Glut4

Heart tissues used for RNA isolation were rapidly excised and frozen in liquid nitrogen. RNA was extracted from heart samples by using TRIzol reagent (Invitrogen, USA) method following manufactory's protocol. The integrity of total RNA was assessed using Nano Drop 2000 Spectrophotometer (Thermo, USA). Total RNA was reverse transcribed by Invitrogen 2-step RT kits Superscript II first strand synthesis kit (Invitrogen, USA) according to the manufacturer's instructions.
The cDNA was 1:20 diluted and used for real-time PCR. Platinum SYBR Green qPCR Super Mix-UDG with ROX kits (Invitrogen, USA) were used to measure gene expression using specific primer sets: pdk4, glut4, ampk, pgc-1α, and gapdh (Invitrogen, USA) (Table 1). Dissociation curves were run following Real-Time PCR reactions to ensure the detection of the desired amplicon and exclude the presence of contaminating products. All reactions were performed in the ABI Prism 7000 Sequence Detection System (Applied Biosystems, USA). Gene expression was normalized to gapdh and the data were analyzed using comparative 2^∧−ΔΔCt method.

Salts, buffers and Bovine serum albumin fraction V (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Colorimetric enzymatic kits were purchased from Sigma Aldrich (St. Louis, MO, USA) for fructose, GS Diagnostics SRL (Guidonia Montecelio, Rome, Italy) for glucose and uric acid and SGM Italia (Rome, Italy) for triglycerides. Elisa kit for insulin determination was purchased from Mercodia AB (Uppsala, Sweden). Chemiluminescent substrate, Immobilon (Millipore Corporation, Billerica, MA 01821, USA), Excellent Chemiluminescent detection Kit (ElabScience, Microtech, Naples, Italy), Polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA) and dye reagent for protein titration (Bio-Rad, Hercules, CA, USA) were used for Western blotting. The antibodies used for Western blot analysis were purchased by Cell Signaling (Danvers, MA, USA) for p-Akt and Akt1, Santa Cruz Biotechnology (Santa Cruz, CA, USA) for p-GSK, GSK-3β, GLUT-4 and VDAC1, Invitrogen (Carlsbad, CA, USA) for GLUT-5, Calbiochem (San Diego, CA, USA) for UCP-3, Abcam, (Cambridge, UK) for Oxphos, Biogenesis Ltd. (England, UK) for ANT, Sigma-Aldrich (St Louis, MO, USA) for actin.

Total protein was extracted from WT and db/db cardiac tissues using RIPA lysis buffer containing a protease and phosphatase inhibitor cocktail (Thermo Fisher, Waltham, MA, USA). The extracted proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. After blocking with 5% skim milk in TBS with 0.1% Tween 20, membranes were incubated with the following primary antibodies overnight at 4 °C: primary antibodies were purchased from Cell Signaling (TGF-β1, PGC1a, NRF1, NRF2, total OXPHOS, ACC, p-ACC Ser⁷⁹, ACSL1, ATGL, FOXO1, p-FOXO1 Ser²⁵⁶, AMPK, p-AMPK Thr¹⁷², Akt, p-Akt Ser⁴⁷³, mTOR, p-mTOR Ser²⁴⁴⁸, PPARγ1/2, NF-κB p65, β-Actin, and GAPDH); Abcam (GLUT4, Col1a1, and TFAM); Invitrogen (FABP3, IGFBP7, and p-SREBP1 Ser³³⁸); and Santa Cruz Biotechnology (CD36, SREBP1, MFN1, OPA1, and DRP1). After washing, the membranes were incubated with the HRP–conjugated secondary antibody (Jackson ImmunoResearch, USA), diluted in 5% skim milk, and incubated for 1 h at room temperature. Finally, the membranes were washed with Tris-buffered saline (TBS) containing 0.1% Tween 20. Immunodetection was performed using an enhanced luminol-based chemiluminescent substrate (WESTSAVE Up, AbFrontier, Korea). GAPDH and β-Actin were used as loading controls. Quantification of each band was performed using ImageJ software.