Momordica charantia fruit juice stimulates glucose and amino acid uptakes in L6 myotubes

The fruit of Momordica charantia (family: Cucurbitacea) is used widely as a hypoglycaemic agent to treat diabetes mellitus (DM). The mechanism of the hypoglycaemic action of M. charantia in vitro is not fully understood. This study investigated the effect of M. charantia juice on either ³H-2-deoxyglucose or N-methyl-amino-a-isobutyric acid (¹⁴C-Me-AIB) uptake in L6 rat muscle cells cultured to the myotube stage. The fresh juice was centrifuged at 5000 rpm and the supernatant lyophilised. L6 myotubes were incubated with either insulin (100 nM), different concentrations (1–10 g ml^–1) of the juice or its chloroform extract or wortmannin (100 nM) over a period of 1–6 h. The results were expressed as pmol min^–1 (mg cell protein)^–1, n= 6–8 for each value. Basal ³H-deoxyglucose and ¹⁴C-Me-AIB uptakes by L6 myotubes after 1 h of incubation were (means ± S.E.M.) 32.14 ± 1.34 and 13.48 ± 1.86 pmol min^–1 (mg cell protein)^–1, respectively. Incubation of L6 myotubes with 100 nM insulin for 1 h resulted in significant (ANOVA, p < 0.05) increases in ³H-deoxyglucose and ¹⁴C-Me-AIB uptakes. Typically, ³H-deoxyglucose and ¹⁴C-Me-AIB uptakes in the presence of insulin were 58.57 ± 4.49 and 29.52 ± 3.41 pmol min^–1 (mg cell protein^–1), respectively. Incubation of L6 myotubes with three different concentrations (1, 5 and 10 g ml^–1) of either the lyophilised juice or its chloroform extract resulted in time-dependent increases in ³H-deoxy-D-glucose and ¹⁴C-Me-AIB uptakes, with maximal uptakes occurring at a concentration of 5 g ml^–1. Incubation of either insulin or the juice in the presence of wortmannin (a phosphatidylinositol 3-kinase inhibitor) resulted in a marked inhibition of ³H-deoxyglucose by L6 myotubes compared to the uptake obtained with either insulin or the juice alone. The results indicate that M. charantia fruit juice acts like insulin to exert its hypoglycaemic effect and moreover, it can stimulate amino acid uptake into skeletal muscle cells just like insulin. (Mol Cell Biochem 261: 99–104, 2004)     

Increased Skeletal Muscle 11βHSD1 mRNA Is Associated with Lower Muscle Strength in Ageing

Background

Sarcopenia, the loss of muscle mass and function with age, is associated with increased morbidity and mortality. Current understanding of the underlying mechanisms is limited. Glucocorticoids (GC) in excess cause muscle weakness and atrophy. We hypothesized that GC may contribute to sarcopenia through elevated circulating levels or increased glucocorticoid receptor (GR) signaling by increased expression of either GR or the GC-amplifying enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11βHSD1) in muscle.

Methods

There were 82 participants; group 1 comprised 33 older men (mean age 70.2years, SD 4.4) and 19 younger men (22.2years, 1.7) and group 2 comprised 16 older men (79.1years, 3.4) and 14 older women (80.1years, 3.7). We measured muscle strength, mid-thigh cross-sectional area, fasting morning plasma cortisol, quadriceps muscle GR and 11βHSD1 mRNA, and urinary glucocorticoid metabolites. Data were analysed using multiple linear regression adjusting for age, gender and body size.

Results

Muscle strength and size were not associated with plasma cortisol, total urinary glucocorticoids or the ratio of urinary 5β-tetrahydrocortisol +5α-tetrahydrocortisol to tetrahydrocortisone (an index of systemic 11βHSD activity). Muscle strength was associated with 11βHSD1 mRNA levels (β -0.35, p = 0.04), but GR mRNA levels were not significantly associated with muscle strength or size.

Conclusion

Although circulating levels of GC are not associated with muscle strength or size in either gender, increased cortisol generation within muscle by 11βHSD1 may contribute to loss of muscle strength with age, a key component of sarcopenia. Inhibition of 11βHSD1 may have therapeutic potential in sarcopenia.     

Constitutive Expression of Yes-Associated Protein (Yap) in Adult Skeletal Muscle Fibres Induces Muscle Atrophy and Myopathy

The aim of this study was to investigate the function of the Hippo pathway member Yes-associated protein (Yap, gene name Yap1) in skeletal muscle fibres in vivo. Specifically we bred an inducible, skeletal muscle fibre-specific knock-in mouse model (MCK-tTA-hYAP1 S127A) to test whether the over expression of constitutively active Yap (hYAP1 S127A) is sufficient to drive muscle hypertrophy or stimulate changes in fibre type composition. Unexpectedly, after 5–7 weeks of constitutive hYAP1 S127A over expression, mice suddenly and rapidly lost 20–25% body weight and suffered from gait impairments and kyphosis. Skeletal muscles atrophied by 34–40% and the muscle fibre cross sectional area decreased by ≈40% when compared to control mice. Histological analysis revealed evidence of skeletal muscle degeneration and regeneration, necrotic fibres and a NADH-TR staining resembling centronuclear myopathy. In agreement with the histology, mRNA expression of markers of regenerative myogenesis (embryonic myosin heavy chain, Myf5, myogenin, Pax7) and muscle protein degradation (atrogin-1, MuRF1) were significantly elevated in muscles from transgenic mice versus control. No significant changes in fibre type composition were detected using ATPase staining. The phenotype was largely reversible, as a cessation of hYAP1 S127A expression rescued body and muscle weight, restored muscle morphology and prevented further pathological progression. To conclude, high Yap activity in muscle fibres does not induce fibre hypertrophy nor fibre type changes but instead results in a reversible atrophy and deterioration.     

Disassociation between the effects of amino acids and insulin on signaling, ubiquitin ligases, and protein turnover in human muscle

We determined the effects of intravenous infusion of amino acids (AA) at serum insulin of 5, 30, 72, and 167 mU/l on anabolic signaling, expression of ubiquitin-proteasome components, and protein turnover in muscles of healthy young men. Tripling AA availability at 5 mU/l insulin doubled incorporation of [1-(13)C]leucine [i.e., muscle protein synthesis (MPS), P < 0.01] without affecting the rate of leg protein breakdown (LPB; appearance of d(5)-phenylalanine). While keeping AA availability constant, increasing insulin to 30 mU/l halved LPB (P < 0.05) without further inhibition at higher doses, whereas rates of MPS were identical to that at 5 mU/l insulin. The phosphorylation of PKB Ser(473) and p70(S6k) Thr(389) increased concomitantly with insulin, but whereas raising insulin to 30 mU/l increased the phosphorylation of mTOR Ser(2448), 4E-BP1 Thr(37/46), or GSK3beta Ser(9) and decreased that of eEF2 Thr(56), higher insulin doses to 72 and 167 mU/l did not augment these latter responses. MAFbx and proteasome C2 subunit proteins declined as insulin increased, with MuRF-1 expression largely unchanged. Thus increasing AA and insulin availability causes changes in anabolic signaling and amounts of enzymes of the ubiquitin-proteasome pathway, which cannot be easily reconciled with observed effects on MPS or LPB.     

Concentrations of signal transduction proteins mediating exercise and insulin responses in rat extensor digitorum longus and soleus muscles

Differences in the concentrations of signal transduction proteins often alter cellular function and phenotype, as is evident from numerous, heterozygous knockout mouse models for signal transduction proteins. Here, we measured signal transduction proteins involved in the adaptation to exercise and insulin signalling in fast rat extensor digitorum longus (EDL; 3% type I fibres) and the slow soleus muscles (84% type I fibres). The EDL and soleus were excised from four rats, the proteins extracted and subjected to Western blots for various signal transduction proteins. Our results show major differences in signal transduction protein concentrations between EDL and soleus. The EDL to soleus concentration ratios were: Calcineurin: 1.43 ± 0.10; ERK1: 0.38 ± 0.18; ERK2: 0.61 ± 0.16; p38, : 1.36 ± 0.15; p38/ERK6: 0.95 ± 0.11; PKB/AKT: 1.44 ± 0.08; p70S6k: 6.86 ± 3.58; GSK3: 0.69 ± 0.03; myostatin: 1.95 ± 0.43; NF-B: 0.32 ± 0.10 (values >1 indicate higher expression in the EDL, and values <1 indicate higher expression in the soleus). With the exception of p38/ERK6, the concentration of each signal transduction protein was uniformly higher in one muscle than in the other in all four animals. These experiments show that signal transduction protein concentrations vary between fast and slow muscles, presumably reflecting a concentration difference on a fibre level. Proteins that promote particular functions such as growth or slow phenotype are not necessarily higher in muscles with that particular trait (e.g. higher in larger fibres or slow muscle). Interindividual differences in fibre composition might explain variable responses to training and insulin. (Mol Cell Biochem 261: 111–116, 2004)     

Momordica charantia fruit juice stimulates glucose and amino acid uptakes in L6 myotubes

The fruit of Momordica charantia (family: Cucurbitacea) is used widely as a hypoglycaemic agent to treat diabetes mellitus (DM). The mechanism of the hypoglycaemic action of M. charantia in vitro is not fully understood. This study investigated the effect of M. charantia juice on either 3H-2-deoxyglucose or N-methyl-amino-a-isobutyric acid (14C-Me-AIB) uptake in L6 rat muscle cells cultured to the myotube stage. The fresh juice was centrifuged at 5000 rpm and the supernatant lyophilised. L6 myotubes were incubated with either insulin (100 nM), different concentrations (1-10 microg ml(-1)) of the juice or its chloroform extract or wortmannin (100 nM) over a period of 1- 6 h. The results were expressed as pmol min(-1) (mg cell protein)(-1), n = 6-8 for each value. Basal 3H-deoxyglucose and 14C-Me-AIB uptakes by L6 myotubes after 1 h of incubation were (means +/- S.E.M.) 32.14 +/- 1.34 and 13.48 +/- 1.86 pmol min(-1) (mg cell protein)(-1), respectively. Incubation of L6 myotubes with 100 nM insulin for 1 h resulted in significant (ANOVA, p < 0.05) increases in 3H-deoxyglucose and 14C-Me-AIB uptakes. Typically, 3H-deoxyglucose and 14C-Me-AIB uptakes in the presence of insulin were 58.57 +/- 4.49 and 29.52 +/- 3.41 pmol min(-1) (mg cell protein(-1)), respectively. Incubation of L6 myotubes with three different concentrations (1, 5 and 10 microg ml(-1)) of either the lyophilised juice or its chloroform extract resulted in time-dependent increases in 3H-deoxy-D-glucose and 14C-Me-AIB uptakes, with maximal uptakes occurring at a concentration of 5 microg ml(-1). Incubation of either insulin or the juice in the presence of wortmannin (a phosphatidylinositol 3-kinase inhibitor) resulted in a marked inhibition of 3H-deoxyglucose by L6 myotubes compared to the uptake obtained with either insulin or the juice alone. The results indicate that M. charantia fruit juice acts like insulin to exert its hypoglycaemic effect and moreover, it can stimulate amino acid uptake into skeletal muscle cells just like insulin.     

Recovery of free ADP, Pi, and free energy of ATP hydrolysis in human skeletal muscle

We measuredsignificant undershoots of the concentrations of free ADP([ADP]) and P_i([P_i]) and the freeenergy of ATP hydrolysis (G_ATP) belowinitial resting levels during recovery from severe ischemic exercisewith ³¹P-nuclear magneticresonance spectroscopy in 11 healthy sports students. Undershoots ofthe rate of oxidative phosphorylation would be predicted if the rate ofoxidative phosphorylation would depend solely on free[ADP],[P_i], orG_ATP. However,undershoots of the rate of oxidative phosphorylation have not beenreported in the literature. Furthermore, undershoots of the rate ofoxidative phosphorylation are unlikely because there is evidence that a balance between ATP production and consumption cannot be achieved if anundershoot of the rate of oxidative phosphorylation actually occurs.Therefore, oxidative phosphorylation seems to depend not only on free[ADP],[P_i], orG_ATP. Anexplanation is that acidosis-related or other factors control oxidativephosphorylation additionally, at least under some conditions.

     

Concentrations of signal transduction proteins exercise and insulin responses in rat extensor digitorum longus and soleus muscles

Differences in the concentrations of signal transduction proteins often alter cellular function and phenotype, as is evident from numerous, heterozygous knockout mouse models for signal transduction proteins. Here, we measured signal transduction proteins involved in the adaptation to exercise and insulin signalling in fast rat extensor digitorum longus (EDL; 3% type I fibres) and the slow soleus muscles (84% type I fibres). The EDL and soleus were excised from four rats, the proteins extracted and subjected to Western blots for various signal transduction proteins. Our results show major differences in signal transduction protein concentrations between EDL and soleus. The EDL to soleus concentration ratios were: Calcineurin: 1.43 +/- 0.10; ERK1: 0.38 +/- 0.18; ERK2: 0.61 +/- 0.16; p38alpha, beta: 1.36 +/- 0.15; p38gamma/ERK6: 0.95 +/- 0.11; PKB/AKT: 1.44 +/- 0.08; p70S6k: 6.86 +/- 3.58; GSK3beta: 0.69 +/- 0.03; myostatin: 1.95 +/- 0.43; NF-kappaB: 0.32 +/- 0.10 (values >1 indicate higher expression in the EDL, and values < 1 indicate higher expression in the soleus). With the exception of p38gamma/ERK6, the concentration of each signal transduction protein was uniformly higher in one muscle than in the other in all four animals. These experiments show that signal transduction protein concentrations vary between fast and slow muscles, presumably reflecting a concentration difference on a fibre level. Proteins that promote particular functions such as growth or slow phenotype are not necessarily higher in muscles with that particular trait (e.g. higher in larger fibres or slow muscle). Interindividual differences in fibre composition might explain variable responses to training and insulin.