Gregory D. Cartee

Aging skeletal muscle: response to exercise.

The mass of many weight-bearing muscles declines in old rats, secondary to the atrophy of fibers, particularly of type IIb, with relatively little loss of muscle fibers during most of the adult life span. In humans, muscle atrophy is the result of a combination of progressive fiber loss and fiber atrophy. In both species, the proportion of histochemically determined fiber types is relatively stable across the adult portion of the life span. The loss of strength in old age is predominantly accounted for by reduced muscle mass in humans and rats. Resistance training leads to increased muscle mass and strength in old humans and rats, primarily by increasing fiber CSA. Muscle capillarity is unchanged in old rats but decreases in old dogs. Apparently, capillarity declines in truly sedentary older people. Endurance training enhances capillarity, and old rats and humans can attain levels of capillarity comparable to their active young counterparts, even when performing considerably less exercise. Blood flow during contractile activity is reduced in male rats and humans but not in old female rats or dogs. Oxidative capacity declines in many muscles of sedentary old rats and humans. With endurance training, old individuals from both species attain levels of muscle oxidative capacity quite similar to those in identically training young individuals. Muscle insulin-stimulated glucose transport is enhanced in rats after a bout of exercise, regardless of age. Endurance training elevates muscle GLUT-4 levels in young and middle-aged, but not old, rats, perhaps because the old rats trained at slower treadmill speeds. Middle-aged (47-62 yr) men and women can substantially increase muscle GLUT-4 with relatively brief (12-14 wk) endurance training; older humans (> 70-80 yr) have not been studied. Endurance training leads to reduced LDH activity without altering PFK or phosphorylase in old rats and humans. Muscle glycogen depletion, CP depletion, and lactate accumulation during contractile activity are exaggerated in old rats, apparently secondary to reduced muscle oxidative capacity and blood flow. Resting muscle glycogen concentration is diminished in older humans, probably in part because of a more sedentary lifestyle. Although several months of endurance training raises muscle glycogen concentration in older people, it does not restore it to youthful levels. Endurance training can greatly improve endurance in old age, at least in part by the same mechanism originally described in youth, i.e., an increase in muscle oxidative capacity, which contributes to reduced glycogen depletion.(ABSTRACT TRUNCATED AT 400 WORDS)

Calorie restriction increases cell surface GLUT-4 in insulin-stimulated skeletal muscle

Reduced calorie intake [calorie restriction (CR); 60% of ad libitum (AL)] leads to enhanced glucose transport without altering total GLUT-4 glucose transporter abundance in skeletal muscle. Therefore, we tested the hypothesis that CR (20 days) alters the subcellular distribution of GLUT-4. Cell surface GLUT-4 content was higher in insulin-stimulated epitrochlearis muscles from CR vs. AL rats. The magnitude of this increase was similar to the CR-induced increase in glucose transport, and GLUT-4 activity (glucose transport rate divided by cell surface GLUT-4) was unaffected by diet. The CR effect was specific to the insulin-mediated pathway, as evidenced by the observations that basal glucose transport and cell surface GLUT-4 content, as well as hypoxia-stimulated glucose transport, were unchanged by diet. CR did not alter insulins stimulation of insulin receptor substrate (IRS)-1-associated phosphatidylinositol 3-kinase (PI3K) activity. Muscle abundance of IRS-2 and p85 subunit of PI3K were unaltered by diet, but IRS-1 content was lower in CR vs. AL. These data demonstrate that, despite IRS-1-PI3K activity similar to AL, CR specifically increases insulins activation of glucose transport by enhancing the steady-state proportion of GLUT-4 residing on the cell surface.

Interaction Between BTBR and C57BL/6J Genomes Produces an Insulin Resistance Syndrome in (BTBR × C57BL/6J) F1 Mice

Insulin resistance is a common syndrome that often precedes the development of noninsulin-dependent diabetes mellitus (NIDDM). Both diet and genetic factors are associated with insulin resistance. BTBR and C57BL/6J (B6) mice have normal insulin responsiveness and normal fasting plasma insulin levels. However, a cross between these two strains yielded male offspring with severe insulin resistance. Surprisingly, on a basal diet (6.5% fat), the insulin resistance was not associated with fasting hyperinsulinemia. However, a 15% fat diet produced significant hyperinsulinemia in the male mice (twofold at 10 weeks; P < .05). At 10 weeks of age, visceral fat contributed approximately 4.3% of the total body weight in the males versus 1.8% in females. In the males, levels of plasma triacylglycerol and total cholesterol increased 40% and 30%, respectively, compared to females. Plasma free fatty acid concentrations were unchanged. Oral glucose tolerance tests revealed significant levels of hyperglycemia and hyperinsulinemia 15 to 90 minutes after oral glucose administration in the male mice. This was particularly dramatic in males on a 15% fat diet. Glucose transport was examined in skeletal muscles in (BTBR x B6)F1 mice. In the nonhyperinsulinemic animals (females), insulin stimulated 2-deoxyglucose transport 3.5-fold in the soleus and 2.8-fold in the extensor digitorum longus muscles. By contrast, glucose transport was not stimulated in the hyperinsulinemic male mice. Hypoxia stimulates glucose transport through an insulin-independent mechanism. This is known to involve the translocation of GLUT4 from an intracellular pool to the plasma membrane. In the insulin-resistant male mice, hypoxia induced glucose transport as effectively as it did in the insulin-responsive mice. Thus, defective glucose transport in the (BTBR x B6)F1 mice is specific for insulin-stimulated glucose transport. This is similar to what has been observed in muscles taken from obese NIDDM patients. These animals represent an excellent genetic model for studying insulin resistance and investigating the transition from insulin resistance in the absence of hyperinsulinemia to insulin resistance with hyperinsulinemia.

Influence of age on skeletal muscle glucose transport and glycogen metabolism

Age-related alterations in skeletal muscle carbohydrate metabolism can influence both health and performance. Exercising muscle glycogenolysis is accelerated in old, male rats compared with young animals, perhaps secondary to the age-related reduction in muscle oxidative capacity and blood flow during contractile activity. Muscle oxidative capacity and blood flow during exercise are also reduced in untrained older humans. Endurance training enhances muscle oxidative capacity and promotes muscle glycogen sparing during exercise by young and old rats. Resting muscle glycogen concentration is unchanged in old rats, but considerably reduced in untrained, older humans. Exercise training increases the muscle glycogen levels of older people. The concentration of GLUT-4 glucose transporter protein declines in some muscles of rats during growth and development, but remains stable thereafter. Exercise training can elevate the muscle GLUT-4 protein levels of both young and old humans. On the other hand, exercise training has been shown to increase the GLUT-4 values of adult, but not old rats. After one bout of exercise, muscle sensitivity for insulin-stimulated glucose transport is improved in young and old rats. These findings indicate that several age-related changes in muscle carbohydrate metabolism can be minimized by acute or chronic exercise.

RNA interference-mediated reduction in GLUT1 inhibits serum-induced glucose transport in primary human skeletal muscle cells.

Using RNA interference (RNAi), we specifically down-regulate protein expression in differentiated human skeletal myotube cultures. Serum stimulation of myotubes increases glucose uptake. Using a sensitive photolabeling technique, we demonstrate that this increase in glucose uptake is accompanied by increased cell-surface content of glucose transporter (GLUT) 1. Using RNAi, we specifically reduce GLUT1 mRNA and protein expression, leading to inhibition of serum-mediated increase in glucose transport. Thus, we demonstrate the utility of RNAi in a primary human differentiated cell system, and apply this methodology to demonstrate that serum-mediated increase in glucose transport in human skeletal muscle cells is dependent on GLUT1.

Comparison of the effects of 20 days and 15 months of calorie restriction on male Fischer 344 rats.

The aim of this study was to compare, in 19-month-old male Fischer 344 rats, the influence of brief (20 days) and prolonged (∼15 months) calorie restriction (CR; consuming ∼60% of ad libitum, AL, intake) on circulating levels of glucose, insulin, C-peptide, and free fatty acids (FFA); age-matched AL rats were also studied. In the prolonged CR group, there was an ∼85% decline in fat pad masses (epididymal and retroperitoneal) compared to AL and brief CR rats (these latter groups did not differ significantly). Compared to AL levels, glucose was 15% lower with prolonged CR (p <0.05) while the brief CR values tended to be lower (10%) than AL; the CR groups did not differ significantly. Plasma FFA levels were significantly (p <0.05) greater (85–106%) in the brief CR group compared to each of the other groups. Plasma insulin concentrations for the CR groups were lower (p<0.05; ∼50–60%) than AL levels. Plasma concentrations of C-peptide (an indicator of insulin secretion) were also lower for each CR group vs AL levels, and a high correlation was found between plasma insulin and C-peptide concentrations (r2 =0.90; p<0.001). The C-peptide/ insulin ratios for the CR groups were similar, and the value of each CR group exceeded that for the AL rats. These results demonstrate that: the CR-induced reduction in plasma insulin is attributable in large part to reduced insulin secretion; these decreases in insulin secretion and concentration are essentially undiminished when brief CR is initiated rather late in life, and the reductions are independent of substantial reductions in body fat.

Of mice and men: filling gaps in the TBC1D1 story

Skeletal muscle is the major tissue for the increased glucose disposal caused by insulin or exercise. Each stimulus elevates GLUT4 glucose transporter translocation to skeletal muscles cell surface membranes, but distinct signalling pathways lead to this common outcome. Insulins proximal signalling events include activation of the insulin receptor, phosphatidylinositol 3-kinase, and Akt2. The signalling events necessary for exercise-induced glucose transport may involve increased cytosolic calcium, AMP-activated protein kinase and other mechanisms. The prevalence of obesity, insulin resistance and diabetes provides motivation for understanding the exercise pathway in humans because in many insulin resistant conditions, exercise-stimulated glucose transport is normal, presenting an attractive therapeutic target. Recently, two related Rab-GTPase activating proteins (GAPs) known as TBC1D1 and TBC1D4 (also called Akt substrate of 160 kDa or AS160) were recognized to potentially link the proximal signalling of insulin and/or exercise with GLUT4. TBC1D4s phosphorylation in response to insulin was discovered by Gustav Lienhards group using 3T3-L1 adipocytes and was subsequently found in rat skeletal muscle with insulin or contraction (Bruss et al. 2005). Reviewing TBC1D4s relationship with GLUT4 is helpful before considering TBC1D1. Insulins activation of Akt2 causes TBC1D4 phosphorylation on multiple Akt phosphomotifs, thereby inhibiting TBC1D4s activation of Rab-GTPase proteins associated with GLUT4 vesicles and/or causing TBC1D4s release from GLUT4 vesicles. Insulin-induced phosphorylation of TBC1D4 on key insulin-responsive motifs enhances TBC1D4s association with 14-3-3 proteins, which may regulate GLUT4 vesicle traffic. Akt2 is not essential for exercise-stimulated glucose transport, but mutation of four phosphomotifs on TBC1D4 caused a small reduction in contraction-stimulated glucose uptake in mouse muscle. A mutation in TBC1D4s calmodulin binding domain (CBD) also caused a modest decline in glucose uptake with contraction, but not with insulin. Simultaneous mutation of the CBD and phosphomotifs did not reduce contraction-stimulated glucose uptake below the values found with either mutation alone. These results suggest a modest role for TBC1D4 in contraction-stimulated glucose uptake, but TBC1D4-independent mechanisms (potentially involving TBC1D1) are likely to be essential for most of the contractions effect. TBC1D1 and TBC1D4 have significant sequence similarity, including a GAP domain and a CBD. The sequence surrounding a key Akt phospho-site of TBC1D4 (T642) is nearly identical to the sequence surrounding TBC1D1s T596, but TBC1D4 includes a greater number of predicted Akt phosphomotifs. Although AMPK can phosphorylate both proteins, TBC1D1 includes an important AMPK phosphomotif (Ser237) that TBC1D4 lacks. In L6 cells, Ser237 phosphorylation of TBC1D1 is increased with AMPK activation, but not by insulin. Preventing the increase in Ser237 phosphorylation by expressing mutated TBC1D1 in HEK-293 cells blocks the AMPK-associated increase in 14-3-3 binding by TBC1D1 (Chen et al. 2008). Insulin or electrically simulated contractions enhances TBC1D1 phosphorylation on various sites in rodent muscle, but only contraction elevates phosphorylation on Ser237 (Funai et al. 2009). Contraction-stimulated (but not insulin-stimulated) glucose uptake was partially reduced in muscle of mice expressing TBC1D1 mutated on four phosphomotifs (including Ser231, homologous to human Ser237) (An et al. 2010). Evidence from cells and rodent muscle links TBC1D1 Ser237 phosphorylation to both 14-3-3 binding and contraction-stimulated (but not insulin-stimulated) glucose transport. But is this relevant to humans? The results of the study by Frosig et al. (2010) in this issue of The Journal of Physiology fill an important gap in the TBC1D1 story by demonstrating that in vivo exercise (cycle ergometery) by humans can increase the phosphorylation of skeletal muscle TBC1D1 on the key Ser237 site. The increased Ser237 phosphorylation occurred with each of three protocols which involved a nearly 3-fold range of work-rates (222–658 W) and 40-fold range of duration (0.5–20 min). Furthermore, TBC1D1s capacity for 14-3-3 binding was increased in muscle by each exercise protocol. The exercise effects on Ser237 phosphorylation and 14-3-3 binding were rapid and sustained, as would also be expected for exercise-stimulated glucose transport. What is the mechanism for increased phosphorylation of Ser237-TBC1D1 after exercise? There is not a straightforward experimental approach for directly answering this question in humans undergoing in vivo exercise. Accordingly, Frosig et al. used mice to probe the specific roles of AMPK isoforms in contraction-induced Ser237 phosphorylation. Studying α1- and α2-AMPK knockout mice and wild-type controls, they demonstrated that contraction-stimulated Ser237 phosphorylation was unaltered in mice deficient in α1, but greatly diminished in muscles from mice lacking α2. Earlier research demonstrated that α2-AMPK knockout mice compared to wild-type controls have normal contraction-stimulated glucose transport, but it is uncertain if the residual contraction effect on Ser237 in α2-knockout mice plays a role in contraction-mediated glucose transport. It is also unknown if the residual Ser237 phosphorylation in α2-knockout mice is attributable to a compensatory increase in α1-AMPK activity or to another kinase. A recent publication found that electrically stimulated muscle contractions activated an AMPK-related kinase known as sucrose non-fermenting AMPK-related kinase (SNARK), and that a mutation of SNARK that attenuated contraction-stimulated SNARK activity was accompanied by decreased contraction-stimulated glucose transport (Koh et al. 2010). Furthermore, exercise by humans similar to the 2 and 20 min protocols used by Frosig et al. also activated skeletal muscle SNARK. However, the effect of SNARK on TBC1D1 Ser237 phosphorylation remains to be assessed. What will future chapters of the TBC1D1 story reveal? Do α2-AMPK and/or SNARK regulate Ser237 phosphorylation in human skeletal muscle? Is Ser237 phosphorylation necessary for exercise-induced glucose transport in human skeletal muscle? Does TBC1D1s CBD participate in exercise-stimulated glucose transport? Do exercise effects on TBC1D1 have functional roles other than increased glucose transport? The TBC1D1 story is far from finished.