FTO is increased in muscle during type 2 diabetes, and its overexpression in myotubes alters insulin signaling, enhances lipogenesis and ROS production, and induces mitochondrial dysfunction

Abstract

Objective: A strong association between genetic variants and obesity was found for the fat mass and obesity-associated gene (FTO). However, few details are known concerning the expression and function of FTO in skeletal muscle of patients with metabolic diseases.

Research design and methods: We investigated basal FTO expression in skeletal muscle from obese nondiabetic subjects and type 1 and type 2 diabetic patients, compared with age-matched control subjects, and its regulation in vivo by insulin, glucose, or rosiglitazone. The function of FTO was further studied in myotubes by overexpression experiments.

Results: We found a significant increase of FTO mRNA and protein levels in muscle from type 2 diabetic patients, whereas its expression was unchanged in obese or type 1 diabetic patients. Moreover, insulin or glucose infusion during specific clamps did not regulate FTO expression in skeletal muscle from control or type 2 diabetic patients. Interestingly, rosiglitazone treatment improved insulin sensitivity and reduced FTO expression in muscle from type 2 diabetic patients. In myotubes, adenoviral FTO overexpression increased basal protein kinase B phosphorylation, enhanced lipogenesis and oxidative stress, and reduced mitochondrial oxidative function, a cluster of metabolic defects associated with type 2 diabetes.

Conclusions: This study demonstrates increased FTO expression in skeletal muscle from type 2 diabetic patients, which can be normalized by thiazolidinedione treatment. Furthermore, in vitro data support a potential implication of FTO in oxidative metabolism, lipogenesis and oxidative stress in muscle, suggesting that it could be involved in the muscle defects that characterize type 2 diabetes.

FIG. 1.

Regulation of FTO expression in human skeletal muscle. A: Basal FTO mRNA levels were measured by real-time RT-PCR in skeletal muscle of age-matched control, obese, type 2 diabetic patients, and type 1 diabetic subjects. Values are means ± SEM (n = 5–10). *P < 0.05 versus age-matched control subjects. The mRNA level of the reference gene HPRT did not differ among groups (3.9 ± 0.5, 2.9 ± 0.1, 3.8 ± 0.5, 3.3 ± 0.3, and 4.2 ± 0.5 amo/μg total RNA, in control 50, obese, type 2 diabetic patients [T2DM], control 25, and type 1 diabetic patients [T1DM], respectively, not significant [NS]). B: Representative Western blot illustrating FTO protein levels in skeletal muscle of age-matched control, obese, and type 2 diabetic patients. Data of the histogram are means ± SEM (n = 3). *P < 0.05 versus age-matched control subjects. C and D: FTO mRNA levels were measured by real-time RT-PCR in skeletal muscle of age-matched control and type 2 diabetic patients, before and after a 3-h euglycemic hyperinsulinemic clamp (C) or a 3-h hyperglycemic euinsulinemic clamp (D). Values are means ± SEM (n = 6). E: FTO mRNA levels were measured by real-time RT-PCR in skeletal muscle of type 2 diabetic patients, before and after a 12-week rosiglitazone treatment. Values are means ± SEM (n = 6). *P < 0.05 versus before treatment. The mRNA level of the reference gene HPRT did not differ before and after rosiglitazone treatment (5 ± 0.6 and 6.6 ± 0.5 amo/μg total RNA, respectively, NS). a.u., arbitrary units.

FIG. 2.

Effect of FTO overexpression on insulin signaling in HEK293 cells. HEK293 were transiently transfected with pcDNA3-FTO or empty pcDNA3 vector (control). Forty-eight h posttransfection, cells were depleted in serum for 3 h and stimulated with insulin (10⁻⁷ M, 10 min). A: Representative Western blots of FTO, pSer473PKB, pThr308PKB, and total PKB. B: Histogram illustrates the quantification and normalization of the phosphorylation of PKB in control and FTO-overexpressing cells. Values are means ± SEM (n = 3). *P < 0.001 versus control cells, #P < 0.001 FTO versus GFP. Ins, insulin.

FIG. 3.

Adenoviral overexpression of FTO in differentiated myotubes. Human myotubes or C₂C₁₂ cells were infected with recombinant adenovirus encoding human FTO or GFP (control) for 48 h. A: Representative Western blots of FTO, pSer473PKB, PKB, pPDK1, PDK1, and tubulin, in GFP- or FTO-overexpressing C₂C₁₂ myotubes. B: Representative Western blots of pSer473PKB and PKB total, in GFP- or FTO-overexpressing C₂C₁₂ myotubes (210 [7] ifu/well), under basal conditions or after insulin stimulation. Histogram represents means ± SEM (n = 4). *P < 0.05 versus basal situation, ^#P < 0.05 FTO versus GFP. C: Representative Western blots of FTO, pSer473PKB, PKB, p70/85S6K, and actin in human myotubes overexpressing either GFP or FTO (210 [7] ifu/well). D and E: Myotubes were transfected with siRNA control or specific for FTO for 48 h. D: Validation of FTO silencing in muscle cells. E: Representative Western blots of pSer473PKB and PKB in myotubes silencing for FTO (50 nmol/l of siRNA). Histogram illustrates the quantification and normalization of the phosphorylation of PKB in myotubes silenced for FTO. Values are means ± SEM (n = 3). ifu, inclusion-forming units.

FIG. 4.

Effect of FTO overexpression on de novo lipogenesis. Human myotubes were infected with recombinant adenovirus encoding human FTO genome or GFP (control) for 48 h (210 [7] ifu/well). A: De novo lipogenesis was measured with [2-¹⁴C]acetate during 24 h. Values are means ± SEM (n = 3). *P < 0.05. B: mRNA levels of FAS, ACC1, GPAT, and PPARγ were measured by real-time RT-PCR. Data represent means ± SEM (n = 4).*P < 0.05. C: Palmitate-induced ROS production either in human myotubes overexpressing either GFP or FTO, or in myotubes silencing for FTO (siRNA, 50 nmol/l). After 48 h of infection/transfection, myotubes were incubated with BSA or palmitate (750 μmol/l) for 16 h, and ROS production was measured using nitro-blue tetrazolium chloride assay. Values represent means ± SEM (n = 3).*P < 0.05. FAS, fatty acid synthase; ACC1, acetyl-CoA carboxylase 1; GPAT, glycerol-3 phosphate acyltransferase; PPARγ, peroxisome proliferator–activated receptor γ; a.u., arbitrary units; ifu, inclusion-forming units.

FIG. 5.

Effect of FTO overexpression on mitochondria structure and function in human myotubes. Human myotubes were infected with recombinant adenovirus encoding human FTO genome or GFP (control) for 48 h (210 [7] ifu/well). A: Electronic microscopy analysis of human myotubes overexpressing GFP or FTO. B: Analysis of the expression of respiratory chain complexes in human myotubes overexpressing GFP or FTO. It should be noted that complex IV was not detected in our conditions. Histograms represent the means ± SEM (n = 4). *P < 0.05. NS, not significant. C: Analysis of complex I (pyruvate + malate)- and complex II (succinate + rotenone)-mediated ATP synthesis in GFP- and FTO-overexpressing myotubes. Histograms represent the means ± SEM (n = 4). *P = 0.02. a.u., arbitrary units; ifu, inclusion-forming units. (A high-quality color representation of this figure is available in the online issue.)

FIG. 6.

Reduced OXPHOS and antioxidant genes and increased oxidative stress in skeletal muscle of type 2 diabetic patients. A: mRNA levels of ATP5B, UQCR, SOD2, and PGC1α were measured by real-time RT-PCR in skeletal muscle of control and type 2 diabetic patients. Data represent means ± SEM (n = 10). *P < 0.05. B: Immunoblot showing total protein carbonylation in skeletal muscle of control and type 2 diabetic patients. Histogram represents means ± SEM (n = 4).*P < 0.05. ATP5B, ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide; UQCR, ubiquinol-cytochrome c reductase; SOD2, superoxide dismutase 2; a.u., arbitrary units; T2DM, type 2 diabetes.