G6pase

Dulbecco's Modified Eagle's Medium (DMEM), trypsin-EDTA, penicillin-streptomycin solution, and fetal bovine serum (FBS) were acquired from Hyclone (South Logan, UT, USA). Antibodies against NKAα1, NKAα2, NKAα3, P110α, P110β, PEPCK, G6pase, β-actin, β-tubulin, GAPDH, and the secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Alexa Fluor 568 conjugated goat anti-rat IgG (H+L) was purchased from Invitrogen Corporation (Carlsbad, USA). Antibodies against pan-cadherin, phospho-GS (Ser641), phospho-GSK3 (Ser9), GS and GSK3 were obtained from Abcam (Cambridge, MA, USA). The generation of a DR-region-specific antibody (⁸⁹⁷DVEDSYGQQWTYEQR⁹¹¹) was performed as our previous reports 32 (link), 33 (link). Hematoxylin and eosin (H&E) staining kit, G6pase and PEPCK activity kits, periodic acid-schiff (PAS) kit, glucose assay kit, insulin assay kit, and glycogen assay kit were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The specific primers were provided by Integrated DNA Technologies Pte. Ltd. (Singapore). LY294002 and MK2206 were bought from Selleck Chemicals (Houston, TX, USA). D12492 60 kcal% fat was obtained from Research Diets (New Brunswick, NJ, USA). Glucosamine (GlcN), insulin, glucose, and phosphate colorimetric kit were obtained from Sigma (St. Louis, USA).

Protein was extracted from the frozen tissues of animals, including islet, liver, skeletal muscle and white adipose tissue. Protein concentrations were determined by BCA method. Equal amounts of protein (20 μg per lane) were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore). After blocking, the filters were incubated with the primary antibodies. Antibodies including, Akt, p-Akt, InsR (insulin receptor), p-InsR, Acc, p-Acc, HK2 (hexokinase 2), PFKL (6-phosphofructokinase, liver type), PKM1/2 (pyruvate kinase muscle isozyme), PDH (pyruvate dehydrogenase), PDHK1 (pyruvate dehydrogenase kinase isozyme 1) were purchased from Cell Signaling Technology. Antibodies, including PKLR (pyruvate kinase, liver and RBC), PEPCK (phosphoenolpyruvate carboxykinase), CPT1, PTBP1 were purchased from Abcam. Antibodies including GCK (glucokinase), PFK1 (phosphofructokinase 1), G6Pase (glucose-6-Phosphatase), PPARα, PPARδ, PPARγ were purchased from Santa cruz biotechnology. After washes and incubation with the appropriate horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology), the immune complexes were visualized using a chemiluminescence reagent. Western blot results were densitometrically quantified with Quantity One software (Bio-Rad), and the intensity values were normalized to β-actin.

Proteins were isolated using lysis buffer, incubated in SDS buffer, separated on SDS-polyacrylamide gels, and electroblotted onto PVDF membranes. Immunoreactive protein bands were detected using an Odyssey Scanning System (LI-COR Inc., Superior St., Lincoln, NE, USA). The following antibodies were used for Western blotting: G6Pase, FBPase, PEPCK, COX2, c-Myc, p-AKT, p-p38, PDHK, MCT1, β-actin (Santa Cruz, CA, USA) at 1:400 dilution; MCT4, p-ERK, GLUT 4, (Bioworld Technology, Inc., MN) at 1:800 dilution; HIF-1α, Hexokinase II, Hydroxy-HIF-1α, PFKFB 3 (Cell Signaling Technology, Inc., MA) at 1:800 dilution.

NXT was kindly provided by Xianyang Buchang Pharmaceutical Co. Ltd (Shan’xi, China). Rabbit anti-VEGFA, PCK1, GCK, INSR, PI3K (p110), IRS1 and pi-IRS1 polyclonal antibodies were purchased from Proteintech Group (Chicago, IL). Goat anti-AGE polyclonal antibody was purchased from Novus Biologicals (Littleton, CO). Rabbit anti-AKT, pi-AKT, pi-IRS1, IRS2, PI3K (P85), AMPKα and pi-AMPKα polyclonal antibodies were purchased from Cell Signaling Technology Inc (Danvers, MA). The following antibodies were purchased from Santa Cruz Inc.: rabbit anti-MMP2, fibronectin, G6Pase, FGF21, WT1, TGFβ1, TGFβR2 and Smad2/3 polyclonal antibodies; goat anti-MMP9, collagen type I α2 (COL1A2), collagen type IV α1/3 (COL4A1/3), pi-Smad2/3 and CTGF polyclonal antibodies; and mouse anti-GLUT4 monoclonal antibody. The mouse insulin ELISA assay kit was purchased from ABclonal Inc. (Wuhan, China). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) except as indicated.