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. 2016 Apr;65(4):1009-21.
doi: 10.2337/db15-1334. Epub 2016 Jan 28.

Sorcin Links Pancreatic β-Cell Lipotoxicity to ER Ca2+ Stores

Alice Marmugi  1 , Julia Parnis  1 , Xi Chen  2 , LeAnne Carmichael  1 , Julie Hardy  1 , Naila Mannan  1 , Piero Marchetti  3 , Lorenzo Piemonti  4 , Domenico Bosco  5 , Paul Johnson  6 , James A M Shapiro  7 , Céline Cruciani-Guglielmacci  8 , Christophe Magnan  8 , Mark Ibberson  9 , Bernard Thorens  10 , Héctor H Valdivia  2 , Guy A Rutter  11 , Isabelle Leclerc  11
Affiliations

Sorcin Links Pancreatic β-Cell Lipotoxicity to ER Ca2+ Stores

Alice Marmugi et al. Diabetes. 2016 Apr.

Abstract

Preserving β-cell function during the development of obesity and insulin resistance would limit the worldwide epidemic of type 2 diabetes. Endoplasmic reticulum (ER) calcium (Ca(2+)) depletion induced by saturated free fatty acids and cytokines causes β-cell ER stress and apoptosis, but the molecular mechanisms behind these phenomena are still poorly understood. Here, we demonstrate that palmitate-induced sorcin downregulation and subsequent increases in glucose-6-phosphatase catalytic subunit-2 (G6PC2) levels contribute to lipotoxicity. Sorcin is a calcium sensor protein involved in maintaining ER Ca(2+) by inhibiting ryanodine receptor activity and playing a role in terminating Ca(2+)-induced Ca(2+) release. G6PC2, a genome-wide association study gene associated with fasting blood glucose, is a negative regulator of glucose-stimulated insulin secretion (GSIS). High-fat feeding in mice and chronic exposure of human islets to palmitate decreases endogenous sorcin expression while levels of G6PC2 mRNA increase. Sorcin-null mice are glucose intolerant, with markedly impaired GSIS and increased expression of G6pc2 Under high-fat diet, mice overexpressing sorcin in the β-cell display improved glucose tolerance, fasting blood glucose, and GSIS, whereas G6PC2 levels are decreased and cytosolic and ER Ca(2+) are increased in transgenic islets. Sorcin may thus provide a target for intervention in type 2 diabetes.

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Figures

Figure 1
Figure 1
Sorcin deletion impairs glucose tolerance whereas sorcin overexpression in β-cells improves glucose tolerance during HFD. IPGTTs (1 g glucose/kg) were performed in SD-fed 2-month-old (A) and 9-month-old (B) Sri−/− male mice and sex-, weight-, and aged-matched WT controls (n = 4–10). IPGTTs (1 g glucose/kg) were performed in HFD-fed SRI-tg1 (n = 8–9, 16 weeks old) (C) and SRI-tg10 (n = 9–11, 8 weeks old) (D) male mice and littermate controls. Right panels represent AUC of blood glucose concentration during IPGTTs. IPITTs were performed in HFD-fed SRI-tg1 (n = 8–9, 17 weeks old, 1 unit insulin/kg) (E) and SRI-tg10 (n = 9–11, 9 weeks old, 0.5 units insulin/kg) (F) male mice and littermate controls. Right panels represent body weights for each group at the time of IPITTs. Values are means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (two-way ANOVA). CTRL, control.
Figure 2
Figure 2
Sorcin overexpression enhances GSIS without expansion of β-cell mass, whereas sorcin deletion impairs GSIS. Plasma insulin concentration during 3 g glucose/kg IPGTTs were assessed in HFD-fed SRI-tg10 male mice (n = 5–7, 11 weeks old) (A) and SD-fed Sri−/− male mice (n = 4–6, 9 months old) (B) and their respective controls. Top left panels represent plasma insulin values and bottom panels represent corresponding blood glucose measurements. Top right panels represent AUC of plasma insulin concentrations. C–F: Pancreatic β-cell mass was evaluated in HFD-fed SRI-10 mice. Five consecutive pancreatic sections from 9-week-old SRI-tg10 and littermate control mice (n = 3–4) were immunostained for insulin, glucagon, and DAPI (scale bars = 50 μm) (C) to quantify for mean pancreas and islet size (D), and individual β-cell and α-cell area and β-cell to α-cell ratio as described in research design and methods (E). Ex vivo insulin secretion assays were performed in response to 3 or 17 mmol/L glucose (G3; G17) on isolated islets from HFD-fed transgenic (TG) SRI-tg10 male mice (n = 3, 27 weeks old) (F), human cadaveric donors (n = 3, see Supplementary Table 2 for donors characteristics) (G), and SD-fed WT C57BL/6 mice (n = 4–5, 10 weeks old) (H) transduced with an adenovirus encoding sorcin-GFP or GFP only as indicated. *P < 0.05 (in vivo GSIS/IPGTT: two-way ANOVA; β-cell mass and ex vivo GSIS: two-tailed Student t tests). CTRL, control.
Figure 3
Figure 3
Sorcin overexpression increases intracellular Ca2+ fluxes and ER Ca2+ stores. Cytosolic Ca2+ levels ([Ca2+]c) were measured in isolated and dissociated islets loaded with Fura-2 from HFD-fed SRI-tg1 (n = 3–4, 14 weeks old) (A) and SRI-tg10 (n = 3, 9 weeks old) (B) male mice incubated sequentially with low (3 mmol/L, G3) and high (17 mmol/L, G17) glucose concentrations as indicated. Right panels represent AUC of [Ca2+]c. Acetylcholine (Ach)-induced ER Ca2+ release was measured in clusters of dissociated islets from HFD-fed SRI-tg1 (n = 4, 14 weeks old) (C) and SRI-tg10 (n = 3, 9 weeks old) (D) male mice transduced with Ad-RIP-D4ER adenovirus to measure [Ca2+]ER as stated in research design and methods. The islets were incubated in 17 mmol/L glucose in the presence of Diaz (Dz; 250 μmol/L) to prevent extracellular Ca2+ influx. Left panels, representative calcium traces; right panels, quantification of the amplitude (∆) of ER Ca2+ depletion after treatment with Ach. *P < 0.05; **P < 0.01; ***P < 0.001 ([Ca2+]c: two-way ANOVA; [Ca2+]ER ∆: two-tailed Student t tests). au, arbitrary units; CTRL, control.
Figure 4
Figure 4
Sorcin regulates G6pc2 expression and decreases fasting blood glucose in vivo. Quantitative RT-PCR analysis of Sri (A–C) and G6pc2 (D–F) expression was performed in isolated islets from HFD-fed SRI-tg1 male (n = 3 mice/genotype, 27 weeks old) (A and D), SRI-tg10 male (n = 5 mice/genotype, 8 weeks old) (B and E), and SD-fed SRI−/− male (n = 4–7 mice/genotype, 9 months old) (C and F) mice and their respective controls. G: Fasting blood glucose levels were measured in HFD-fed SRI-tg10 male and their littermate control mice aged 8, 16, and 24 weeks, as indicated (n = 9–11 mice per group, HFD from 4 weeks old). *P < 0.05; **P < 0.01; ***P < 0.001 (two-tailed Student t tests). CTRL, control.
Figure 5
Figure 5
Lipotoxicity decreases endogeneous sorcin expression whereas sorcin overexpression prevents palmitate-induced G6PC2 and ER stress marker induction. A: Quantitative RT-PCR analysis of SRI, G6PC2, CHOP, and GRP78/BiP expression was performed on human islets (n = 3 donors) transduced for 48 h with either sorcin (GFP-SRI) or null (GFP) adenoviruses, as indicated. B: Quantitative RT-PCR analysis of Sri expression was performed in isolated islets from chow or HFD-fed WT mice (n = 4–5, 19-week-old females, HFD from 5 weeks old). Quantitative RT-PCR analysis of SRI, G6PC2, CHOP, and/or GRP78/BiP expression was performed on human islets (n = 3 donors) treated for 72 h with 0.5 mmol/L BSA-conjugated palmitate or BSA only (C) or on MIN6 β-cells transduced for 24 h with an adenovirus encoding GFP or sorcin-GFP followed by 48 h treatment with 0.5 mmol/L BSA-conjugated palmitate or BSA only (D and E). Values are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (two-tailed Student t tests).
Figure 6
Figure 6
Sorcin inhibits G6PC2 promoter activity. Promoter luciferase reporter studies were performed in MIN6 β-cells cotransfected with either GFP (control), sorcin (SRI), or NFAT-GFP cDNAs and −1075+124hG6PC2-Luci (A) or three tandem repeats of NFAT binding sites (3× NFAT-Luci) and pRL-CMV (B) and treated with DMSO (0.1%), Diaz (100 μmol/L), or CsA (0.2 μmol/L) for an additional 24 h before cell lysis, as indicated (n = 3–4 independent experiments). Values are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (two-tailed Student t tests).
Figure 7
Figure 7
Sorcin (SRI) activates ATF6 transcriptional activity. Promoter luciferase reporter studies were performed in clonal β-cell lines MIN6 (A), 1.1B4 (B), INS1(832/13) (C), and human embryonic kidney HEK cells (D). Cells were cotransfected with an artificial promoter containing five ATF6 binding sites (p5xATF6-GL3), pRL-CMV, and either GFP or SRI cDNA for 24 h and treated with DMSO (1%), 4-PBA (10 mmol/L), tunicamycin (10 mg/mL), or thapsigargin (10 mmol/L) for an additional 16–20 h before cell lysis and luciferase assay. a, P < 0.005 for the effect of treatment vs. DMSO; b, P < 0.05 for the effect of SRI overexpression (unpaired two-tailed Student t tests). E: Western blot showing the efficiency of two different SRI short hairpin RNA constructs (shSRI-144 and shSRI-457) after 3 and 5 days in culture in MIN6 cells. Cells were transfected with either pLKO.1-shScrambled, -shSRI144, or -shSRI457 and incubated with 1 μg/mL puromycin for 3 or 5 days before cell lysis and Western blotting using polyclonal anti-SRI (1:300) and monoclonal anti-tubulin (1:10,000). Dashed vertical white lines have been added for clarity. F: SRI silencing reduces the activity of an artificial promoter containing five ATF6 binding sites in MIN6 cells. MIN6 were cotransfected with p5xATF6-GL3, pRL-CMV, and either pLKO.1-shScrambled, -shSRI144, or -shSRI457 for 48 or 72 h before cell lysis and luciferase assay. Results are expressed as mean ± SEM; n = 3 independent experiments. *P < 0.005, paired two-tailed Student t tests. Quantitative RT-PCR analysis of Sri (G) and spliced Xbp1:total Xbp1 (H) was performed in MIN6 cells transduced for 24 h with adenoviruses encoding SRI-GFP or GFP only and treated with DMSO (0.1%) or thapsigargin (Thaps, 100 μmol/L) for an additional 24 h. Results are expressed as mean ± SEM; n = 3 independent experiments. *P < 0.05; ***P < 0.005 (paired two-tailed Student t tests).
Figure 8
Figure 8
Sorcin lies on a pathway linking β-cell lipotoxicity to ER calcium and ER stress, representing a mechanism for dysregulation of β-cell function under conditions of metabolic stress. A: In pancreatic β-cells, sorcin is downregulated under conditions of lipotoxic stress such as exposure to HFD and palmitate or proinflammatory cytokines, as shown by others (20). The inverse relationship between sorcin and G6PC2 expression levels observed in islets suggests that an important mechanism of action of sorcin is to regulate G6PC2 expression to influence both ER stress and GSIS. B: Sorcin overexpression is sufficient to protect against β-cell dysfunction during HFD. In stressed β-cells, sorcin overexpression increases ER and cytosolic [Ca2+], decreasing G6PC2, through the NFAT signaling pathway, which would stimulate GSIS. By maintaining a high concentration of Ca2+ in the ER lumen, sorcin prevents ER stress and maintains long-term capacities for GSIS during HFD. FFA, free fatty acid.
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