Fig 2: Gq-meditated stimulation of AMPK phosphorylation, GLUT4 translocation, and phosphorylation of HSL (S565) in 3T3F442A mouse adipocytes.a Western blotting studies with GqD-expressing mouse 3T3F442A cells (GqD-3T3F442A cells) treated with CNO (10 µM) for different periods of time. b Quantitative analysis of pAMPK/AMPK and pAS160/AS160 protein expression levels shown in (a). Data shown in (b) were derived from four of five independent experiments. Protein expression levels were normalized relative to protein expression levels at time ‘0’. c Western blotting studies carried out in the presence of a Gq/11 inhibitor. GqD-3T3F442A cells) were incubated with CNO (10 µM) for 30 min either in the absence or presence of FR900359 (FR; 1 µM), a Gq/11 inhibitor. d Quantitative analysis of pAMPK/AMPK and pAS160/AS160 protein expression levels shown in (c). e Immunoblotting analysis of GLUT4 plasma membrane localization. After treatment of GqD-3T3F442A cells with CNO (10 µM) or insulin (10 nM) for 30 min, the plasma membrane fraction was isolated and subjected to immunoblotting analysis using an anti-GLUT4 antibody. Na + /K + ATPase served as a marker for plasma membrane proteins. f Quantitative analysis of GLUT4 expression levels shown in (e). g Western blot analysis of HSL phosphorylation at position S565. GqD-3T3F442A cells were treated with CNO (10 µM), CL316,243 (CL; 100 nM), a ß3-adrenergic receptor agonist, or a combination of both drugs. To demonstrate the specificity of the HSL antibody used, GqD-3T3F442A cells were treated with HSL-specific siRNA or scrambled control siRNA. h Quantitative analysis of pHSL(S565)/HSL protein expression levels shown in (g). Data are presented as means ± s.e.m. b, d, f, h: one-way ANOVA followed by Bonferroni’s post-hoc test). Source data are provided as a Source data file.

Fig 3: Effect of PHLPP1 silencing on PHLPP isoforms, Akt isoforms and AS160 in insulin signaling and insulin resistant condition in neuronal cells (N2A). Three days post-proliferation, PHLPP1 was silenced using PHLPP1 specific siRNA. N2A cells were differentiated in serum-free medium in the absence of (MF) or chronic presence of 100 nM insulin (MFI) for 3 days. Cells were lysed and subjected to western blotting, followed by probing with relevant primary antibodies. A Bar represents relative change in PHLPP1 probed with anti-PHLPP1 antibody. B Bar represents relative change in PHLPP2 probed with anti-PHLPP2 antibody. C Bar represents relative change in pAkt1 (Ser-473) probed with anti-Akt1 antibody. D Bar represents relative change in pAkt2 (Ser-474) probed with anti-Akt2 antibody. E Post-insulin stimulation, lysates were subjected to immunoprecipitation using anti-Akt3 antibody. Bar represents relative change in pAkt (Ser-473) probed with anti-Akt3 antibody. F Bar represents relative change in pAS160 (Ser-588) when probed with anti-AS160 antibody. G Bar represents relative change in pAS160 (Thr-642) when probed with anti-AS160 antibody. GAPDH has been used as a loading control. Experiments were executed three times and a representative result is shown. Data expressed are mean ± SE. ***P < 0.001, **P < 0.01, *P < 0.05 compared to lane 1, ###P < 0.001, #P < 0.05 as compared to lane 2, $$P < 0.01, $$P < 0.05 compared to lane 6. IB Immunoblot; IP Immunoprecipitation

Fig 4: Effects of insulin and wortmannin on subcellular localization and phosphorylation of AS160 (pAS160) Thr642 in cytosol and membrane fractions of muscle.(a) AS160 in the cytosol fraction. (b) AS160 in the membrane fraction. (c) pAS160 Thr642 in the cytosol fraction. There was no detectable pAS160 Thr642 in the membrane fraction, regardless of insulin concentration. *Significantly different from the all other treatment groups (P < 0.05). Values are expressed as mean ± SD; n = 10 per treatment.

Fig 5: Lactate-induced hypertrophic and oxidative signaling pathways activation in skeletal muscle. The working hypothesis for signaling pathways activated by in vivo lactate administration in mixed/fast (A) and slow (B) skeletal muscles. ERK1/2: extracellular signal-regulated protein kinases 1 and 2; Akt: protein kinase B; TSC2: tuberous sclerosis complex 2; mTORC1: mammalian target of rapamycin complex 1; p70S6K: ribosomal S6 kinase; rpS6: ribosomal protein S6; AMPK: 5’-AMP-activated protein kinase; TBC1D4: TBC1 domain family member 4; ACC: Acetyl-CoA carboxylase; PDH: pyruvate dehydrogenase. ? represents activation; ···I represents inhibition. +P: increased phosphorylation; -P: decreased phosphorylation. ? represents unknown mechanisms.