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Pancreatic β-cell glutaminase 2 maintains glucose homeostasis under conditions of hyperglycemia

Pancreatic β-cell glutaminase 2 maintains glucose homeostasis under conditions of hyperglycemia

 


mouse

Animals were cared for according to Chiba University guidelines. All animal experiments were approved by the Chiba University Animal Care Review Board. This study was reported according to the ARRIVE 2.0 Essential 10 guidelines (https://arriveguidelines.org).pancreatic β-cell specific Gls2 Conditional knockout mice (CKO) were constructed from rat insulin II promoter (RIP)-Cre transgenic mice. [9]By using a gene targeting strategy involving FLP and loxP, Gls2 Exons 2 through 7 were knocked out in pancreatic β-cells. Mice were housed in a pathogen-free environment. From 6 weeks of age, Gls2 CKO and RIP-Cre mice were maintained on a 60 kcal% high-fat diet (#D12492, Research Diets, NJ, USA) for up to 32 weeks. For mouse genotyping, the primers used to sequence RIP, FLP, and Neo are shown below. RIP: 5′-ACCTGAAGATGTTCGCGATTATCT-3′ and 5′-ACCGTCAGTACGTGAGATATCTT-3′, FLP1: 5′-TATGTGCCTACTAACGCTTGT-3′ and 5′-AGAGCCACATTCATGAGCTAT-3′, mGls2 dNeo: 5′-CACCTGAGTGAGGTAGACAATCCTA-3′ and 5′- GTAGAAAAGCAGGTCACCAAGTCG-3′.

cell culture

A pancreatic β-cell line of MIN6 cells provided by ATCC was supplemented with 10% fetal bovine serum (FBS) and 71 μmol/l 2-mercaptoethanol (Sigma).

Provocation test and hormone assay

Provocation tests were performed after 16 hours of fasting. Oral glucose tolerance test (OGTT), insulin tolerance test (ITT), and pyruvate tolerance test (PTT) were performed by measuring glucose (1 g/kg body weight, dextrose, anhydrous, Wako Pure Chemical Industries, Osaka, Japan), insulin intraperitoneally. intraperitoneal injection (0.75 U/kg body weight, Humulin R, Eli Lilly, Indianapolis, In, USA) and intraperitoneal injection of pyruvic acid (2 g/kg body weight, 20% sodium pyruvate, FUJIFILM Wako Pure Chemical Co., Osaka, Japan). , Japan), respectively. Blood glucose, plasma insulin, and plasma glucagon levels were measured using a glucose monitor (Glutest Mint, Sanwa Kagaku Kenkyusho, Aichi, Japan) and Ultra Sensitive Mouse Insulin ELISA Kit (Morinaga Biological Science Laboratory, Inc.). Did. , Kanagawa, Japan), and the Mercodia Glucagon ELISA-10 μL Kit (Mercodia AB, Uppsala, Sweden), respectively.

Mouse islet isolation

A mixed anesthetic was prepared with 0.3 mg/kg medetomidine, 4.0 mg/kg midazolam, and 5.0 mg/kg butorphanol. Anesthetics were administered to mice by intraperitoneal injection and collagenase P (Cat. No. 11 249 002 001, Roche, Basel, Switzerland) was used to extract islets from mouse pancreases with minor modifications to the manufacturer’s protocol. separated from32.

RNA isolation and quantitative RT–qPCR

Total RNA was extracted from each sample, followed by reverse transcription as previously described33The cDNA products were then analyzed by RT-qPCR using the primers listed below using the 7500 Fast Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). All gene-specific mRNA expression values ​​were normalized to the internal housekeeping gene 18S.meters18S: 5′-CTTAGAGGGACAAGTGGCG-3′ and 5′-ACGCTGAGCCAGTCAGTGTA-3′, mGls2: 5′-AACTATGACAACCTGCGGC-3′ and 5′-GCCGACAATGCAAACCTTC-3′.

Immunoblotting analysis and antibodies

Immunoblotting analysis was performed as previously described1GLS2, p53 and β-actin proteins were detected by anti-GLS2 antibody (ab113509, Abcam), anti-p53 antibody (FL-393: sc-6243, Santa Cruz) and anti-β-actin antibody (A1978, Sigma). I was. Each.

Immunohistochemistry and image analysis

Immunohistochemical methods were applied to formalin-fixed paraffin-embedded tissue. Insulin and glucagon antibodies were obtained from Santa Cruz Biotechnology (sc-9168 and sc-514592, respectively). Gls2 and 8-OHdG antibodies were obtained from Gene Tex (GTX31828) and the Japanese Institute for the Control of Aging (MOG), respectively. Histochemical image analysis of β-cell/islet and α-cell/islet regions was performed using a Keyence fluorescence microscope BZ-X700. Histochemical image analysis of Gls2 and 8-OHdG was performed using ImageJ (National Institutes of Health).

Measurement of cellular ATP levels

Intracellular ATP was measured by luciferin-luciferase bioluminescence assay using ATP assay reagent (Toyo Ink). Briefly, 100 μL of ATP reagent was injected into 96-well samples and their relative light intensities were recorded at room temperature on a Lumat LB 9501 luminometer (Berthold).

Human data acquisition and data analysis

Raw results of single-cell RNA-sequencing data of human islets were downloaded from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) at GSE81608.14. Generally applicable gene set enrichment (GAGE)34 Using integrated differential expression and pathway analysis (iDEP)35 Applied for differential pathway enrichment analysis between β-cells with and without GLS2 Expression. GAGE ignored gene sets containing less than 15 genes or more than 2000 genes and considered FDR < 0.2. KEGG pathway36 It was used as the input gene set for GAGE. For each of the significant KEGG pathways, fold changes of associated genes were displayed in pathway diagrams using the Pathview Bioconductor package.37.

statistics

Results are presented as mean ± SEM. Two-group comparisons were analyzed by Mann-Whitney test or Wilcoxon test. All measurements were obtained from different individual samples and not repeated measurements of the same sample. Experiments were performed several times and representative data are shown.

ethics

All animal studies were conducted in accordance with the International Guiding Principles for Biomedical Research Involving Animals and were approved by the Animal Care and Use Committee of Chiba University and the National Institute for Physiological Sciences of Japan.

Sources

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2/ https://www.nature.com/articles/s41598-023-34336-z

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