Fig 1: Expression of TCA cycle enzymes in H9C2KE2 cells. (A): Fumarase protein expression (N = 6); (B): Pyruvate carboxylase protein expression (N = 8); (C): Succinate Dehydrogenase B protein expression (N = 8); (D): Fumarate levels (N = 6). Representative Western blots are also shown. Data are shown as mean ± SD values described as KE220L vs. KE230L and WT30L vs. KE230L with * p < 0.05, **** p < 0.0001 for showing the significance. A Shapiro-Wilk test was applied for testing the data normality distribution. For t-test analysis, an unpaired test for all normally distributed data and a Mann-Whitney for all abnormally distributed data was used. (WTL–Wild-type cells exposed for long-term/KE2L–GLUT4 overexpressing cells exposed for long-term).
Fig 2: EFR3A is the major isoform in 3T3-L1 adipocytes and is localized to the plasma membrane(A) shows RT-PCR analysis of the expression of EFR3A and EFR3B normalized to GAPDH in 3T3-L1 fibroblasts and adipocytes. Data presented is from triplicate biological repeats each with at least three technical replicates (mean and S.D.) EFR3A is the predominant isoform in 3T3-L1 fibroblasts and adipocytes. (B) shows a subcellular analysis of 3T3-L1 adipocytes treated with or without 100 nM insulin for 20 min and separated into PM (PM)-enriched, high density microsomes (HDM) and low density microsomes (LDM), which were then immunoblotted for the proteins indicated; figures at right are approximate positions of MW markers in kDa. In each case, 30 µg of protein was loaded in each fraction. Data from a representative experiment is shown, replicated four times with qualitatively similar results. (C–E) shows quantification of GLUT4 (C), EFR3 (D) and PI4K-IIIa (E) signals in PM and LDM fractions in the four fraction experiments. GLUT4 levels in the PM increase 1.7-fold (C, *P=0.002) and decrease in the LDM by 25% (**P=0.003) consistent with similar studies. EFR3 and PI4K-IIIa levels in the PM-enriched fractions increase in response to insulin (D, 2.1-fold, ***P=0.012 and E, 1.9-fold, ****P=0.03, respectively). Modest decreases in the LDM fraction for these proteins do not reach statistical significance (n.s.). Syntaxin4 (Sx4) is used as a marker for a protein known to be enriched in the PM [55,56].
Fig 3: EFR3 knockdown does not impair insulin action or adipocyte differentiation(A) 3T3-L1 adipocytes were treated with siRNA designed to knockdown EFR3A or scrambled control siRNA (Scr) at day 6 post-differentiation as described. Cells were incubated in serum-free media for 2 h and 100 nM insulin added for the final 15 min. Cell lysates were prepared and immunoblotted for total Akt or phospho-Akt as shown. Data from a representative experiment are presented. (B) Lysates prepared in a similar manner were immunoblotted for EFR3, acetyl coenzyme A carboxylase (ACC), fatty acid synthase (FAS) or GLUT4; representative immunoblots are presented. Data from a typical experiment is shown, with quantification from four experiments of this type presented in (C) Data shown as a % change comparing unstimulated samples; *statistically significant decrease, P=0.035. Similar data are obtained when the insulin-stimulated lysates are quantified (not shown). ACC, FAS and GLUT4 levels were not affected by EFR3 depletion.
Fig 4: Lack of OGT in skeletal muscle affects glucose homeostasis and insulin signaling. (A–B) Blood glucose levels before and during a hyperinsulinemic euglycemic clamp performed using 4 mU/kg/min of insulin. (C–D) Glucose infusion rates (GIR) during clamp. (E) Plasma insulin levels before and during steady state of the clamp. (F) Glucose uptake in skeletal muscles after steady state. (G) Glucose uptake in adipose tissues after steady state. (H) Glucose uptake in BAT, heart, and brain after steady state. (I) Glycogen in gastrocnemius, triceps, quadriceps and liver from clamped mice. (J) Glycogen in gastrocnemius and liver from 5-hour fasted mice. (K) Immunoblot of Akt and Akt phosphorylation of pS473 and pT308 in gastrocnemius. (L) Immunoblot of Akt1 in gastrocnemius from clamped mice. (M) Immunoblot of TBC1D4 and TBC1D4 phosphorylation of, pS324, pT642, and pS711 in gastrocnemius. (N) Immunoblot of GLUT4 in gastrocnemius. Data represent means ± SEM from n = 8–10 mice in each genotype. Mice were 18-week-old males. *p < 0.05; **p < 0.01; ***p < 0.001 compared with WT mice.
Fig 5: Representative Western blots for a subject in the high-intensity intermittent swimming training group, including the molecular weight of band migration. Muscle tissue samples were obtained before (Pre) and after (Post) the intervention period and the protein expression was determined in duplicate when possible (see “muscle sampling” in the methods section for details). VL, vastus lateralis muscle; D, deltoideus muscle; MCT4, monocarboxylate transporter 4; NHE1, Na+/H+ exchanger 1. Pump a1, a2, and ß1 = Na+/K+ pump a1, a2, and ß1 subunit, respectively. FXYD1, phospholemman; SOD1 and SOD2, superoxide dismutase 1 and 2, respectively. CAT, catalase; GLUT4, glucose transporter 4; GS, glycogen synthase.
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