Skeletal muscle-selective knockout of LKB1 increases insulin sensitivity, improves glucose homeostasis, and decreases TRB3 - PubMed (original) (raw)

. 2006 Nov;26(22):8217-27.

doi: 10.1128/MCB.00979-06. Epub 2006 Sep 11.

David E Arnolds, Nobuharu Fujii, Thien T Tran, Marc J Rogers, Niels Jessen, Yangfeng Li, Chong Wee Liew, Richard C Ho, Michael F Hirshman, Rohit N Kulkarni, C Ronald Kahn, Laurie J Goodyear

Affiliations

Skeletal muscle-selective knockout of LKB1 increases insulin sensitivity, improves glucose homeostasis, and decreases TRB3

Ho-Jin Koh et al. Mol Cell Biol. 2006 Nov.

Abstract

LKB1 is a tumor suppressor that may also be fundamental to cell metabolism, since LKB1 phosphorylates and activates the energy sensing enzyme AMPK. We generated muscle-specific LKB1 knockout (MLKB1KO) mice, and surprisingly, found that a lack of LKB1 in skeletal muscle enhanced insulin sensitivity, as evidenced by decreased fasting glucose and insulin concentrations, improved glucose tolerance, increased muscle glucose uptake in vivo, and increased glucose utilization during a hyperinsulinemic-euglycemic clamp. MLKB1KO mice had increased insulin-stimulated Akt phosphorylation and a > 80% decrease in muscle expression of TRB3, a recently identified Akt inhibitor. Akt/TRB3 binding was present in skeletal muscle, and overexpression of TRB3 in C2C12 myoblasts significantly reduced Akt phosphorylation. These results demonstrate that skeletal muscle LKB1 is a negative regulator of insulin sensitivity and glucose homeostasis. LKB1-mediated TRB3 expression provides a novel link between LKB1 and Akt, critical kinases involved in both tumor genesis and cell metabolism.

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Figures

FIG. 1.

FIG. 1.

High-efficiency ablation of the LKB1 protein selectively in skeletal muscle. (A) LKB1 expression was not significantly altered for wild-type (WT) mice compared to that for mice harboring one (lox/+) or two (lox/lox) alleles in gastrocnemius muscle lysates. Heterozygotes (+/−) showed approximately 50% of control LKB1 expression, while MLKB1KO (−/−) mice showed nearly complete loss of LKB1 expression. (B) LKB1 activity was assessed in an in vitro kinase activity assay using LKB1tide as the substrate. Consistent with protein data, +/− and −/− animals showed reduced LKB1 activity compared to controls. (C and D) Muscles from MLKB1KO animals (−/−) show nearly complete depletion of the LKB1 protein (C), while expression is not affected in nonmuscle tissue (D). Gastroc, gastrocnemius; TA, tibialis anterior; Sol, soleus. Data are means ± SEM; n = 3 to 6/group. *, P < 0.05; ***, P < 0.001 versus control results (+/+).

FIG. 2.

FIG. 2.

MLKB1KO mice are AICAR intolerant and have lower skeletal muscle glycogen content and improved whole-body glucose homeostasis. (A) Body weights of male mice (n = 5 to 10/group) were tracked for 12 weeks, beginning at 6 weeks of age. (B) Mice were injected i.p. with AICAR (0.5 g/kg), and blood from the tail vein was used to measure glucose. (C and D) Gastrocnemius (gastroc) and tibialis anterior (TA) muscles were dissected from fed (C) and 14-h-fasted (D) mice and assayed for glycogen. (E) Fasting blood glucose and serum insulin concentrations were lower in MLKB1KO. (F) Mice were injected i.p. with glucose (2 g/kg), and blood from the tail vein used to measure glucose. For panels B to F, data are means ± SEM; n = 5 to 12/group. , P < 0.05; **, P < 0.01; ***, P < 0.001 versus results with corresponding +/+ mice.

FIG. 3.

FIG. 3.

MLKB1KO mice have improved skeletal muscle glucose uptake in vivo and in vitro. (A and B) Mice were injected i.p. with glucose (2 g/kg) containing 3H-2-deoxyglucose and

d

-[14C(U)]glucose as radioactive tracers and followed for 2 h. Tibialis anterior muscles were processed and assayed for glucose uptake (A) and glycogen incorporation (B). (C) Glucose disposal rate before and during the hyperinsulinemic-euglycemic clamp (5 mU/kg/min). EDL (D and E) or soleus (F and G) muscles were dissected and incubated in the absence of insulin or with submaximal (450 μU/ml) (Submax) or maximal (50 mU/ml) (Max) insulin. (D) Basal glucose transport was decreased and insulin-stimulated glucose transport was normal in the EDL. (E) Expressed as an increase above the basal, level, submaximal insulin-stimulated transport was significantly greater in EDL muscle from MLKB1KO mice. (F and G) There were no differences between the genotypes in glucose transport in soleus. Data are means ± SEM; n = 6 to 12/group. *, P < 0.05 versus basal level of same genotype. †, P < 0.05; †††, P < 0.001 versus results with corresponding +/+ mice.

FIG. 4.

FIG. 4.

Submaximal insulin stimulation of Akt Thr308 phosphorylation. EDL and soleus muscles were dissected and incubated with submaximal insulin (450 μU/ml). (A and B) Insulin-stimulated Akt Thr308 phosphorylation in EDL and soleus muscle was greater in MLKB1KO mice than in controls. Data are means ± SEM; n = 5 to 6/group. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus basal level of same genotype. †, P < 0.05 versus results with corresponding +/+ mice.

FIG. 5.

FIG. 5.

Protein expression of PGC1α, PPARα, and TRB3 is decreased in MLKB1KO mice. PGC1α (A), PPARα (B), or TRB3 (C) protein was assessed by immunoblotting with muscle lysates obtained from tibialis anterior muscle. There were significant decreases in PGC1α, PPARα, and TRB3 protein in muscles from MLKB1KO mice. Data are means ± SEM, and the blots shown are representative of six mice per group. *, P < 0.05; **, P < 0.001 versus results with control (+/+) mice; A.U., arbitary units.

FIG. 6.

FIG. 6.

TRB3 binds Akt and inhibits Akt phosphorylation in response to insulin. (A) Western blot analysis of TRB3 in immunoprecipitates prepared from gastrocnemius and tibialis anterior muscles in the absence or presence of Akt antibody. A band of the appropriate molecular weight for TRB3 was recovered from muscle lysates, but no band was detected in immunoprecipitates prepared in the absence of muscle lysates or Akt antibody. (B to D) Overexpression of TRB3 blunted insulin-stimulated Akt Thr308 (C) or Ser473 (D) phosphorylation without altering the Akt protein level in C2C12 cells. (E) Akt activity was assayed by in vitro immune complex assay as described in Materials and Methods. Data are means ± SEM, and the blots shown are representative of three per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus control results. Submax, submaximal insulin; Max, maximal insulin; A.U., arbitrary units.

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