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. 2016 Oct;65(10):3039-52.
doi: 10.2337/db16-0084. Epub 2016 Aug 3.

SERCA2 Deficiency Impairs Pancreatic β-Cell Function in Response to Diet-Induced Obesity

Xin Tong  1 , Tatsuyoshi Kono  2 , Emily K Anderson-Baucum  2 , Wataru Yamamoto  1 , Patrick Gilon  3 , Djamel Lebeche  4 , Richard N Day  1 , Gary E Shull  5 , Carmella Evans-Molina  6
Affiliations

SERCA2 Deficiency Impairs Pancreatic β-Cell Function in Response to Diet-Induced Obesity

Xin Tong et al. Diabetes. 2016 Oct.

Abstract

The sarcoendoplasmic reticulum (ER) Ca(2+) ATPase 2 (SERCA2) pump is a P-type ATPase tasked with the maintenance of ER Ca(2+) stores. Whereas β-cell SERCA2 expression is reduced in diabetes, the role of SERCA2 in the regulation of whole-body glucose homeostasis has remained uncharacterized. To this end, SERCA2 heterozygous mice (S2HET) were challenged with a high-fat diet (HFD) containing 45% of kilocalories from fat. After 16 weeks of the HFD, S2HET mice were hyperglycemic and glucose intolerant, but adiposity and insulin sensitivity were not different between HFD-fed S2HET mice and HFD-fed wild-type controls. Consistent with a defect in β-cell function, insulin secretion, glucose-induced cytosolic Ca(2+) mobilization, and the onset of steady-state glucose-induced Ca(2+) oscillations were impaired in HFD-fed S2HET islets. Moreover, HFD-fed S2HET mice exhibited reduced β-cell mass and proliferation, altered insulin production and proinsulin processing, and increased islet ER stress and death. In contrast, SERCA2 activation with a small molecule allosteric activator increased ER Ca(2+) storage and rescued tunicamycin-induced β-cell death. In aggregate, these data suggest a critical role for SERCA2 and the regulation of ER Ca(2+) homeostasis in the β-cell compensatory response to diet-induced obesity.

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Figures

Figure 1
Figure 1
S2HET mice exhibit impaired glucose tolerance in response to HFD. SERCA2 HET and WT littermate controls were fed an HFD containing 45% of kilocalories from fat for 16 weeks starting at 8 weeks of age. A–C: Protein and RNA were isolated from HET and WT islets before (WT-PreHFD and HET-PreHFD) and after 16 weeks of HFD (WT-HFD and HET-HFD). A: Immunoblot analysis was performed using antibodies against SERCA2, SERCA3, and actin. B: Quantitative SERCA2 protein levels are shown graphically. C: Reverse-transcribed RNA was subjected to real-time quantitative PCR to measure SERCA2b and SERCA3 transcript levels (normalized to Actb). D and E: Longitudinal changes in body weight were measured, and DEXA analysis was performed in HET and WT mice at the start and after 16 weeks of the HFD. F: Blood glucose in 6-h fasted WT and HET mice before and after 16 weeks of HFD. G: Random-fed serum insulin levels after 16 weeks of the HFD. H–K: IPGTT or OGTT were performed before or after 16 weeks of HFD treatment in HET and WT mice; area under the curve (AUC) analysis is shown graphically. Results are displayed as means ± SEM (n = at least 8 per group, except panel F, where n = 19 for S2HET group, and G, where n is indicated by the scatterplot). Indicated comparisons are significantly different: *P < 0.05; **P < 0.01; ***P < 0.001; n.s. indicates that no significant differences were observed between groups.
Figure 2
Figure 2
HFD-fed S2HET and WT mice exhibit comparable levels of insulin sensitivity. A and B: Insulin tolerance tests were performed before (WT-PreHFD and HET-PreHFD) and after 14 weeks of HFD feeding (WT-HFD and HET-HFD), with area under the curve (AUC) analysis shown graphically. Protein homogenates from adipose (C), liver (D), or skeletal muscle (E) were obtained from saline (basal) or insulin-injected HET and WT mice fed the HFD for 16 weeks. Immunoblot analysis was performed using antibodies against phosphorylated (p)AKT (ser473), total AKT, and actin. Relative protein levels are shown graphically. Results are displayed as means ± SEM (n = 6 per group). Indicated comparisons are significantly different: **P < 0.01; n.s. indicates that no significant differences were observed between groups.
Figure 3
Figure 3
SERCA2 deficiency leads to decreased insulin secretion. A: Serum insulin levels were measured in S2HET (HET) and WT mice fed the HFD for 16 weeks at time 0 after a 5-h fast and 10 min after an intraperitoneal injection of glucose (2 mg/kg total body weight). B: Islets were isolated from HFD-fed S2HET and WT mice, and GSIS was measured and normalized to total insulin content. Results are displayed as the means ± SEM from three or more independent experiments for each group. Indicated comparisons are significantly different: *P < 0.05; ***P < 0.001; n.s. indicates that no significant differences were observed between groups.
Figure 4
Figure 4
Islets isolated from S2HET mice exhibit impaired Ca2+ homeostasis. Islets were isolated from 8-week-old PreHFD-WT mice or WT mice fed the HFD for 16 weeks and 8-week-old PreHFD-S2HET mice or S2HET mice fed the HFD for 16 weeks. Isolated islets were loaded with Fura-2 AM, and Ca2+ imaging was performed. A and C: Representative cytosolic Ca2+ recording of islets after stimulation with 15 mmol/L glucose. B: Quantification of the relative basal cytosolic Ca2+ (F0) in WT and HET islets. D: Quantification of the phase 1 duration. E: Quantification of the phase 1 cytosolic Ca2+ amplitude. F: Quantification of the average cytosolic Ca2+ oscillatory amplitude from five continuous oscillatory cycles per islet. G: Representative cytosolic Ca2+ recording from WT-HFD and HET-HFD islets after stimulation with 30 mmol/L KCl. H: Quantification of the phase 1 cytosolic Ca2+ amplitude in response to KCl (n = at least 26 islets from 3 biological replicates per group for A–H). I and J: Dispersed islets were transduced with a D4ER adenovirus, and FLIM was used to measure ER Ca2+. Representative lifetime map with lookup table indicating donor lifetime in ns (scale bar = 10 µm) and average donor lifetime in PreHFD-WT and S2HET β-cells treated under control (CTR) or GLT conditions (n = at least 10 cells per condition). Results are displayed as means ± SEM. Indicated comparisons are significantly different: *P < 0.05; **P < 0.01; ***P < 0.001; n.s. indicates that no significant differences were observed between groups.
Figure 5
Figure 5
HFD-fed S2HET mice exhibit impaired insulin production, processing, and granule maturation. Islets were isolated from HFD-fed S2HET (HET) and WT mice. A: Total islet insulin content was normalized to islet DNA content. B: Ins1 and Ins2 transcript levels were measured by quantitative real-time PCR and normalized to Actb levels. C: Serum proinsulin levels were measured after 16 weeks of HFD in WT and S2HET mice. Results are expressed as the ratio of serum proinsulin to insulin (n = 5 biological replicates). D and E: Islets from WT or S2HET mice (n = 3 each) before and after 16 weeks of HFD were pooled and analyzed by electron microscopy. Representative electron microscopic images of β-cells and insulin granule morphology from HFD-fed WT and HET mice are shown. Panel E shows quantitative analysis of 20 images per group (scale bar = 2 µm). F–H: Protein and RNA were isolated from HFD-fed WT and HET, and immunoblot analysis was performed using antibodies against PC1/3 and actin. Quantitative PC1/3 protein and transcripts levels are shown graphically. Results are displayed as the means ± SEM. The indicated comparisons are significantly different: *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 6
Figure 6
HFD-fed S2HET mice exhibit decreased β-cell mass and proliferation and increased β-cell death. A: Representative images of insulin immunohistochemistry in pancreata harvested from HFD-fed S2HET (HET) and WT mice. B: β-Cell mass was quantified in WT and HET mice before and after 16 weeks of HFD treatment. C and D: Representative images of insulin and glucagon immunofluorescence in pancreata harvested from HFD-fed WT and HET mice. Quantification of the number of α-cells per islet is shown graphically. E and F: Quantification of the percentage of β-cells positive for proliferating cell nuclear antigen (PCNA). G: Randomly fed serum was collected from WT and HET mice before and during HFD feeding at indicated time points. Levels of circulating unmethylated mouse insulin DNA levels were measured by droplet digital PCR and are depicted as Log (copies/µL). Results are displayed as means ± SEM (n = at least 4 biological replicates per group in A–F, and n values are indicated in the scatter plots in panel G). Scale bars = 50 μm. The indicated comparisons were significantly different: *P < 0.05; **P < 0.01; n.s. indicates that no significant differences were observed between groups.
Figure 7
Figure 7
S2HET islets and SERCA2-deficient β-cells exhibit increased ER stress. A: Islets from WT or S2HET (HET) mice (n = 3 each) fed the HFD for 16 weeks were pooled, fixed, and analyzed by electron microscopy. Representative images of the β-cell ER structure are shown and indicated by arrows (scale bar = 1 µm). B: Islets were isolated from S2HET mice and WT controls fed HFD for 16 weeks (n = 5–6). Reverse-transcribed RNA was subjected to quantitative real-time PCR to measure Grp78, Dnajc3, Hsp90b1, Pdia4, spliced (S)/total (T) Xbp-1, and Actb transcript levels. C: Freshly isolated islets from 10-week-old male S2HET and WT mice fed normal chow were treated with or without 25 mmol/L glucose + 500 μmol/L BSA-conjugated palmitate (GLT) for 24 h. Reverse-transcribed RNA was subjected to quantitative real-time PCR to measure indicated gene transcript levels (n = 9 biological replicates analyzed over three individual experiments). D: Immunoblot analysis was performed using antibodies against SERCA2, SERCA3, and actin in S2KO INS-1 cells. E: Reverse transcribed RNA isolated from S2KO and WT INS-1 cells was subjected to quantitative real-time PCR for quantification of SERCA2b, SERCA3, RyR2, and IP3R3, and results were normalized to Actb transcript levels. F: Reverse-transcribed RNA isolated from S2KO and WT INS-1 cells treated with or without GLT for 24 h was subjected to quantitative real-time PCR for quantification of indicated transcript levels. G–I: Protein and RNA were isolated from WT and S2KO INS-1 cells. Immunoblot analysis was performed using antibodies against PC1/3 and actin. Quantitative protein and transcripts levels are shown graphically. Results are displayed as means ± SEM. Indicated comparisons are significantly different: *P < 0.05; **P < 0.01; ***P < 0.001; n.s. indicates that no significant differences were observed between groups.
Figure 8
Figure 8
SERCA2 KO β-cells manifest impaired Ca2+ storage and increased susceptibility to stress-induced death. A and B: WT and S2KO INS-1 cells were treated with or without 10 μmol/L TM for 24 h. Immunoblot analysis was performed using antibodies against cleaved caspase-3 (Casp3), total caspase-3, and actin. Quantitative protein levels are shown graphically. C: WT and S2KO INS-1 cells were treated under control (CTR) conditions or exposed to GLT or TM for 24 h, and viability was measured using the CellTiter-Glo assay. Results were normalized to results obtained in WT cells under control conditions. D–F: SERCA2b was overexpressed (S2OE) via adenoviral (Adv) transduction in WT and S2KO INS-1 cells and then treated with GLT or TM for 24 h. D: Immunoblot analysis was performed using antibodies against SERCA2 and actin. CellTiter-Glo viability tests were performed in WT (E) and S2KO cells (F). Results were normalized to cells transduced with LacZ virus under control conditions. SERCA2b was overexpressed via adenoviral transduction in WT (WT-OE) and S2KO (KO-OE) INS-1 cells, followed by exposure to GLT for 24 h. G and H: To assess cytosolic Ca2+ levels, calcium 6 fluorescence was measured under Ca2+-free conditions. I and J: INS-1 cells were transduced with a D4ER adenovirus, and FLIM was used to measure ER Ca2+. Shown is the average donor lifetime in WT and S2KO INS-1 cells (I) or WT INS-1 cells treated with 10 μmol/L CDN1163 for 2 or 24 h (J) (n = at least 10 regions of interest per cell type or treatment). K and L: WT and S2KO INS-1 cells were treated with or without TM combined with or without 10 μmol/L CDN1163 for 24 h. Immunoblot analysis was performed using antibodies against cleaved caspase-3 and actin, and quantitative protein levels are shown graphically. Results are displayed as means ± SEM. Indicated comparisons were significantly different: *P < 0.05; **P < 0.01; ***P < 0.001.
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