FOXO1, a member of the FOXO forkhead type transcription factors, is markedly up-regulated in skeletal muscle in energy-deprived states such as fasting and severe diabetes, but its functions in skeletal muscle have remained poorly understood. In this study, we created transgenic mice specifically overexpressing FOXO1 in skeletal muscle. These mice weighed less than the wildtype control mice, had a reduced skeletal muscle mass, and the muscle was paler in color. Microarray analysis revealed that the expression of many genes related to the structural proteins of type I muscles (slow twitch, red muscle) was decreased. Histological analyses showed a marked decrease in size of both type I and type II fibers and a significant decrease in the number of type I fibers in the skeletal muscle of FOXO1 mice. Enhanced gene expression of a lysosomal proteinase, cathepsin L, which is known to be up-regulated during skeletal muscle atrophy, suggested increased protein degradation in the skeletal muscle of FOXO1 mice. Running wheel activity (spontaneous locomotive activity) was significantly reduced in FOXO1 mice compared with control mice. Moreover, the FOXO1 mice showed impaired glycemic control after oral glucose and intraperitoneal insulin administration. These results suggest that FOXO1 negatively regulates skeletal muscle mass and type I fiber gene expression and leads to impaired skeletal muscle function. Activation of FOXO1 may be involved in the pathogenesis of sarcopenia, the age-related decline in muscle mass in humans, which leads to obesity and diabetes.

Intracellular neutral lipid storage droplets are essential organelles of eukaryotic cells, yet little is known about the proteins at their surfaces or about the amino acid sequences that target proteins to these storage droplets. The mammalian proteins Perilipin, ADRP, and TIP47 share extensive amino acid sequence similarity, suggesting a common function. However, while Perilipin and ADRP localize exclusively to neutral lipid storage droplets, an association of TIP47 with intracellular lipid droplets has been controversial. We now show that GFP-tagged TIP47 co-localizes with isolated intracellular lipid droplets. We have also detected a close juxtaposition of TIP47 with the surfaces of lipid storage droplets using antibodies that specifically recognize TIP47, further indicating that TIP47 associates with intracellular lipid storage droplets. Finally, we show that related proteins from species as diverse as Drosophila and Dictyostelium can also target mammalian or Drosophila lipid droplet surfaces in vivo. Thus, sequence and/or structural elements within this evolutionarily ancient protein family are necessary and sufficient to direct association to heterologous intracellular lipid droplet surfaces, strongly indicating that they have a common function for lipid deposition and/or mobilization.

Adrenergic receptor (AR) activation increases expression of peroxisome proliferator-activated receptor (PPAR)-gamma coactivator 1alpha (PGC-1alpha) mRNA, which may promote mitochondrial biogenesis in skeletal muscles. An AR-activated increase in PGC-1alpha mRNA was observed in exercise. PGC-1alpha mRNA is considered a single transcript (PGC-1alpha-a); however, a transcript search of PGC-1alpha in expressed sequence tag libraries revealed that two novel isoforms of PGC-1alpha mRNA, named PGC-1alpha-b and PGC-1alpha-c, were expressed in mice tissues. Compared with PGC-1alpha-a mRNA (a previously described isoform), PGC-1alpha-b or PGC-1alpha-c mRNA was transcribed by a different exon 1 of the PGC-1alpha gene and produced slightly smaller-sized proteins. PGC-1alpha-b or PGC-1alpha-c protein was functional; both isoforms possessed transcriptional activity and could coactivate PPARs, similar to those in PGC-1alpha-a in vitro. Transgenic mice overexpressing PGC-1alpha-b or PGC-1alpha-c in skeletal muscles showed increased gene expression related to mitochondrial biogenesis and fatty acid oxidation. In C57BL/6J mice, injection of the beta2-AR agonist clenbuterol increased PGC-1alpha-b and PGC-1alpha-c mRNA expression more than 350-fold, but not PGC-1alpha-a, in skeletal muscle. A single bout of exercise also increased PGC-1alpha-b and PGC-1alpha-c mRNAs, but not PGC-1alpha-a, in skeletal muscles. The increases in skeletal muscles in response to exercise were inhibited by pretreatment with the beta2-AR-specific inhibitor ICI 118,551. However, in liver, fasting increased PGC-1alpha-a mRNA, but not PGC-1alpha-b and PGC-1alpha-c mRNAs. These data indicate that AR activation is a major mechanism of an increase in PGC-1alpha expression in skeletal muscles, and the increase in PGC-1alpha mRNAs was isoform specific.

A single bout of exercise increases expression of peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha mRNA, which may promote mitochondrial biogenesis in skeletal muscle. In brown adipose tissue, cold exposure up-regulates PGC-1alpha expression via adrenergic receptor (AR) activation. Because exercise also activates the sympathetic nervous system, we examined whether exercise-induced increase in PGC-1alpha mRNA expression in skeletal muscle was mediated via AR activation. In C57BL/6J mice, injection of the beta2-AR agonist clenbuterol, but not alpha-, beta1-, or beta3-AR agonists, increased PGC-1alpha mRNA expression more than 30-fold in skeletal muscle. The clenbuterol-induced increase in PGC-1alpha mRNA expression in mice was inhibited by pretreatment with the beta-AR antagonist propranolol. In ex vivo experiments, direct exposure of rat epitrochlearis to beta2-AR agonist, but not alpha-, beta1-, and beta3-AR agonist, led to an increase in levels of PGC-1alpha mRNA. Injection of beta2-AR agonist did not increase PGC-1alpha mRNA expression in beta1-, beta2-, and beta3-AR knockout mice (beta-less mice). PGC-1alpha mRNA in gastrocnemius was increased 3.5-fold in response to running on a treadmill for 45 min. The exercise-induced increase in PGC-1alpha mRNA was inhibited by approximately 70% by propranolol or the beta2-AR-specific inhibitor ICI 118,551. The exercise-induced increase in PGC-1alpha mRNA in beta-less mice was also 36% lower than that in wild-type mice. These data indicate that up-regulation of PGC-1alpha expression in skeletal muscle by exercise is mediated, at least in part, by beta-ARs activation. Among ARs, beta2-AR may mediate an increase in PGC-1alpha by exercise.

Green tea contains various antioxidative flavan-3ols (tea catechins), such as (-)-epigallocatechin gallate (EGCg, the major catechin), which exert potent inhibitory effects on LDL oxidation in vitro and ex vivo in humans. In this study, the antiatherogenic effects of tea catechins were examined in atherosclerosis-susceptible C57BL/6J, apoprotein (apo)E-deficient mice. Male apoE-deficient mice (10 wk old) were fed an atherogenic diet for 14 wk; during that time, one group (tea) was supplied drinking water supplemented with green tea extract (0.8 g/L), and another group (control) was offered the vehicle only. The tea extract consisted of the following (g/100 g): EGCg, 58.4; (-)-epigallocatechin (EGC), 11.7; (-)-epicatechin (EC), 6.6; (-)-gallocatechingallate (GCg), 1.6; (-)-epicatechin gallate (ECg), 0.5; and caffeine, 0.4. The estimated actual intake of tea catechin was 1.7 mg/(d. mouse). Tea ingestion did not influence plasma cholesterol or triglyceride concentrations. Plasma lipid peroxides were reduced in the tea group at wk 8, suggesting that the in vivo oxidative state is improved by tea ingestion. Atheromatous areas in the aorta from the arch to the femoral bifurcation and aortic weights were both significantly attenuated by 23% in the tea group compared with the control group. Aortic cholesterol and triglyceride contents were 27 and 50% lower, respectively, in the tea group than in the control group. These results suggest that chronic ingestion of tea extract prevents the development of atherosclerosis without changing the plasma lipid level in apoE-deficient mice, probably through the potent antioxidative activity of the tea.