Ki67-positive cells with the total number of nuclei counted. **, p < 0.01 Sel1l^+/+ versus Sel1l^+/− mice (n = 4 per genotype). All values are expressed as the mean ± S.E.

FIGURE 3.

Endoplasmic reticulum stress in pancreatic islets and liver of Sel1l^+/− mice fed with a high fat diet. A and B, shown is a comparison of UPR gene expression in Sel1l^+/+ and Sel1l^+/− pancreatic islets. Islets were isolated from 30-week-old Sel1l^+/+ and Sel1l^+/− mice and cultured overnight in media containing 10 mm (A) or 30 mm (B) glucose. The mRNA expression of five UPR genes, including Bip, Herp, Chop, Xbp1s, and p58IPK, were analyzed by real-time RT-PCR. C, real-time PCR analysis of UPR gene expression in Sel1l^+/− islets treated with and without the chemical chaperon, 4-PBA is shown. For each gene, the expression in the vehicle (DMSO) treated islets was set to 1 and used to calculate a relative expression of the gene in 4-PBA treated islets. D, shown is UPR gene expression at the protein level in livers of Sel1l^+/− and Sel1l^+/+ mice on HFD for 20 weeks. Liver lysates from 5 Sel1l^+/+ and 7 Sel1l^+/− mice (30 μg per sample) were immunoblotted and probed with antibodies against the indicated gene products. HSP90 was used as a loading control. E, shown is quantification of protein expression in Sel1l^+/+ and Sel1l^+/− mice indicated in D. For each protein the expression in Sel1l^+/+ liver was set to 1 and used to calculate a relative expression in Sel1l^+/− liver. *, p < 0.05, **, p < 0.01 Sel1l^+/− mice (n = 7) versus Sel1l^+/+ (n = 5). All values are expressed as the mean ± S.E.

FIGURE 4.

Perturbation of SEL1L function in insulinoma cell lines impairs glucose-stimulated insulin secretion and β-cell proliferation. A, G-luc secretion in Min6 cells transiently expressing a dominant-negative form of SEL1L is shown. Mins6 cells were transiently co-transfected with the indicated expression plasmids. Conditioned media were sampled at 24, 36, and 48 h post-transfection and analyzed by luciferase assay. Expression of dSel1l-GFP markedly suppresses G-luc secretion in Min6 cells. B, G-luc secretion in Min6 cells overexpressing SEL1L. Min6 cells were transiently co-transfected with the indicated expression plasmids. Conditioned media were sampled at 24 h post-transfection and analyzed by luciferase assay. Expression of SEL1L-GFP restores protein secretion inhibited by NHK-GFP. **, p < 0.01. NHK-GFP + SEL1L-GFP versus NHK-GFP- expressing cells. C, shown is an immunoblotting analysis of dSEL1L-GFP fusion protein expression in Min6 cells stably integrated with a Tet-inducible Sel1l-GFP transgene. dSEL1L-GFP was detected using an anti-GFP antibody. Dox, doxycycline. D, GSIS of Min6 cells stably expressing dSEL1L-GFP is shown. *, p < 0.05 dSEL1L-GFP-negative versus dSEL1L-GFP-positive cells. Expression of dSEL1L-GFP markedly inhibits glucose-stimulated insulin secretion in Min6 cells. E, shown is an immunoblotting analysis of SEL1L-GFP and dSEL1L-GFP expression in INS-1 cells stably integrated with Tet-inducible Sel1l-GFP and dSel1l-GFP transgenes. F, shown is an immunoblotting analysis of GRP78/BIP expression in INS-1 cells stably expressing dSEL1L-GFP. Ectopic expression of dSEL1L-GFP in INS-1 cells results in up-regulation of GRP78/BIP. Tub, tubulin G and H, shown are growth profiles of INS-1 cells stably expressing dSEL1L-GFP. Cell proliferation was analyzed through cell counting (G) and by thymidine incorporation assay (H). The data were derived from three independent experiments. *, p < 0.05; **, p < 0.01, vehicle versus dSEL1L-GFP expressing cells. All values are expressed as the mean ± S.E.