A distinct role of STING in regulating glucose homeostasis through insulin sensitivity and insulin secretion

Significance The role of STING in maintaining glucose homeostasis remains unknown. Herein, using global and β-cell–specific STING knockout mouse models, we revealed a distinct role of STING in the regulation of glucose homeostasis through β-cells and peripheral tissues. Specially, while global STING knockout beneficially alleviated insulin resistance and glucose intolerance induced by high-fat diet, it surprisingly impaired islet glucose-stimulated insulin secretion (GSIS). Further analyses revealed that STING deficiency down-regulated expression of β-cell key transcription factor Pax6, impairing Pax6 nuclear localization and binding activity to the promoters of its target genes, including Glut2 and Abcc8, causing impaired GSIS. These data highlight pathophysiological significance of fine-tuned STING signaling in β-cells and insulin target tissues for maintaining glucose homeostasis.


Supplemental Materials and Methods
Intraperitoneal Glucose and Insulin Tolerance Tests. The intraperitoneal glucose tolerance testing (IPGTT) was performed by intraperitoneally injection of 2 g/kg body weight of glucose after 6-hour fasting. Tail vein blood glucose was monitored at 0, 15, 30, 60, 90, and 120 min. Blood glucose levels were determined using an automatic glucometer (Roche, ACCU-CHEK, Germany) and glucose test results higher than 33.3 mmol/L were recorded as 33.3 mmol/L. The area under the curve (AUC) was calculated using the trapezoidal rule. At the same time, blood samples were collected at individual time points and serum insulin levels were determined with insulin ELISA test kits (EZassay, China) according to the manufacturer's protocol. For intraperitoneal insulin tolerance test (IPITT), the mice were intraperitoneally injected with 0.75 U/kg body weight of insulin after a 4-hour fasting. All experiments described above were performed on age-and sex-matched cohorts.
Islet perifusion assays. For islet perifusion assays, islets were isolated and incubated overnight in RPMI medium containing 10% FBS, 50 μg/mL streptomycin, and 50 U/mL penicillin at 37°C in a 5% CO2 humidified environment. For each assay, 150 islet equivalents [IEQ] (approximately 100 size-matched islets) were used. Islets loaded in the perifusion chamber were first perifused with KREB's buffer containing 2.8 mmol/L glucose for 30 min, followed by perifusion with KREB's buffers containing 16.7 mmol/L glucose, and 2.8 mmol/L glucose. Flow rate of fluid input (2.8 mmol/L glucose and 16.8 mmol/L glucose in KREB's buffer) was 1 ml/min and fractions were collected every minute. Insulin secretion at each time point was quantified using HTRF insulin assay kit (62INSPEC, Cisbio) according to manufacturer's instructions, and normalized to the insulin content.
Whole islet calcium imaging. After being washed twice with KRBH buffer plus 2 mmol/L glucose, islets were loaded with calcium indicator Fluo-4 AM (Thermo Fisher Scientific, F14201) in KRBH buffer containing 5.6 µmol/L Fluo-4 AM, 0.4‰ Pluronic F127 (Sigma-Aldrich, P2443), and 2 mmol/L glucose for 45 min. Islets were then washed three times with KRBH buffer plus 2 mmol/L glucose, and seeded on 35 mm Glass-Bottom Dish (MatTek) for 15 min. The dish was placed into a 37°C humidified cell culture chamber containing 5% CO2 and mounted on a Zeiss LSM 800 inverted confocal microscope. A solid-state laser provided wavelengths of 488 nm to excite Fluo-4. Images were acquired every 10 seconds before and after adding glucose to the final concentration of 16.8 mmol/L to monitor the dynamic changes of intracellular calcium levels, and analyzed with Image J 1.52q software (NIH).
Biochemical Assays. Blood samples were obtained from mice, and serum was separated by centrifugation. Serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein-cholesterol (LDL-C) and high-density lipoprotein-cholesterol (HDL-C) were determined by assay kits (Nanjing Jiancheng Bioengineering Institute, China) using microplate reader (Thermo Scientific). Serum glucagon levels were determined using glucagon ELISA test kits (Ruixin Biotechnology Co., Ltd., China) according to the manufacturer's protocol.
Immunofluorescence and Immunohistochemical Staining. Paraffin-embedded pancreases were cut into 5 μm sections. Immunohistochemical staining were performed using appropriate antibodies as previously described (25) Transmission electron microscopy (TEM). Fresh pancreases from 8-week-old mice were fixed with 2.5% glutaraldehyde. The pancreases were cut into pieces and then fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 16 h and subsequently treated with 1% osmium tetroxide at 4°C for 2 h. The specimens were dehydrated in a graded ethanol series and then embedded in EMBed 812. Ultrathin sections were obtained using the ultra microtome (Leica EM UC7, Germany) and stained with 2% uranium acetate saturated alcohol solution and 2.6% Lead citrate and observed by TEM (HT7700, Japan).

RNA Extraction, cDNA Synthesis, and Real-Time Quantitative PCR
(RT-QPCR). Total RNA of islets was extracted using TRIzol reagent (Invitrogen, USA) and cDNA was synthesized using a PrimeScriptTMRT reagent Kit with gDNA Eraser (Takara, Japan, Cat: RR047A) following the manufacturer's instructions.

RT-QPCR was conducted with the TB Green Premix Ex TaqTM (Tli RNaseH Plus)
(Takara, Japan, Cat: RR420A). GAPDH was used as standards. We normalized the data to Control = 1 for fold change which were calculated by the 2 -△△ Ct method.
Primers were designed and chemically synthesized by TSINGKE Biological Technology (Beijing, China) Co., Ltd. The forward and reverse primer sequences used in this study were listed in supplemental Table S1. pmol siRNA mixed with transfection reagent. After 6 h incubation, the supernatant was discarded and was replaced with normal medium. The efficiency of RNA knockdown was evaluated by RT-QPCR and western blotting. All RNAi experiments were repeated at least 3 times.

Protein Extraction and Western Blot
Statistics. All of the experiments above were performed independently at least three times. All data are presented as the means ± SEM. Graph-Pad Prism 8 software was used for all statistical analyses. We analyzed data using unpaired Student's t test and used the traditional threshold P < 0.05 to declare statistical significance.

Supplemental figures
Supplemental Figure S1. Global deficiency of STING alleviated HFD-induced hyperlipidemia. Blood samples were used to measure the levels of lipids, including triglyceride (TG) (A), total cholesterol (TC) (B), Low-Density Lipoprotein Cholesterol (LDL-C) (C), and High-Density Lipoprotein Cholesterol (HDL-C) (D). n ≥ 5 mice/group. Values are shown as mean ± SEM. WT + ND vs WT + HFD, * P < 0.05, ** P < 0.01, WT + HFD vs STING -/-+ HFD, # P < 0.05, ## P < 0.01, ns, not significant. Figure S2. The expression of insulin gene and amount of insulin content in islets of global STING knockout mice. (A) mRNA levels of Ins1 and Ins2 in islets from 16-week-old male mice fed with ND or HFD were measured by RT-QPCR. (B) Freshly isolated islets from 16-week-old male mice were directly lysed. Western blots were performed with anti-insulin antibody. (C) The quantification of insulin content from at least three independent experiments shown in Fig. S2B. The relative level of insulin content in islets of control mice fed with ND were set as 1. n ≥ 3 mice/group. Values were shown as the mean ± SEM. WT + ND vs WT + HFD, *** P < 0.001, WT + HFD vs STING -/-+ HFD, # P < 0.05, ns, not significant.