Mammalian Tribbles homolog 3 impairs insulin action in skeletal muscle: role in glucose-induced insulin resistance

Abstract

Tribbles homolog 3 (TRIB3) was found to inhibit insulin-stimulated Akt phosphorylation and modulate gluconeogenesis in rodent liver. Currently, we examined a role for TRIB3 in skeletal muscle insulin resistance. Ten insulin-sensitive, ten insulin-resistant, and ten untreated type 2 diabetic (T2DM) patients were metabolically characterized by hyperinsulinemic euglycemic glucose clamps, and biopsies of vastus lateralis were obtained. Skeletal muscle samples were also collected from rodent models including streptozotocin (STZ)-induced diabetic rats, db/db mice, and Zucker fatty rats. Finally, L6 muscle cells were used to examine regulation of TRIB3 by glucose, and stable cell lines hyperexpressing TRIB3 were generated to identify mechanisms underlying TRIB3-induced insulin resistance. We found that 1) skeletal muscle TRIB3 protein levels are significantly elevated in T2DM patients; 2) muscle TRIB3 protein content is inversely correlated with glucose disposal rates and positively correlated with fasting glucose; 3) skeletal muscle TRIB3 protein levels are increased in STZ-diabetic rats, db/db mice, and Zucker fatty rats; 4) stable TRIB3 hyperexpression in muscle cells blocks insulin-stimulated glucose transport and glucose transporter 4 (GLUT4) translocation and impairs phosphorylation of Akt, ERK, and insulin receptor substrate-1 in insulin signal transduction; and 5) TRIB3 mRNA and protein levels are increased by high glucose concentrations, as well as by glucose deprivation in muscle cells. These data identify TRIB3 induction as a novel molecular mechanism in human insulin resistance and diabetes. TRIB3 acts as a nutrient sensor and could mediate the component of insulin resistance attributable to hyperglycemia (i.e., glucose toxicity) in diabetes.

Fig. 1.

Real-time RT-PCR analysis of Tribbles homolog 3 (TRIB3) mRNA levels in human tissues collected from adipose (n = 5), appendix (n = 2), artery (n = 7), bladder (n = 4), colon (n = 10), esophagus (n = 8), gallbladder (n = 5), heart (n = 8), liver (n = 5), lung (n = 10), lymph node (n = 5), skeletal muscle (n = 9), small intestine (n = 8), spleen (n = 2), stomach (n = 8), and trachea (n = 5), respectively. Data are means ± SE.

Fig. 2.

A: real-time RT-PCR analysis of TRIB3 mRNA levels in human skeletal muscle collected from insulin-sensitive (IS) and insulin-resistant (IR) subjects and from patients with type 2 diabetes (T2DM). B: TRIB3 protein levels in human skeletal muscle from IS (n = 10), IR (n = 10), and T2DM (n = 10) subjects. TRIB3 protein was detected by Western blotting and was quantified by densitometry. Data are means ± SE. *P < 0.05.

Fig. 3.

Relationships between muscle TRIB3 protein expression and fasting plasma glucose (top) and glucose disposal rate normalized by lean body mass (GDR/LBM) as a measure of insulin sensitivity (bottom). TRIB3 protein level was determined by Western blotting. Data were based on studies of individuals over a broad range of insulin sensitivity, including subgroups of IS and IR and T2DM subjects. Correlation coefficient (r) and P values are shown for each graph.

Fig. 4.

Levels of TRIB3 protein expression in skeletal muscle from control rats (n = 5) and streptozotocin (STZ)-induced diabetics rat (n = 5) as determined by Western blotting. Results were quantified by densitometry, and data are means ± SE. *P < 0.05.

Fig. 5.

A: mean body weight and fasting plasma glucose levels in lean control mice (n = 3) and 3 db/db mice (n = 3). B: TRIB3 protein levels in skeletal muscle from lean control mice (n = 3) and from db/db mice (n = 3) as determined by Western blotting. Results were quantified by densitometry, and data are means ± SE. *P < 0.05.

Fig. 6.

A: plasma glucose and serum triglyceride concentrations in lean control rats (white bar) and Zucker fatty rats with (gray bar) or without (hatched bar) pioglitazone treatment. Data are means ± SE. *P < 0.05 compared with lean controls; ‡P < 0.05, compared with Zucker fatty rats without pioglitazone treatment. n = 6/group. B: serum free fatty acid (FFA) and insulin concentrations in lean control (white bar) and Zucker fatty rats with (gray bar) or without (hatched bar) pioglitazone treatment. Data are means ± SE. *P < 0.05, compared with lean controls, ‡P < 0.05, compared with Zucker fatty rats without pioglitazone treatment. n = 6/group. C: TRIB3 protein expression in skeletal muscle from lean rats (n = 6) and Zucker fatty rats without pioglitazone treatment (n = 6) and with pioglitazone treatment (n = 6) as determined by Western blotting. Results were quantified by densitometry, and data are means ± SE. *P < 0.05.

Fig. 7.

A: basal (open bar) and maximally insulin-stimulated (solid bar) glucose transport rates in L6-GLUT4myc cells stably transduced with either a lentiviral myc expression vector as a control or with a lentiviral TRIB3 expression vector. Data are means ± SE of 3 separate experiments. 2-DG, 2-deoxy-d-glucose. *P < 0.05. B: quantitative analyses of cell-surface GLUT4myc content with or without insulin treatment. Open bar represents cell surface glucose transporter 4 (GLUT4) under basal conditions (without insulin treatment); solid bar represents insulin-stimulated GLUT4 translocation to cell surface. Data are means ± SE of 3 separate experiments. *P < 0.05.

Fig. 8.

A: effects of TRIB3 hyperexpression on insulin-induced phosphorylation of Akt in L6 muscle cells. L6 cells were stably transfected with a lentivirus construct for hyperexpression of myc as control or TRIB3 and were then treated with (+) or without (−) 10 nM insulin for 45 min. pAkt and total Akt were measured by Western blotting. B: effects of TRIB3 hyperexpression on insulin-induced phosphorylation of ERK in L6 muscle cells. C: effects of TRIB3 hyperexpression on insulin-induced phosphorylation of insulin receptor substrate-1 (IRS-1) in L6 muscle cells. Representative blots are shown, and data are means ± SE of 3 separate experiments. *P < 0.05.

Fig. 9.

Changes in TRIB3 mRNA levels (A) and TRIB3 protein levels (B) in L6 myotubes in response to different d-glucose media concentrations (0, 2.5, 5, 10, and 15 mM). mRNA was measured by real-time RT-PCR, while protein was analyzed by Western blotting and quantified by densitometry. Representative blot is shown, and data are means ± SE of 3 separate experiments. C: pAMPK protein levels in L6 myotubes in response to different d-glucose concentrations (0, 5, and 15 mM) and l-glucose (15 mM). Representative blot is shown, and data are means ± SE of 3 separate experiments. *P < 0.05.