Limited total arc range of motion increases sheer stress in the shoulder while pitching in collegiate baseball pitchers.

CONTEXT: Upper extremity injuries in baseball pitchers cause significant time-loss from competing and decreased quality of life. Although shoulder range of motion (ROM) is reported as a key factor to prevent potential injury, it remains unclear how limited glenohumeral ROM affects pitching biomechanics which may contribute to upper extremity injuries. OBJECTIVE: To investigate how pitchers with decreased total arc glenohumeral ROM of the throwing arm differed in upper extremity pitching kinematics and kinetics as well as ball velocity compared to pitchers with greater levels of glenohumeral ROM. DESIGN: Cross-sectional Study. SETTING: Laboratory. PATIENTS OR OTHER PARTICIPANTS: Fifty-seven baseball pitchers (ages 18-24) were divided into either control (≧160° total arc) or lower ROM (<160° total arc) groups. MAIN OUTCOME MEASURE(S): The mean glenohumeral ROM deficits, pitching kinematic and kinetic outcomes, and ball velocity were compared between groups. RESULTS: The control group demonstrated significantly less deficit in total arc ROM between arms than the lower ROM (Control: -1.5±10.0°, Lower ROM: -12.4±13.9°, p<.001). While, the lower ROM group displayed less maximal shoulder external rotation (ER) while pitching, the control group had significantly less difference in ROM between maximal shoulder ER while pitching and clinically-measured ER (Lower ROM: 64.4±12.1°, Control: 55.8±16.6°, p=.025). The control group had significantly faster ball velocity compared to the lower ROM group (Control: 85.0 ± 4.3mph, Lower ROM: 82.4 ± 4.8mph, p=.024). CONCLUSION: Pitchers with decreased total arc glenohumeral ROM (<160° total arc) may undergo over-stretching toward ER in the shoulder during the late cocking phase. Pitchers with higher total arc ROM can pitch the same or faster ball without increasing loading in the upper extremity. Total arc glenohumeral ROM measurement can be a clinical screening tool to monitor shoulder condition over the time, and pitchers with limited total arc ROM might be at higher risk of shoulder injury.

Activation of AMP-activated protein kinase (AMPK) through inhibiting interaction with prohibitins.

5'-Adenosine monophosphate-activated protein kinase (AMPK) is a potential therapeutic target for various medical conditions. We here identify a small-molecule compound (RX-375) that activates AMPK and inhibits fatty acid synthesis in cultured human hepatocytes. RX-375 does not bind to AMPK but interacts with prohibitins (PHB1 and PHB2), which were found to form a complex with AMPK. RX-375 induced dissociation of this complex, and PHBs knockdown resulted in AMPK activation, in the cultured cells. Administration of RX-375 to obese mice activated AMPK and ameliorated steatosis in the liver. High-throughput screening based on disruption of the AMPK-PHB interaction identified a second small-molecule compound that activates AMPK, confirming the importance of this interaction in the regulation of AMPK. Our results thus indicate that PHBs are previously unrecognized negative regulators of AMPK, and that compounds that prevent the AMPK-PHB interaction constitute a class of AMPK activator.

A novel JNK2/SREBP-1c pathway involved in insulin-induced fatty acid synthesis in human adipocytes.

Insulin plays important roles in apoptosis and lipid droplet (LD) formation, and it is one of the determinants involved in increasing fat mass. However, the mechanisms underlying insulin-induced enlargement of fat mass remain unclear. Our previous study suggested that insulin-induced increases in LDs are related to c-Jun N-terminal kinase (JNK)2-mediated upregulation of cell death-inducing DNA fragmentation factor-α-like effector (CIDE)C in human adipocytes. However, other genes involved in insulin/JNK2-induced LD formation are unknown. Here, we explored insulin/JNK2-regulated genes to clarify the mechanism of enlargement of LDs. Microarray analysis revealed that an insulin/JNK2 pathway mostly regulates expression of genes involved in lipid metabolism, including sterol regulatory element binding protein (SREBP)-1, a key transcription factor of lipogenesis. The JNK inhibitor SP600125 blocked insulin-induced upregulation of SREBP-1c expression. Small interfering RNA-mediated depletion of JNK2 suppressed insulin-induced nuclear accumulation of the active form of SREBP-1 protein and upregulation of SREBP-1c. Furthermore, depletion of JNK2 attenuated insulin-induced upregulation of SREBP-1c target lipogenic enzymes, leading to reduced de novo fatty acid synthesis. In addition, JNK2 coimmunoprecipitated with SREBP-1, reinforcing the correlation between JNK2 and SREBP-1. These results suggest that SREBP-1c is a novel insulin/JNK2-regulated gene and that the JNK2/SREBP-1c pathway mediates insulin-induced fatty acid synthesis, which may lead to enlargement of LDs in human adipocytes.

Suppression of FoxO1/cell death-inducing DNA fragmentation factor α-like effector A (Cidea) axis protects mouse ß-cells against palmitic acid-induced apoptosis.

Chronic exposure to free fatty acid (FFA) induces pancreatic ß-cell apoptosis, which may contribute to the development of type 2 diabetes. The cell death-inducing DNA fragmentation factor α-like effector (CIDE) family is involved in type 2 diabetes with obesity. In the present study, we found that only apoptosis-inducing FFA upregulated Cidea, and both apoptosis and Cidea were upregulated most strongly by palmitic acid, suggesting that the expression of Cidea is positively correlated with apoptosis. In contrast, there were weak correlations between Cideb and Cidec expression, and apoptosis. Furthermore, suppression of Cidea inhibited palmitic acid-induced apoptosis. Finally, suppression of FoxO1 inhibited palmitic acid-induced Cidea upregulation and apoptosis. These results indicate that Cidea is a critical regulator of FFA-induced apoptosis as a novel downstream target for FoxO1 in ß-cells, suggesting that suppression of Cidea is a potentially useful therapeutic approach for protecting against ß-cell loss in type 2 diabetes.

Differential regulation of CIDEA and CIDEC expression by insulin via Akt1/2- and JNK2-dependent pathways in human adipocytes.

Both insulin and the cell death-inducing DNA fragmentation factor-α-like effector (CIDE) family play important roles in apoptosis and lipid droplet formation. Previously, we reported that CIDEA and CIDEC are differentially regulated by insulin and contribute separately to insulin-induced anti-apoptosis and lipid droplet formation in human adipocytes. However, the upstream signals of CIDE proteins remain unclear. Here, we investigated the signaling molecules involved in insulin regulation of CIDEA and CIDEC expression. The phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and PI-103 blocked both insulin-induced downregulation of CIDEA and upregulation of CIDEC. The Akt inhibitor API-2 and the c-Jun N-terminal kinase (JNK) inhibitor SP600125 selectively inhibited insulin regulation of CIDEA and CIDEC expression, respectively, whereas the MAPK/ERK kinase inhibitor U0126 and the p38 inhibitor SB203580 did not. Small interfering RNA-mediated depletion of Akt1/2 prevented insulin-induced downregulation of CIDEA and inhibition of apoptosis. Depletion of JNK2, but not JNK1, inhibited insulin-induced upregulation of CIDEC and lipid droplet enlargement. Furthermore, insulin increased both Akt and JNK phosphorylation, which was abrogated by the PI3K inhibitors. These results suggest that insulin regulates CIDEA and CIDEC expression via PI3K, and it regulates expression of each protein via Akt1/2- and JNK2-dependent pathways, respectively, in human adipocytes.

Impaired insulin signaling in endothelial cells reduces insulin-induced glucose uptake by skeletal muscle.

In obese patients with type 2 diabetes, insulin delivery to and insulin-dependent glucose uptake by skeletal muscle are delayed and impaired. The mechanisms underlying the delay and impairment are unclear. We demonstrate that impaired insulin signaling in endothelial cells, due to reduced Irs2 expression and insulin-induced eNOS phosphorylation, causes attenuation of insulin-induced capillary recruitment and insulin delivery, which in turn reduces glucose uptake by skeletal muscle. Moreover, restoration of insulin-induced eNOS phosphorylation in endothelial cells completely reverses the reduction in capillary recruitment and insulin delivery in tissue-specific knockout mice lacking Irs2 in endothelial cells and fed a high-fat diet. As a result, glucose uptake by skeletal muscle is restored in these mice. Taken together, our results show that insulin signaling in endothelial cells plays a pivotal role in the regulation of glucose uptake by skeletal muscle. Furthermore, improving endothelial insulin signaling may serve as a therapeutic strategy for ameliorating skeletal muscle insulin resistance.

Direct evidence for leptin-induced lipid oxidation independent of long-form leptin receptor.

Leptin administration has been shown to enhance muscle lipid oxidation in relation to the energy expenditure. Both long-form (Ob-R(L)) and short-form leptin receptors (Ob-R(S)) are expressed in skeletal muscle, but the role of Ob-R(S) is unclear. In the present study, the role of Ob-R(S) in leptin-induced lipid oxidation in skeletal muscles was investigated using primary murine myotubes from m/m and db/db mice. Primary myotubes were treated with leptin (0.1, 1, 10, 100nM) for 24h. Lipid oxidation was determined by (14)CO(2) production rate from [1-(14)C] palmitate. Leptin was found to increase lipid oxidation in a dose- and time-dependent manner in db/db myotubes as well as in m/m myotubes. Leptin significantly increased phosphorylation of JAK2 and STAT3 in both types of myotube. Leptin-induced lipid oxidation was abolished by STAT3 siRNA. To investigate the mechanism underlying leptin-induced lipid oxidation, the effects of pharmacological inhibitors were examined. JAK2 or p38 MAPK inhibitor suppressed leptin-induced lipid oxidation and decreased STAT3 phosphorylation in both types of myotube, respectively. Leptin significantly increased phosphorylation of p38 MAPK, and leptin-induced lipid oxidation was abolished by treatment with p38 MAPK siRNA in both types of myotube. These results suggest that leptin induces lipid oxidation in skeletal muscle through the JAK2/p38 MAPK/STAT3 signaling pathway via not only Ob-R(L) but also Ob-R(S).

Differential roles of CIDEA and CIDEC in insulin-induced anti-apoptosis and lipid droplet formation in human adipocytes.

Both insulin and the cell death-inducing DNA fragmentation factor-alpha-like effector (CIDE) family play important roles in apoptosis and lipid droplet formation. However, regulation of the CIDE family by insulin and the contribution of the CIDE family to insulin actions remain unclear. Here, we investigated whether insulin regulates expression of the CIDE family and which subtypes contribute to insulin-induced anti-apoptosis and lipid droplet formation in human adipocytes. Insulin decreased CIDEA and increased CIDEC but not CIDEB mRNA expression. Starvation-induced apoptosis in adipocytes was significantly inhibited when insulin decreased the CIDEA mRNA level. Small interfering RNA-mediated depletion of CIDEA inhibited starvation-induced apoptosis similarly to insulin and restored insulin deprivation-reduced adipocyte number, whereas CIDEC depletion did not. Lipid droplet size of adipocytes was increased when insulin increased the CIDEC mRNA level. In contrast, insulin-induced enlargement of lipid droplets was markedly abrogated by depletion of CIDEC but not CIDEA. Furthermore, depletion of CIDEC, but not CIDEA, significantly increased glycerol release from adipocytes. These results suggest that CIDEA and CIDEC are novel genes regulated by insulin in human adipocytes and may play key roles in the effects of insulin, such as anti-apoptosis and lipid droplet formation.

Identification of a functional peroxisome proliferator-activated receptor (PPAR) response element (PPRE) in the human apolipoprotein A-IV gene.

Peroxisome proliferator-activated receptor-alpha (PPARalpha) is a key regulator in hepatic lipid metabolism and is a potential therapeutic target for dyslipidaemia. We reported previously that human hepatic apoA-IV is a highly sensitive gene up-regulated by the PPARalpha agonist KRP-101 (KRP), suggesting that induction of apoA-IV expression is one of the mechanisms underlying the decrease in triglycerides and elevation of HDL observed with PPARalpha agonist treatment. However, the mechanism of transcriptional regulation of apoA-IV by PPARalpha activation remains unclear. To clarify whether the apoA-IV promoter is regulated directly by PPARalpha, we analysed the apoA-IV promoter region by transient transfection assay in the human hepatocellular carcinoma cell line, HepG2. Co-transfection assay of unilateral deletions of apoA-IV promoter construct with human PPARalpha/RXRalpha showed that the region from -3279 to -2261 of the apoA-IV promoter includes key sites for transactivation by PPARalpha/RXRalpha. Sequence analysis suggested three putative PPAR response elements (PPREs) in this region. Electrophoretic mobility shift assay (EMSA) showed that a PPRE located from -2979 to -2967 can bind to PPARalpha/RXRalpha. Moreover, site-directed mutagenesis experiments indicated that the -2979/-2967 PPRE plays an essential role in transcriptional regulation of apoA-IV by PPARalpha. Chromatin immunoprecipitation (ChIP) assay confirmed that ligand-induced binding of PPARalpha to endogenous -2979/-2967 PPRE. These results indicate that human apoA-IV is regulated directly by PPARalphavia the -2979/-2967 PPRE.

Chronic leptin treatment stimulates lipid oxidation in immortalized and primary mouse skeletal muscle cells.

Leptin administration enhances lipid oxidation in skeletal muscle. Nevertheless, direct and chronic effect of leptin has not been well characterized. Here, we measured the effect of leptin on skeletal muscles and their signaling pathways using differentiated C(2)C(12) myotubes and primary myotube cultures. Differentiated myotubes expressed both the short and long forms of leptin receptors. Leptin increased lipid oxidation in myotubes in a concentration- and time-dependent manner, with significant induction of lipid oxidation occurring after 6 h. Actinomycin D completely blocked leptin-induced lipid oxidation. Leptin significantly increased phosphorylation of JAK2 and STAT3 in myotubes, and leptin-induced lipid oxidation was abolished by treatment with a JAK2 inhibitor or STAT3 siRNA. We then used mouse myotubes to measure these effects under physiological conditions. Leptin increased lipid oxidation, which again was blocked by a JAK2 inhibitor and STAT3 siRNA. These results suggest that the JAK2/STAT3 signaling pathway may underlie the chronic effects of leptin on lipid oxidation in skeletal muscles.

The death effector domain-containing DEDD supports S6K1 activity via preventing Cdk1-dependent inhibitory phosphorylation.

Cell cycle regulation and biochemical responses upon nutrients and growth factors are the major regulatory mechanisms for cell sizing in mammals. Recently, we identified that the death effector domain-containing DEDD impedes mitotic progression by inhibiting Cdk1 (cyclin-dependent kinase 1) and thus maintains an increase of cell size during the mitotic phase. Here we found that DEDD also associates with S6 kinase 1 (S6K1), downstream of phosphatidylinositol 3-kinase, and supports its activity by preventing inhibitory phosphorylation of S6K1 brought about by Cdk1 during the mitotic phase. DEDD(-/-) cells showed reduced S6K1 activity, consistently demonstrating decreased levels in activating phosphorylation at the Thr-389 site. In addition, levels of Cdk1-dependent inhibitory phosphorylation at the C terminus of S6K1 were enhanced in DEDD(-/-) cells and tissues. Consequently, as in S6K1(-/-) mice, the insulin mass within pancreatic islets was reduced in DEDD(-/-) mice, resulting in glucose intolerance. These findings suggest a novel cell sizing mechanism achieved by DEDD through the maintenance of S6K1 activity prior to cell division. Our results also suggest that DEDD may harbor important roles in glucose homeostasis and that its deficiency might be involved in the pathogenesis of type 2 diabetes mellitus.

A novel PPARalpha agonist ameliorates insulin resistance in dogs fed a high-fat diet.

Agonism of peroxisome proliferator-activated receptor (PPAR) alpha, a key regulator of lipid metabolism, leads to amelioration of lipid abnormalities in dyslipidemic patients. However, whether PPARalpha agonism is an effective form of therapy for obesity-related insulin resistance associated with lipid abnormalities is unclear. The present study investigated the effects of a potent and subtype-selective PPARalpha agonist, KRP-101, in a nonrodent insulin-resistant animal model under pair-fed conditions. Beagle dogs were fed a high-fat diet for 24 wk to induce insulin resistance. During the final 12 wk, 0.03 mg x kg(-1) x day(-1) KRP-101 (n = 5) or vehicle (n = 5) was administered orally once a day. KRP-101 administration resulted in a significantly lower weight of overall visceral fat, which is associated with increased adiponectin and decreased leptin in serum. KRP-101 administration improved hyperglycemia and hyperinsulinemia as well as dyslipidemia in dogs fed a high-fat diet. Oral glucose tolerance test showed that KRP-101 administration improved glucose intolerance. The KRP-101 group showed a markedly lower hepatic triglyceride concentration. Lipid oxidation was increased in the liver and skeletal muscles of the KRP-101 group. These findings in the dog model suggest that the use of potent and subtype-selective PPARalpha agonists as a potentially relevant therapeutic approach to treat human insulin resistance associated with visceral obesity.

Highly sensitive upregulation of apolipoprotein A-IV by peroxisome proliferator-activated receptor alpha (PPARalpha) agonist in human hepatoma cells.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a key regulator in hepatic lipid metabolism and a potential therapeutic target for dyslipidemia. However, in humans hepatic PPARalpha-regulated genes remain unclear. To investigate the effect of PPARalpha agonism on mRNA expressions of lipid metabolism-related genes in human livers, a potent PPARalpha agonist, KRP-101 (KRP), was used to treat the human hepatoma cell line, HepaRG cells. KRP did not affect AOX or L-PBE, which are involved in peroxisomal beta-oxidation. KRP increased L-FABP, CPT1A, VLCAD, and PDK4, which are involved in lipid transport or oxidation. However, the EC(50) values (114-2500 nM) were >10-fold weaker than the EC(50) value (10.9 nM) for human PPARalpha in a transactivation assay. To search for more sensitive genes, we determined the mRNA levels of apolipoproteins, apoA-I, apoA-II, apoA-IV, apoA-V, and apoC-III. KRP had no or little effect on apoA-I, apoC-III, and apoA-II. Interestingly, KRP increased apoA-IV (EC(50), 0.99 nM) and apoA-V (EC(50), 0.29 nM) with high sensitivity. We identified apoA-IV as a PPARalpha-upregulated gene in a study using PPARalpha siRNA. Moreover, when administered orally to dogs, KRP decreased the serum triglyceride level and increased the serum apoA-IV level in a dose-dependent manner. These findings suggest that apoA-IV, newly identified as a highly sensitive PPARalpha-regulated gene in human livers, may be one of the mechanisms underlying PPARalpha agonist-induced triglyceride decrease and HDL elevation.

Targeted disruption of AdipoR1 and AdipoR2 causes abrogation of adiponectin binding and metabolic actions.

Adiponectin plays a central role as an antidiabetic and antiatherogenic adipokine. AdipoR1 and AdipoR2 serve as receptors for adiponectin in vitro, and their reduction in obesity seems to be correlated with reduced adiponectin sensitivity. Here we show that adenovirus-mediated expression of AdipoR1 and R2 in the liver of Lepr(-/-) mice increased AMP-activated protein kinase (AMPK) activation and peroxisome proliferator-activated receptor (PPAR)-alpha signaling pathways, respectively. Activation of AMPK reduced gluconeogenesis, whereas expression of the receptors in both cases increased fatty acid oxidation and lead to an amelioration of diabetes. Alternatively, targeted disruption of AdipoR1 resulted in the abrogation of adiponectin-induced AMPK activation, whereas that of AdipoR2 resulted in decreased activity of PPAR-alpha signaling pathways. Simultaneous disruption of both AdipoR1 and R2 abolished adiponectin binding and actions, resulting in increased tissue triglyceride content, inflammation and oxidative stress, and thus leading to insulin resistance and marked glucose intolerance. Therefore, AdipoR1 and R2 serve as the predominant receptors for adiponectin in vivo and play important roles in the regulation of glucose and lipid metabolism, inflammation and oxidative stress in vivo.

Overexpression of monocyte chemoattractant protein-1 in adipose tissues causes macrophage recruitment and insulin resistance.

Adipose tissue expression and circulating concentrations of monocyte chemoattractant protein-1 (MCP-1) correlate positively with adiposity. To ascertain the roles of MCP-1 overexpression in adipose, we generated transgenic mice by utilizing the adipocyte P2 (aP2) promoter (aP2-MCP-1 mice). These mice had higher plasma MCP-1 concentrations and increased macrophage accumulation in adipose tissues, as confirmed by immunochemical, flow cytometric, and gene expression analyses. Tumor necrosis factor-alpha and interleukin-6 mRNA levels in white adipose tissue and plasma non-esterified fatty acid levels were increased in transgenic mice. aP2-MCP-1 mice showed insulin resistance, suggesting that inflammatory changes in adipose tissues may be involved in the development of insulin resistance. Insulin resistance in aP2-MCP-1 mice was confirmed by hyperinsulinemic euglycemic clamp studies showing that transgenic mice had lower rates of glucose disappearance and higher endogenous glucose production than wild-type mice. Consistent with this, insulin-induced phosphorylations of Akt were significantly decreased in both skeletal muscles and livers of aP2-MCP-1 mice. MCP-1 pretreatment of isolated skeletal muscle blunted insulin-stimulated glucose uptake, which was partially restored by treatment with the MEK inhibitor U0126, suggesting that circulating MCP-1 may contribute to insulin resistance in aP2-MCP-1 mice. We concluded that both paracrine and endocrine effects of MCP-1 may contribute to the development of insulin resistance in aP2-MCP-1 mice.

TFE3 transcriptionally activates hepatic IRS-2, participates in insulin signaling and ameliorates diabetes.

Using an expression cloning strategy, we have identified TFE3, a basic helix-loop-helix protein, as a transactivator of metabolic genes that are regulated through an E-box in their promoters. Adenovirus-mediated expression of TFE3 in hepatocytes in culture and in vivo strongly activated expression of IRS-2 and Akt and enhanced phosphorylation of insulin-signaling kinases such as Akt, glycogen synthase kinase 3beta and p70S6 kinase. TFE3 also induced hexokinase II (HK2) and insulin-induced gene 1 (INSIG1). These changes led to metabolic consequences, such as activation of glycogen and protein synthesis, but not lipogenesis, in liver. Collectively, plasma glucose levels were markedly reduced both in normal mice and in different mouse models of diabetes, including streptozotocin-treated, db/db and KK mice. Promoter analyses showed that IRS2, HK2 and INSIG1 are direct targets of TFE3. Activation of insulin signals in both insulin depletion and resistance suggests that TFE3 could be a therapeutic target for diabetes.

Sterol regulatory element-binding proteins activate insulin gene promoter directly and indirectly through synergy with BETA2/E47.

Insulin gene expression is regulated by pancreatic beta cell-specific factors, PDX-1 and BETA2/E47. Here we have demonstrated that the insulin promoter is a novel target for SREBPs established as lipid-synthetic transcription factors. Promoter analyses of rat insulin I gene in non-beta cells revealed that nuclear SREBP-1c activates the insulin promoter through three novel SREBP-binding sites (SREs), two of which overlap with E-boxes, binding sites for BETA2/E47. SREBP-1c activation of the insulin promoter was markedly enhanced by co-expression of BETA2/E47. This synergistic activation by SREBP-1c/BETA2/E47 was not mediated through SREs but through the E-boxes on which BETA2/E47 physically interacts with SREBP-1c, suggesting a novel function of SREBP as a co-activator. These two cis-DNA regions, E1 and E2, with an appropriate distance separating them, were mandatory for the synergism, which implicates formation of SREBP-1c.BETA2.E47 complex in a DNA looping structure for efficient recruitment of CREB-binding protein/p300. However, in the presence of PDX1, the synergistic action of SREBP-1c with BETA2/E47 was canceled. SREBP-1c-mediated activation of the insulin promoter and expression became overt in beta cell lines and isolated islets when endogenous PDX-1 expression was low. This cryptic SREBP-1c action might play a compensatory role in insulin expression in diabetes with beta cell lipotoxicity.

Enhancement of insulin signaling through inhibition of tissue lipid accumulation by activation of peroxisome proliferator-activated receptor (PPAR) alpha in obese mice.

BACKGROUND: The aim of the present study was to investigate the effect of PPARalpha activation on insulin signaling and lipid accumulation in the liver and skeletal muscle of insulin-resistant (ob/ob) mice. MATERIAL/METHODS: A known subtype-selective PPARalpha agonist, Wy-14,643, was administered to lean and ob/ob mice at 30 mg/kg/day for 4 weeks. Insulin (100 units/kg) or saline was injected into the portal vein of anesthetized mice. The liver and skeletal muscles were used for the detection of tyrosine phosphorylation of the insulin receptor (IR) and insulin receptor substrates (IRSs), as well as for the determination of both IRS-associated PI3-K activity and lipid content; in addition, the measurement of mRNA levels of PPAR-regulated genes was carried out. RESULTS: The PPARalpha agonist lowered plasma levels of glucose, insulin, triglycerides, and free fatty acids in ob/ob mice. Several PPARalpha-upregulated genes related to the transport and oxidation of fatty acids in the liver were increased by treatment with the agonist. The PPARalpha agonist significantly increased IR- and IRS-tyrosine phosphorylation and IRS-associated PI3-K activity in the liver and muscle of ob/ob mice, without exerting the same effects in lean mice. Moreover, these effects in ob/ob mice were accompanied by decreased triglyceride and fatty acyl-CoA contents in the liver and skeletal muscle. CONCLUSIONS: The present results suggest that inhibition of lipid accumulation by hepatic PPARalpha activation leads to an improvement in impaired insulin signaling in muscle tissue as well as in the liver of insulin-resistant mice.

Pharmacological characterization of a human-specific peroxisome proliferater-activated receptor alpha (PPARalpha) agonist in dogs.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a key regulator in lipid metabolism and a potential therapeutic target for lipid-related metabolic diseases. It has been shown that there are species differences between human and mouse in response to several PPARalpha agonists in a transactivation assay. In the present study, we cloned a full length of dog PPARalpha and investigated the effects of a novel and potent agonist (KCL) for human PPARalpha. In a transactivation assay using the full length of PPARalpha, agonistic activity of KCL for dog PPARalpha (EC(50): 0.007 microM) was comparable to that for human PPARalpha (EC(50): 0.003 microM), but not that for rat PPARalpha (EC(50): 11.49 microM). Similar results were obtained from a transactivation assay using a GAL4/PPARalpha ligand-binding domain (LBD) chimera. A point-mutation study showed that I272 on PPARalphaLBD is a major contributor to species differences in response to KCL between human, dog, and rat PPARalpha. KCL also induced mRNA levels of HMG-CoA synthase in dog hepatocytes. When administered orally to dogs and rats, KCL significantly decreased plasma triglyceride levels in a dose-dependent manner. The triglyceride-lowering effects of KCL in dogs were >100-fold more potent than those in rats. These results suggest that KCL may induce activation of highly potent PPARalpha in humans as well as dogs, and that dog is a suitable animal model for studying and predicting the biological actions of potent agonists for human PPARalpha.

SREBPs suppress IRS-2-mediated insulin signalling in the liver.

Insulin receptor substrate 2 (IRS-2) is the main mediator of insulin signalling in the liver, controlling insulin sensitivity. Sterol regulatory element binding proteins (SREBPs) have been established as transcriptional regulators of lipid synthesis. Here, we show that SREBPs directly repress transcription of IRS-2 and inhibit hepatic insulin signalling. The IRS-2 promoter is activated by forkhead proteins through an insulin response element (IRE). Nuclear SREBPs effectively replace and interfere in the binding of these transactivators, resulting in inhibition of the downstream PI(3)K/Akt pathway, followed by decreased glycogen synthesis. These data suggest a molecular mechanism for the physiological switching from glycogen synthesis to lipogenesis and hepatic insulin resistance that is associated with hepatosteatosis.