Peroxisome proliferator-activated receptor γ coactivator 1α regulates downstream of tyrosine kinase-7 (Dok-7) expression important for neuromuscular junction formation.

The neuromuscular junction (NMJ)-formed between a motor nerve terminal and skeletal muscle fiber-plays an important role in muscle contraction and other muscle functions. Aging and neurodegeneration worsen NMJ formation and impair muscle function. Downstream of tyrosine kinase-7 (Dok-7), expressed in skeletal muscle fibers, is essential for the formation of NMJ. Exercise increases the expression of the transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) in skeletal muscles and restores NMJ formation. In this study, we used skeletal muscle-specific PGC1α knockout or overexpression mice to examine the role of PGC1α in regulating Dok-7 expression and NMJ formation. Our findings revealed that Dok-7 expression is regulated by PGC1α, and luciferase activity of the Dok-7 promoter is greatly increased by coexpressing PGC1α and estrogen receptor-related receptor α. Thus, we suggest PGC1α is involved in exercise-mediated restoration of NMJ formation.

Vitamin D Activates Various Gene Expressions, Including Lipid Metabolism, in C2C12 Cells.

Vitamin D is a fat-soluble molecule, well known for its role in regulating calcium homeostasis in bone. It has become increasingly clear that it also has important effects in many other organs, including the skeletal muscle. In order to gain insight into the role of vitamin D in the skeletal muscle, we performed microarray analysis using C2C12 myoblasts treated with 1,25-dihydroxyvitamin D (1,25(OH)2D), active form of vitamin D. We found multiple genes upregulated by 1,25(OH)2D. Some of them, i.e., vitamin D receptor (Vdr), diacylglycerol O-acyltransferase (Dgat1 and Dgat2, the rate limiting steps of triacylglycerol acylation), and vascular endothelial growth factor A (Vegfa), were previously reported to be upregulated by 1,25(OH)2D in C2C12 cells. RT-qPCR analysis confirmed increased mRNA levels of Rarres2, Dio2, Tgm2, Lpl, Mdfi, Igfbp3, Dgat1, Crabp2, Gadd45a, Vagfa, Dgat2, C3, Ldhb, Cebpa, Igfbp5, Mrc2, Vdr. Thus, many genes, including lipid metabolism genes as well as genes related to muscle functions, appear to be upregulated by 1,25(OH)2D in muscle cells.

The Steroidal Alkaloid Tomatidine and Tomatidine-Rich Tomato Leaf Extract Suppress the Human Gastric Cancer-Derived 85As2 Cells In Vitro and In Vivo via Modulation of Interferon-Stimulated Genes.

The steroidal alkaloid tomatidine is an aglycone of α-tomatine, which is abundant in tomato leaves and has several biological activities. Tomatidine has been reported to inhibit the growth of cultured cancer cells in vitro, but its anti-cancer activity in vivo and inhibitory effect against gastric cancer cells remain unknown. We investigated the efficacy of tomatidine using human gastric cancer-derived 85As2 cells and its tumor-bearing mouse model and evaluated the effect of tomatidine-rich tomato leaf extract (TRTLE) obtained from tomato leaves. In the tumor-bearing mouse model, tumor growth was significantly inhibited by feeding a diet containing tomatidine and TRTLE for 3 weeks. Tomatidine and TRTLE also inhibited the proliferation of cultured 85As2 cells. Microarray data of gene expression analysis in mouse tumors revealed that the expression levels of mRNAs belonging to the type I interferon signaling pathway were altered in the mice fed the diet containing tomatidine and TRTLE. Moreover, the knockdown of one of the type I interferon-stimulated genes (ISGs), interferon α-inducible protein 27 (IFI27), inhibited the proliferation of cultured 85As2 cells. This study demonstrates that tomatidine and TRTLE inhibit the tumor growth in vivo and the proliferation of human gastric cancer-derived 85As2 cells in vitro, which could be due to the downregulation of ISG expression.

FOXO1 cooperates with C/EBPδ and ATF4 to regulate skeletal muscle atrophy transcriptional program during fasting.

Catabolic conditions, such as starvation, inactivity, and cancer cachexia, induce Forkhead box O (FOXO) transcription factor(s) expression and severe muscle atrophy via the induction of ubiquitin-proteasome system-mediated muscle proteolysis, resulting in frailty and poor quality of life. Although FOXOs are clearly essential for the induction of muscle atrophy, it is unclear whether there are other factors involved in the FOXO-mediated transcriptional regulation. As such, we identified FOXO-CCAAT/enhancer-binding protein δ (C/EBPδ) signaling pathway as a novel proteolytic pathway. By comparing the gene expression profiles of FOXO1-transgenic (gain-of-function model) and FOXO1,3a,4-/- (loss-of-function model) mice, we identified several novel FOXO1-target genes in skeletal muscle including Redd1, Sestrin1, Castor2, Chac1, Depp1, Lat3, as well as C/EBPδ. During starvation, C/EBPδ abundance was increased in a FOXOs-dependent manner. Notably, knockdown of C/EBPδ prevented the induction of the ubiquitin-proteasome system and decrease of myofibers in FOXO1-activated myotubes. Conversely, C/EBPδ overexpression in primary myotubes induced myotube atrophy. Furthermore, we demonstrated that FOXO1 enhances the promoter activity of target genes in cooperation with C/EBPδ and ATF4. This research comprehensively identifies novel FOXO1 target genes in skeletal muscle and clarifies the pathophysiological role of FOXO1, a master regulator of skeletal muscle atrophy.

Effects of fenofibrate and its combination with lovastatin on the expression of genes involved in skeletal muscle atrophy, including FoxO1 and its targets.

Fibrates and statins have been widely used to reduce triglyceride and cholesterol levels, respectively. Besides its lipid-lowering effect, the side effect of muscle atrophy after fibrate administration to humans has been demonstrated in some studies. Combination therapy with fibrates and statins also increases the risk of rhabdomyolysis. FoxO1, a member of the FoxO forkhead type transcription factor family, is markedly upregulated in skeletal muscle in energy-deprived states and induces muscle atrophy via the expression of E3-ubiquitine ligases. In this study, we investigated the changes in FoxO1 and its targets in murine skeletal muscle with fenofibrate treatment. High doses of fenofibrate (greater than 0.5% (wt/wt)) over one week increased the expression of FoxO1 and its targets in the skeletal muscles of mice and decreased skeletal muscle weight. These fenofibrate-induced changes were diminished in the PPARα knockout mice. When the effect of combination treatment with fenofibrate and lovastatin was investigated, a significant increase in FoxO1 protein levels was observed despite the lack of deterioration of muscle atrophy. Collectively, our findings suggest that a high dose of fenofibrate over one week causes skeletal muscle atrophy via enhancement of FoxO1, and combination treatment with fenofibrate and lovastatin may further increase FoxO1 protein level.

Skeletal muscle-specific forkhead box protein-O1 overexpression suppresses atherosclerosis progression in apolipoprotein E-knockout mice.

Calorie restriction (CR) reportedly prevents atherosclerotic diseases. Furthermore, CR induces forkhead box protein-O1 (FOXO-1) expression in the skeletal muscle, altering the character of the skeletal muscle. We previously reported that the change in skeletal muscle character, induced by the overexpression of peroxisome proliferator-activated receptor γ coactivator-1α, suppresses atherosclerotic progression in an atherosclerotic apolipoprotein E-knockout (ApoE-KO) mouse model. Thus, we hypothesized that skeletal muscle alternation induced by FOXO-1 may also have an anti-atherosclerotic effect in ApoE-KO mice. In this study, we investigated whether skeletal muscle-specific FOXO-1 overexpression suppresses the progression of atherosclerosis in ApoE-KO mice. We generated ApoE-KO/FOXO-1 mice, in which an ApoE-KO mouse was crossbred with a mouse presenting skeletal muscle-specific FOXO-1 overexpression (FOXO-1Tg). The mice were sacrificed at 20 weeks of age, and atherosclerotic plaque area and protein expression in the plaque were measured. Additionally, we measured the tumor necrosis factor α (TNFα)- induced mRNA expression in human umbilical vein endothelial cells (HUVECs), using serum collected from the FOXO-1Tg mice. Accordingly, ApoE-KO/FOXO-1 mice showed a 65% reduced atherosclerotic plaque area when compared with the ApoE-KO mice, with concomitantly reduced vascular cell adhesion molecule-1 (VCAM-1) and macrophage infiltration. As compared to serum from wild-type mice, the serum collected from the FOXO-1Tg mice significantly suppressed the mRNA expression of VCAM-1, an atherosclerosis initiation factor, in TNFα-treated HUVECs. Therefore, these data suggest that skeletal muscle-specific FOXO-1 overexpression suppresses the progression of atherosclerosis in ApoE-KO mice. In part, the CR-induced anti-atherosclerotic effect could be attributed to FOXO-1 upregulation in the skeletal muscle.

Vitamin D and Sarcopenia: Potential of Vitamin D Supplementation in Sarcopenia Prevention and Treatment.

Skeletal muscle, the largest organ in the human body, accounting for approximately 40% of body weight, plays important roles in exercise and energy expenditure. In the elderly, there is often a progressive decline in skeletal muscle mass and function, a condition known as sarcopenia, which can lead to bedridden conditions, wheelchair confinement as well as reducing the quality of life (QOL). In developed countries with aging populations, the prevention and management of sarcopenia are important for the improvement of health and life expectancy in these populations. Recently, vitamin D, a fat-soluble vitamin, has been attracting attention due to its importance in sarcopenia. This review will focus on the effects of vitamin D deficiency and supplementation on sarcopenia.

FOXO1 suppresses PGC-1ß gene expression in skeletal muscles.

Peroxisome proliferator-activated receptor-gamma coactivator-1ß (PGC-1ß) is a transcriptional regulator whose increased expression activates energy expenditure-related genes in skeletal muscles. However, how PGC-1ß is regulated remains largely unclear. Here, we show that PGC-1ß gene expression is negatively correlated with the expression of a transcription factor, forkhead box protein O1 (FOXO1), whose expression is increased during muscle atrophy. In the skeletal muscles of FOXO1-overexpressing transgenic mice, PGC-1ß gene expression is decreased. Denervation or plaster cast-based unloading, as well as fasting, increases endogenous FOXO1 expression in skeletal muscles, with decreased PGC-1ß expression. In the skeletal muscles of FOXO1-knockout mice, the decrease in PGC-1ß expression caused by fasting was attenuated. Tamoxifen-inducible FOXO1 activation in C2C12 myoblasts causes a marked decrease of PGC-1ß expression. These findings together reveal that FOXO1 activation suppresses PGC-1ß expression. During atrophy with FOXO1 activation, decreased PGC-1ß may decrease energy expenditure and avoid wasting energy.

Health Promotion Effects of Soy Isoflavones.

Soybeans contain several physiologically active ingredients, such as soy phytosterol, soyasaponin, soy protein, and lecithin, and are therefore expected to express the functionalities of said ingredients. Among them, soy isoflavones have been studied in recent years for their various functions, including their obesity-preventing effect, blood glucose level reducing effect, osteoporosis and breast cancer risk reduction, and anti-oxidative effect, and several health promoting effects and disease preventing effects are expected. For example, it has been determined that soy isoflavones reduce body and fat weight in experiments in which mice were fed a diet containing soy isoflavones in studies on anti-obesity. Epidemiologic studies with humans have also shown that women who consume more soybeans have lower BMI than those who consume less. We previously found that soy isoflavones may have anti-obesity effects in myoblasts through the activation of transcriptional coactivator PGC-1ß, which increases energy expenditure. In recent studies, a decrease in blood glucose level due to soy isoflavone was seen in an experiment in which diabetic model mice were fed a diet containing soy isoflavone. It has also been suggested that soy isoflavone intake may increase bone mineral density in postmenopausal women and reduce the risk of breast cancer. This review focuses on the actions of soy isoflavones known to date, including their anti-obesity and anti-diabetic effects, bone loss preventing effects, and cancer risk reduction effects, and introduces reports on the health promotion and disease prevention effects of soy isoflavones.

Metabolomic analysis of C2C12 myoblasts induced by the transcription factor FOXO1.

The transcription factor FOXO1 is considered to play roles in the regulation of energy metabolism in various tissues. To determine the metabolic changes occurring due to FOXO1 activation, we analyzed the metabolic profile of C2C12 myoblasts expressing a FOXO1-estrogen receptor fusion protein using capillary electrophoresis with electrospray ionization time-of-flight mass spectrometry (CE-TOFMS). In FOXO1-activated cells, the metabolite levels during glycolysis are higher and the gene expression of pyruvate dehydrogenase kinase, an enzyme that inhibits glucose utilization, is increased. In addition, the metabolite levels of numerous amino acids are decreased, with increased gene expression of branched chain amino acid metabolism enzymes. Our results suggest that FOXO1 suppresses glucose utilization and promotes the use of proteins/amino acids as energy sources in muscle cells, potentially during starvation.

Genistein, daidzein, and resveratrols stimulate PGC-1ß-mediated gene expression.

PGC-1ß is a transcriptional co-activator of nuclear receptors such as the estrogen receptor-related receptor (ERR). Transgenic overexpression of PGC-1ß in mice increases energy expenditure and suppresses high-fat diet-induced obesity. In this study, we screened various food-derived and natural compounds using a reporter assay system to measure the transcriptional activity of PGC-1ß. Soy-derived isoflavones, genistein and daidzein, and several resveratrols activated PGC-1ß. Genistein, daidzein, and trans-oxyresveratrol activated ERR-responsive element-mediated reporter activity in the presence of PGC-1ß. Stable overexpression of PGC-1ß in C2C12 myoblasts increased the expression of medium-chain acyl-CoA dehydrogenase (MCAD), an important enzyme in fatty acid ß-oxidation. Genistein and daidzein increased MCAD mRNA levels and mitochondrial content in PGC-1ß-expressing C2C12 cells. These compounds activated ERR/PGC-1ß complex-mediated gene expression, and our findings may be a practical foundation for developing functional foods targeting obesity.

Vitamin D Attenuates FOXO1-Target Atrophy Gene Expression in C2C12 Muscle Cells.

Vitamin D is known to be effective for the prevention of muscle atrophy, such as age-related sarcopenia. However, vitamin D action in skeletal muscle tissue and muscle cells is largely unknown. We previously found that a transcription factor, FOXO1 gene expression, was induced in various muscle atrophy conditions causing muscle atrophy by upregulating atrophy-related genes, including atrogin 1 (ubiquitin ligase) and cathepsin L (lysosomal proteinase). In this study, we found that vitamin D inhibited FOXO1-mediated transcriptional activity in a reporter gene assay. Moreover, vitamin D suppressed the glucocorticoid-induced gene expression of atrogin 1 and cathepsin L in C2C12 myoblasts. Thus, vitamin D may prevent muscle atrophy via the FOXO1-mediated pathway in muscle cells.

FOXO1 delays skeletal muscle regeneration and suppresses myoblast proliferation.

Unloading stress, such as bed rest, inhibits the regenerative potential of skeletal muscles; however, the underlying mechanisms remain largely unknown. FOXO1 expression, which induces the upregulated expression of the cell cycle inhibitors p57 and Gadd45α, is known to be increased in the skeletal muscle under unloading conditions. However, there is no report addressing FOXO1-induced inhibition of myoblast proliferation. Therefore, we induced muscle injury by cardiotoxin in transgenic mice overexpressing FOXO1 in the skeletal muscle (FOXO1-Tg mice) and observed regeneration delay in skeletal muscle mass and cross-sectional area in FOXO1-Tg mice. Increased p57 and Gadd45α mRNA levels, and decreased proliferation capacity were observed in C2C12 myoblasts expressing a tamoxifen-inducible active form of FOXO1. These results suggest that decreased proliferation capacity of myoblasts by FOXO1 disrupts skeletal muscle regeneration under FOXO1-increased conditions, such as unloading.

FOXO1 activates glutamine synthetase gene in mouse skeletal muscles through a region downstream of 3'-UTR: possible contribution to ammonia detoxification.

Skeletal muscle is a reservoir of energy in the form of protein, which is degraded under catabolic conditions, resulting in the formation of amino acids and ammonia as a byproduct. The expression of FOXO1, a forkhead-type transcription factor, increases during starvation and exercise. In agreement, transgenic FOXO1-Tg mice that overexpress FOXO1 in skeletal muscle exhibit muscle atrophy. The aim of this study was to examine the role of FOXO1 in amino acid metabolism. The mRNA and protein expressions of glutamine synthetase (GS) were increased in skeletal muscle of FOXO1-Tg mice. Fasting induced FOXO1 and GS expression in wild-type mice but hardly increased GS expression in muscle-specific FOXO1 knockout (FOXO1-KO) mice. Activation of FOXO1 also increased GS mRNA and protein expression in C2C12 myoblasts. Using a transient transfection reporter assay, we observed that FOXO1 activated the GS reporter construct. Mutation of a putative FOXO1-binding consensus sequence in the downstream genomic region of GS decreased basal and FOXO1-dependent reporter activity significantly. A chromatin immunoprecipitation assay showed that FOXO1 was recruited to the 3' region of GS in C2C12 myoblasts. These results suggest that FOXO1 directly upregulates GS expression. GS is considered to mediate ammonia clearance in skeletal muscle. In agreement, an intravenous ammonia challenge increased blood ammonia concentrations to a twofold higher level in FOXO1-KO than in wild-type mice, demonstrating that the capacity for ammonia disposal correlated inversely with the expression of GS in muscle. These data indicate that FOXO1 plays a role in amino acid metabolism during protein degradation in skeletal muscle.

Analysis of DNA methylation change induced by Dnmt3b in mouse hepatocytes.

DNA methylation is a key epigenetic contributor to gene regulation in mammals. We have recently found that in the mouse liver, the promoter region of glycerol-3-phosphate acyltransferase 1, a rate-limiting enzyme of de novo lipogenesis, is regulated by DNA methylation, which is mediated by Dnmt3b, an enzyme required for the initiation of de novo methylation. In this study, using primary cultures of mouse hepatocytes with adenoviral overexpression of Dnmt3b, we characterized Dnmt3b-dependent DNA methylation on a genome-wide basis. A genome-wide DNA methylation analysis, called microarray-based integrated analysis of methylation by isoschizomers, identified 108 genes with Dnmt3b dependent DNA methylation. In DNA expression array analysis, expression of some genes with Dnmt3b-dependent DNA methylation was suppressed. Studies with primary mouse hepatocytes overexpressing Dnmt3b or Dnmt3a revealed that many genes with Dnmt3b-dependent methylation are not methylated by Dnmt3a, whereas those methylated by Dnmt3a are mostly methylated by Dnmt3b. Bioinformatic analysis showed that the CANAGCTG and CCGGWNCSC (N denotes A, T, G, or C; W denotes A or T; and S denotes C or G) sequences are enriched in genes methylated by overexpression of Dnmt3b and Dnmt3a, respectively. We also observed a large number of genes with Dnmt3b-dependent DNA methylation in primary cultures of mouse hepatocytes with adenoviral overexpression of Dnmt3, suggesting that Dnmt3b is an important DNA methyltransferase in primary mouse hepatocytes, targets specific genes, and potentially plays a role in vivo.

Melanocortin 4 receptor-deficient mice as a novel mouse model of nonalcoholic steatohepatitis.

Obesity may be viewed as a state of chronic low-grade inflammation that participates in the development of the metabolic syndrome. Nonalcoholic steatohepatitis (NASH) is considered a hepatic phenotype of the metabolic syndrome and a high risk for progression to cirrhosis and hepatocellular carcinoma. Although the "two hit" hypothesis suggests involvement of excessive hepatic lipid accumulation and chronic inflammation, the molecular mechanisms underlying the development of NASH remain unclear, in part because of lack of appropriate animal models. Herein we report that melanocortin 4 receptor-deficient mice (MC4R-KO) develop steatohepatitis when fed a high-fat diet, which is associated with obesity, insulin resistance, and dyslipidemia. Histologic analysis reveals inflammatory cell infiltration, hepatocyte ballooning, and pericellular fibrosis in the liver in MC4R-KO mice. Of note, all of the MC4R-KO mice examined developed well-differentiated hepatocellular carcinoma after being fed a high-fat diet for 1 year. They also demonstrated enhanced adipose tissue inflammation, ie, increased macrophage infiltration and fibrotic changes, which may contribute to excessive lipid accumulation and enhanced fibrosis in the liver. Thus, MC4R-KO mice provide a novel mouse model of NASH with which to investigate the sequence of events that make up diet-induced hepatic steatosis, liver fibrosis, and hepatocellular carcinoma and to aid in understanding the pathogenesis of NASH, pursuing specific biomarkers, and evaluating potential therapeutic strategies.

Increased expression of macrophage-inducible C-type lectin in adipose tissue of obese mice and humans.

OBJECTIVE: We have provided evidence that saturated fatty acids, which are released from adipocytes via macrophage-induced adipocyte lipolysis, serve as a naturally occurring ligand for the Toll-like receptor (TLR) 4 complex in macrophages, thereby aggravating obesity-induced adipose tissue inflammation. The aim of this study was to identify the molecule(s) activated in adipose tissue macrophages in obesity. RESEARCH DESIGN AND METHODS: We performed a cDNA microarray analysis of coculture of 3T3-L1 adipocytes and RAW264 macrophages. Cultured adipocytes and macrophages and the adipose tissue of obese mice and humans were used to examine mRNA and protein expression. RESULTS: We found that macrophage-inducible C-type lectin (Mincle; also called Clec4e and Clecsf9), a type II transmembrane C-type lectin, is induced selectively in macrophages during the interaction between adipocytes and macrophages. Treatment with palmitate, a major saturated fatty acid released from 3T3-L1 adipocytes, induced Mincle mRNA expression in macrophages at least partly through the TLR4/nuclear factor (NF)-κB pathway. Mincle mRNA expression was increased in parallel with macrophage markers in the adipose tissue of obese mice and humans. The obesity-induced increase in Mincle mRNA expression was markedly attenuated in C3H/HeJ mice with defective TLR4 signaling relative to control C3H/HeN mice. Notably, Mincle mRNA was expressed in bone-marrow cell (BMC)-derived proinflammatory M1 macrophages rather than in BMC-derived anti-inflammatory M2 macrophages in vitro. CONCLUSIONS: Our data suggest that Mincle is induced in adipose tissue macrophages in obesity at least partly through the saturated fatty acid/TLR4/NF-κB pathway, thereby suggesting its pathophysiologic role in obesity-induced adipose tissue inflammation.

Role of transient receptor potential vanilloid 2 in LPS-induced cytokine production in macrophages.

There is considerable evidence indicating that intracellular Ca2+ participates as a second messenger in TLR4-dependent signaling. However, how intracellular free Ca2+ concentrations ([Ca2+]i) is increased in response to LPS and how they affect cytokine production are poorly understood. Here we examined the role of transient receptor potential (TRP), a major Ca2+ permeation pathway in non-excitable cells, in the LPS-induced cytokine production in macrophages. Pharmacologic experiments suggested that TRPV family members, but neither TRPC nor TRPM family members, are involved in the LPS-induced TNFalpha and IL-6 production in RAW264 macrophages. RT-PCR and immunoblot analyses showed that TRPV2 is the sole member of TRPV family expressed in macrophages. ShRNA against TRPV2 inhibited the LPS-induced TNFalpha and IL-6 production as well as IkappaBalpha degradation. Experiments using BAPTA/AM and EGTA, and Ca2+ imaging suggested that the LPS-induced increase in [Ca2+]i involves both the TRPV2-mediated intracellular and extracellular Ca2+ mobilizations. BAPTA/AM abolished LPS-induced TNFalpha and IL-6 production, while EGTA only partially suppressed LPS-induced IL-6 production, but not TNFalpha production. These data indicate that TRPV2 is involved in the LPS-induced Ca2+ mobilization from intracellular Ca2+ store and extracellular Ca2+. In addition to Ca2+ mobilization through the IP3-receptor, TRPV2-mediated intracellular Ca2+ mobilization is involved in NFkappaB-dependent TNFalpha and IL-6 expression, while extracellular Ca2+ entry is involved in NFkappaB-independent IL-6 production.

Insulin-induced ectodomain shedding of heparin-binding epidermal growth factor-like growth factor in adipocytes in vitro.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized as a type I transmembrane protein, which is proteolytically cleaved to release a soluble form via members of the a disintegrin and metalloproteinase (ADAM) family of proteolytic enzymes. This study was designed to elucidate the molecular mechanism underlying insulin-induced HB-EGF shedding in adipocytes in vitro. The 3T3-L1 adipocytes with stable expression of alkaline phosphatase (AP)-tagged proHB-EGF (3T3-L1/HB-EGF-AP adipocytes) were developed and AP activities of conditioned media were determined. Using 3T3-L1/HB-EGF-AP adipocytes, we demonstrated that insulin induces HB-EGF shedding in differentiated 3T3-L1 adipocytes in a dose- and time-dependent manner. There is no significant increase in insulin-induced HB-EGF shedding in undifferentiated 3T3-L1 preadipocytes. Studies with metalloprotease inhibitors suggested that insulin-induced HB-EGF shedding in adipocytes is mediated at least in part via ADAM17. Treatment with recombinant HB-EGF results in a dose- and time-dependent increase in HB-EGF shedding in adipocytes, which is significantly suppressed by pharmacologic blockade of ADAM17 (P < 0.01). Moreover, insulin-induced HB-EGF shedding in adipocytes is significantly inhibited by AG1478, an EGF receptor antagonist (P < 0.01). This study provides in vitro evidence that insulin induces HB-EGF shedding in 3T3-L1 adipocytes. Our data also suggest the role of ADAM17 in insulin-induced HB-EGF shedding in adipocytes.

The cathepsin L gene is a direct target of FOXO1 in skeletal muscle.

FOXO1 (forkhead box O1), a forkhead-type transcription factor whose gene expression is up-regulated in the skeletal muscle during starvation, appears to be a key molecule of energy metabolism and skeletal muscle atrophy. Cathepsin L, a lysosomal proteinase whose expression is also up-regulated in the skeletal muscle during starvation, is induced in transgenic mice overexpressing FOXO1 relative to wild-type littermates. In the present study, we conducted in vivo and in vitro experiments focusing on FOXO1 regulation of Ctsl (cathepsin L gene; CTSL1 in humans) expression in the skeletal muscle. During fasting and refeeding of C57BL/6 mice, Ctsl was regulated in parallel with FOXO1 in the skeletal muscle. Fasting-induced Ctsl expression was attenuated in transgenic mice overexpressing a dominant-negative form of FOXO1 or in skeletal-muscle-specific Foxo1-knockout mice relative to respective wild-type controls. Using C2C12 mouse myoblasts overexpressing a constitutively active form of FOXO1, we showed that FOXO1 induces Ctsl expression. Moreover, we found FOXO1-binding sites in both the mouse Ctsl and human CTSL1 promoters. The luciferase reporter analysis revealed that the mouse Ctsl and human CTSL1 promoters are activated by FOXO1, which is abolished by mutations in the consensus FOXO1-binding sites. Gel mobility-shift and chromatin immunoprecipiation assays showed that FOXO1 is recruited and binds to the Ctsl promoter. The present study provides in vivo and in vitro evidence that Ctsl is a direct target of FOXO1 in the skeletal muscle, thereby suggesting a role for the FOXO1/cathepsin L pathway in fasting-induced skeletal muscle metabolic change and atrophy.