Hepatocyte growth factor is a novel stimulator of glucose uptake and metabolism in skeletal muscle cells

Abstract

Skeletal muscle plays a major role in glucose and lipid metabolism. Active hepatocyte growth factor (HGF) is present in the extracellular matrix in skeletal muscle. However, the effects of HGF on glucose and lipid metabolism in skeletal muscle are completely unknown. We therefore examined the effects of HGF on deoxyglucose uptake (DOGU), glucose utilization, and fatty acid oxidation (FAO) in skeletal muscle cells. HGF significantly enhanced DOGU in mouse soleus muscles in vitro. Furthermore, HGF significantly increased: (i) DOGU in a time- and dose-dependent manner; (ii) glucose utilization; and (iii) plasma membrane expression of Glut-1 and Glut-4 in the rat skeletal muscle model of L6 myotubes. HGF-mediated effect on DOGU was dependent on the activation of phosphatidylinositol 3-kinase signaling pathway. On the other hand, HGF markedly and significantly decreased FAO in L6 myotubes without affecting the activities of carnitine palmitoyltransferase I and II. Collectively, these results indicate that HGF is a potent activator of glucose transport and metabolism and also a strong inhibitor of FAO in rodent myotubes. HGF, through its ability to stimulate glucose transport and metabolism and to impair FAO, may participate in the regulation of glucose disposal in skeletal muscle.

FIGURE 1.

HGF stimulates glucose uptake in mouse soleus muscle. A, Western blot analysis of HGF and c-Met expression in soleus muscle from two C57BL/6J male mice (M1 and M2). Actin expression was determined to ensure similar protein loading. B, effect of 25 ng/ml HGF and/or 1 milliunit/ml insulin on 2-[³H]deoxyglucose (DOG) uptake in mouse soleus muscle. Values are means ± S.E. of 6–8 muscles per condition. *, p < 0.05 versus control uptake; #, p < 0.05 versus uptake in muscles stimulated with HGF or insulin alone.

FIGURE 2.

HGF stimulates glucose uptake and utilization but does not alter glucose incorporation into glycogen in L6 myotubes. A, Western blot analysis of HGF and c-Met expression in two different cultures of L6 myoblasts before (left two lanes) and after differentiation into L6 myotubes (right two lanes). Actin expression was determined to ensure similar protein loading. HGF time (B) and dose (C) dependently stimulated glucose transport in L6 myotubes. Cells were incubated with HGF (25 ng/ml) for several time periods and with various concentrations for 24 h as indicated in the figure. Uptake of 2-[³H]deoxyglucose (DOG) was measured during a 5-min period as described under “Experimental Procedures.” D, HGF (25 ng/ml) increased glucose utilization in L6 myotubes. E, effect of HGF (25 ng/ml) and/or 100 nm insulin on [U-¹⁴C]glucose incorporation into glycogen in L6 myotubes. Values are means ± S.E. of at least four experiments performed in triplicate. *, p < 0.025 and **, p < 0.01 versus control or 0 value; +, p < 0.05 versus uptake in the previous time point or concentration.

FIGURE 3.

HGF increases the presence of glucose transporters in the plasma membrane of L6 myotubes. A, Western blot analysis of Glut-1 and Glut-4 expression in membrane protein extracts from L6 myotubes treated with 25 ng/ml HGF for 1 h (Acute) or 24 h (Chronic) and with 100 nm insulin for 30 min. Expression of the α1-subunit Na⁺/K⁺ ATPase was determined to ensure similar membrane protein loading. Densitometric analysis of Glut-1 (B) and Glut-4 (C) in four different experiments. Western blot analysis of Glut-1 (D) and Glut-4 (E) expression in total protein extracts from L6 myotubes treated with 25 ng/ml HGF for 24 h. Expression of tubulin was determined to ensure similar protein loading. The y-axis represents arbitrary units. Results are means ± S.E. *, p < 0.05 versus control (C).

FIGURE 4.

Activation of PI3K and ERK1/2 is essential for HGF-induced stimulation on glucose transport in L6 myotubes. A, representative Western blot depicting the effects of HGF (25 ng/ml) on Ser-473-Akt, ERK1/2, and p38 MAPK phosphorylation in L6 myotubes. Densitometric analysis of Ser-473-Akt (B), ERK1/2 (C), and p38 MAPK (D) phosphorylation in four different experiments. The y-axis represents the ratio of phosphorylated versus total protein in arbitrary units. Results are means ± S.E. *, p < 0.05 and **, p < 0.01 versus time 0. Gab-1, in response to 25 ng/ml HGF (E), and IRS-1, in response to 100 nm insulin (20 min) (F), recruit the p85 subunit of PI3K. Representative autoradiographs of two independent experiments with identical results. G, uptake of 2-[³H]deoxyglucose (DOG) in L6 myotubes incubated for 24 h with 25 ng/ml HGF with or without 100 nm wortmannin (W), 10 μm PD98059 (PD), or 10 μm SB202190 (SB). H, net DOG uptake was calculated subtracting the corresponding basal from the HGF-stimulated uptake. Values are means ± S.E. of four different experiments in triplicate. *, p < 0.05 and **, p < 0.01 versus corresponding control. #, p < 0.05 versus HGF.

FIGURE 5.

HGF inhibits fatty acid oxidation in L6 myotubes. A, oxidation of palmitate in L6 myotubes treated with 25 ng/ml HGF for 3 h. Values are means ± S.E. of four different experiments in triplicate. *, p < 0.05 versus control. B, CPT-I and CPT-II activity in total cellular extracts of L6 myotubes treated with 25 ng/ml HGF for 3 h. CPT-I activity was equally inhibited by 1 mm malonyl-CoA in protein extracts of L6 myotubes treated with or without HGF. Values are means ± S.E. of three different experiments in triplicate. C, Western blot analysis of acetyl-CoA carboxylase phosphorylation (Ser-79) in protein extracts from L6 myotubes treated with 25 ng/ml HGF for 3 h. The y-axis represents the ratio of phosphorylated acetyl-CoA carboxylase versus tubulin in arbitrary units. Values are means ± S.E. of four different experiments. *, p < 0.05 versus control. D, effect of 25 ng/ml HGF on palmitate-induced lipid accumulation in L6 myotubes. Values are means ± S.E. of four different experiments in triplicate. No significant differences were found following HGF treatment.