Takumi Yokokawa

Mixed lactate and caffeine compound increases satellite cell activity and anabolic signals for muscle hypertrophy

We examined whether a mixed lactate and caffeine compound (LC) could effectively elicit proliferation and differentiation of satellite cells or activate anabolic signals in skeletal muscles. We cultured C2C12 cells with either lactate or LC for 6 h. We found that lactate significantly increased myogenin and follistatin protein levels and phosphorylation of P70S6K while decreasing the levels of myostatin relative to the control. LC significantly increased protein levels of Pax7, MyoD, and Ki67 in addition to myogenin, relative to control. LC also significantly increased follistatin expression relative to control and stimulated phosphorylation of mTOR and P70S6K. In an in vivo study, male F344/DuCrlCrlj rats were assigned to control (Sed, n = 10), exercise (Ex, n = 12), and LC supplementation (LCEx, n = 13) groups. LC was orally administered daily. The LCEx and Ex groups were exercised on a treadmill, running for 30 min at low intensity every other day for 4 wk. The LCEx group experienced a significant increase in the mass of the gastrocnemius (GA) and tibialis anterior (TA) relative to both the Sed and Ex groups. Furthermore, the LCEx group showed a significant increase in the total DNA content of TA compared with the Sed group. The LCEx group experienced a significant increase in myogenin and follistatin expression of GA relative to the Ex group. These results suggest that administration of LC can effectively increase muscle mass concomitant with elevated numbers of myonuclei, even with low-intensity exercise training, via activated satellite cells and anabolic signals.

Modest hypoxia significantly reduces triglyceride content and lipid droplet size in 3T3-L1 adipocytes

BACKGROUND A previous study has demonstrated that endurance training under hypoxia results in a greater reduction in body fat mass compared to exercise under normoxia. However, the cellular and molecular mechanisms that underlie this hypoxia-mediated reduction in fat mass remain uncertain. Here, we examine the effects of modest hypoxia on adipocyte function. METHODS Differentiated 3T3-L1 adipocytes were incubated at 5% O2 for 1 week (long-term hypoxia, HL) or one day (short-term hypoxia, HS) and compared with a normoxia control (NC). RESULTS HL, but not HS, resulted in a significant reduction in lipid droplet size and triglyceride content (by 50%) compared to NC (p<0.01). As estimated by glycerol release, isoproterenol-induced lipolysis was significantly lowered by hypoxia, whereas the release of free fatty acids under the basal condition was prominently enhanced with HL compared to NC or HS (p<0.01). Lipolysis-associated proteins, such as perilipin 1 and hormone-sensitive lipase, were unchanged, whereas adipose triglyceride lipase and its activator protein CGI-58 were decreased with HL in comparison to NC. Interestingly, such lipogenic proteins as fatty acid synthase, lipin-1, and peroxisome proliferator-activated receptor gamma were decreased. Furthermore, the uptake of glucose, the major precursor of 3-glycerol phosphate for triglyceride synthesis, was significantly reduced in HL compared to NC or HS (p<0.01). CONCLUSION We conclude that hypoxia has a direct impact on reducing the triglyceride content and lipid droplet size via decreased glucose uptake and lipogenic protein expression and increased basal lipolysis. Such an hypoxia-induced decrease in lipogenesis may be an attractive therapeutic target against lipid-associated metabolic diseases.

Acute resistance exercise-induced IGF1 expression and subsequent GLUT4 translocation

Acute aerobic exercise (AE) is a major physiological stimulus for skeletal muscle glucose uptake through activation of 5′ AMP‐activated protein kinase (AMPK). However, the regulation of glucose uptake by acute resistance exercise (RE) remains unclear. To investigate the intracellular regulation of glucose uptake after acute RE versus acute AE, male Sprague–Dawley rats were divided into three groups: RE, AE, or nonexercise control. After fasting for 12 h overnight, the right gastrocnemius muscle in the RE group was exercised at maximum isometric contraction via percutaneous electrical stimulation (3 × 10 sec, 5 sets). The AE group ran on a treadmill (25 m/min, 60 min). Muscle samples were taken 0, 1, and 3 h after completion of the exercises. AMPK, Ca2+/calmodulin‐dependent protein kinase II, and TBC1D1 phosphorylation were increased immediately after both forms of exercise and returned to baseline levels by 3 h. Muscle IGF1 expression was increased by RE but not AE, and maintained until 3 h after RE. Additionally, Akt and AS160 phosphorylation were sustained for 3 h after RE, whereas they returned to baseline levels by 3 h after AE. Similarly, GLUT4 translocation remained elevated 3 h after RE, although it returned to the baseline level by 3 h after AE. Overall, this study showed that AMPK/TBC1D1 and IGF1/Akt/AS160 signaling were enhanced by acute RE, and that GLUT4 translocation after acute RE was more prolonged than after acute AE. These results suggest that acute RE‐induced increases in intramuscular IGF1 expression might be a distinct regulator of GLUT4 translocation.

Effect of resistance exercise under conditions of reduced blood insulin on AMPKα Ser485/491 inhibitory phosphorylation and AMPK pathway activation

Insulin stimulates skeletal muscle glucose uptake via activation of the protein kinase B/Akt (Akt) pathway. Recent studies suggest that insulin downregulates AMP-activated protein kinase (AMPK) activity via Ser485/491 phosphorylation of the AMPK α-subunit. Thus lower blood insulin concentrations may induce AMPK signal activation. Acute exercise is one method to stimulate AMPK activation; however, no study has examined the relationship between blood insulin levels and acute resistance exercise-induced AMPK pathway activation. Based on previous findings, we hypothesized that the acute resistance exercise-induced AMPK pathway activation would be augmented by disruptions in insulin secretion through a decrease in AMPKα Ser485/491 inhibitory phosphorylation. To test the hypothesis, 10-wk-old male Sprague-Dawley rats were administered the toxin streptozotocin (STZ; 55 mg/kg) to destroy the insulin secreting β-cells. Three days postinjection, the right gastrocnemius muscle from STZ and control rats was subjected to resistance exercise by percutaneous electrical stimulation. Animals were killed 0, 1, or 3 h later; activation of the Akt/AMPK and downstream pathways in the muscle tissue was analyzed by Western blotting and real-time PCR. Notably, STZ rats showed a significant decrease in basal Akt and AMPKα Ser485/491 phosphorylation, but substantial exercise-induced increases in both AMPKα Thr172 and acetyl-CoA carboxylase (ACC) Ser79 phosphorylation were observed. Although no significant impact on resistance exercise-induced Akt pathway activation or glucose uptake was found, resistance exercise-induced peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1 α (PGC-1α) gene expression was augmented by STZ treatment. Collectively, these data suggest that circulating insulin levels may regulate acute resistance exercise-induced AMPK pathway activation and AMPK-dependent gene expression relating to basal AMPKα Ser485/491 phosphorylation.

Acute bout of exercise induced prolonged muscle glucose transporter-4 translocation and delayed counter-regulatory hormone response in type 1 diabetes

Previous studies have demonstrated that an acute bout of aerobic exercise induces a subsequent delayed onset of hypoglycemia among patients with type 1 diabetes. However, the mechanisms of exercise-induced hypoglycemia in type 1 diabetes are still unclear. Streptozotocin (STZ) was injected to 6-week-old male Wistar rats, and three days after STZ injection, animals were randomly assigned into 2 groups: STZ with insulin only (STZ) and STZ with insulin and exercise (STZ+EX). Normal Wistar rats with exercise were used as control (CON+EX). Insulin was intraperitoneally injected (0.5 U/kg) to both STZ groups (−0.5 h), and a bout of aerobic exercise (15 m/min for 30 min) was conducted at euglycemic conditions (0 h). Blood was collected at 0, 1, 3, and 5 h after exercise from the carotid artery. While the blood glucose level was stable during the post-exercise period (0–5 h) in the STZ and CON+EX groups, it decreased significantly only in the STZ+EX group at 3 h. Plasma glucagon, adrenalin, and noradrenalin levels significantly increased at 1 h in the STZ group, whereas significant hormonal responses were observed at 5 h in the STZ+EX group. In skeletal muscle glucose metabolism-related pathway, the level of glucose transporter-4 (GLUT-4) translocation was significantly higher at 1 h in the CON and STZ groups. However, in the STZ+EX group, these activations were maintained by 5 h, indicating a sustained glucose metabolism in the STZ+EX group. A single bout of aerobic exercise induced a delayed onset of hypoglycemia in STZ-treated rats. A prolonged enhancement of GLUT-4 translocation and delayed counter-regulatory hormone responses may have contributed to the induction of hypoglycemia.

Exercise training increases CISD family protein expression in murine skeletal muscle and white adipose tissue

Mitochondrial function in skeletal muscle and white adipose tissue (WAT) declines with aging and the progression of type 2 diabetes and insulin resistance. Although exercise increases mitochondrial biogenesis and function in both tissues, the molecular mechanisms are not fully understood. CDGSH iron sulfur domain-containing proteins (CISDs) are a novel family of proteins that regulate mitochondrial activity and biogenesis. However, the relationship between exercise and CISD expression is unclear. We addressed this in the present study by examining changes in the expression of CISDs and mitochondrial proteins in skeletal muscle and WAT of mice subjected to chronic exercise training. Mice were randomly assigned to either the sedentary or exercise group and were housed for 4 weeks in a standard cage without or with a running wheel, respectively. CISD and mitochondrial protein levels in the plantaris and soleus muscles and epididymal WAT were evaluated by western blotting. Chronic exercise increased CISD1 and CISD2 as well as mitochondrial protein expression in plantaris muscle and WAT but not soleus muscle. Moreover, this exercise-induced adaptation was strongly correlated with mitochondrial protein expression. Thus, mitochondrial biogenesis induced by chronic exercise coincides with the expression of CISDs in specific tissues, which may be critical for the maintenance of mitochondrial integrity.

Resistance training recovers attenuated APPL1 expression and improves insulin-induced Akt signal activation in skeletal muscle of type 2 diabetic rats

Adapter protein containing Pleckstrin homology (PH) domain, phosphotyrosine-binding (PTB) domain, and leucine zipper motif 1 (APPL1) has been reported as a positive regulator of insulin-stimulated Akt activation. The expression of APPL1 is reduced in skeletal muscles of type 2 diabetic (T2D) animals, implying that APPL1 may be an important factor affecting insulin sensitivity. However, the regulation of APPL1 expression and the physiological interventions modulating these effects are unclear. Accordingly, we first confirmed that APPL1 expression and insulin-induced Akt phosphorylation were significantly attenuated in skeletal muscles of T2D rats. Additionally, we found that APPL1 expression levels were significantly correlated with fasting blood glucose levels. Next, we identified important signals involved in the expression of APPL1. APPL1 mRNA expression increased upon AMP-activated protein kinase, calcium, p38 mitogen-activated protein kinase, and insulin-like growth factor-1 signal activation. Moreover, acute resistance exercise in vivo significantly activated these signaling pathways. Finally, through in vivo experiments, we found that chronic resistance training (RT) increased APPL1 expression and activated insulin-induced Akt signaling in skeletal muscles of rats with T2D. Furthermore, variations in APPL1 expression (i.e., the difference between control and RT muscles) significantly correlated with variations in insulin-stimulated Akt phosphorylation under the same conditions. Therefore, chronic RT recovered attenuated APPL1 expression and improved insulin-stimulated Akt phosphorylation in skeletal muscles of T2D rats. Accordingly, APPL1 may be a key regulator of insulin resistance in skeletal muscle, and RT may be an important physiological treatment increasing APPL1 expression, which is attenuated in T2D.