Total body skeletal muscle mass estimated by magnetic resonance imaging and creatine (methyl-d3 ) dilution in athletes.

Creatine dilution (D3 -cr) is a technique for estimating total skeletal muscle mass (SMM) with practical utility, but has not been applied in athletic populations where body composition may differ to that in the normal population. This study aimed to assess the agreement between SMM derived from both D3 -cr and that obtained from whole-body magnetic resonance imaging (MRI) in 15 male and 5 female national level kayakers (stature: 182.0 ± 13.1 and 170.0 ± 9.0 cm; body mass: 80.6 ± 9.9 and 66.4 ± 6.0 kg; V̇O2 peak: 56.5 ± 7.0 and 49.6 ± 4.4 mL kg-1  min-1 , mean ± SD). SMM was determined following 60 mg of dosed D3 -cr and analysis of expelled urine collected on four subsequent days for creatine, creatinine, D3 -cr, and D3 -creatinine using liquid chromatography/mass spectroscopy. SMM was then estimated by assuming a creatine pool size of 4.3 g/kg. During the same time period, a whole-body MRI was undertaken to derive SMM from the analysis of multiple slices taken across the body. A strong positive correlation (F = 74.32; R = 0.90; P < .0001) between the two methods was observed, but the D3 -cr SMM was found to be significantly higher (43.3 ± 6.8 kg) when compared with MRI (36.3 ± 5.8 kg, P < .0001). However, the difference between the methods was removed when a higher intramuscular creatine pool (5.1 g/kg) was assumed. These data show that D3 -cr has potential utility in athletes, as referenced against MRI, but show that assumptions regarding creatine pool size need to be carefully considered.

Dramatic elevation in urinary amino terminal titin fragment excretion quantified by immunoassay in Duchenne muscular dystrophy patients and in dystrophin deficient rodents.

Enzyme-linked and electrochemiluminescence immunoassays were developed for quantification of amino (N-) terminal fragments of the skeletal muscle protein titin (N-ter titin) and qualified for use in detection of urinary N-ter titin excretion. Urine from normal subjects contained a small but measurable level of N-ter titin (1.0 ± 0.4 ng/ml). A 365-fold increase (365.4 ± 65.0, P = 0.0001) in urinary N-ter titin excretion was seen in Duchene muscular dystrophy (DMD) patients. Urinary N-ter titin was also evaluated in dystrophin deficient rodent models. Mdx mice exhibited low urinary N-ter titin levels at 2 weeks of age followed by a robust and sustained elevation starting at 3 weeks of age, coincident with the development of systemic skeletal muscle damage in this model; fold elevation could not be determined because urinary N-ter titin was not detected in age-matched wild type mice. Levels of serum creatine kinase and serum skeletal muscle troponin I (TnI) were also low at 2 weeks, elevated at later time points and were significantly correlated with urinary N-ter titin excretion in mdx mice. Corticosteroid treatment of mdx mice resulted in improved exercise performance and lowering of both urinary N-ter titin and serum skeletal muscle TnI concentrations. Low urinary N-ter titin levels were detected in wild type rats (3.0 ± 0.6 ng/ml), while Dmdmdx rats exhibited a 556-fold increase (1652.5 ± 405.7 ng/ml, P = 0.002) (both at 5 months of age). These results suggest that urinary N-ter titin is present at low basal concentrations in normal urine and increases dramatically coincident with muscle damage produced by dystrophin deficiency. Urinary N-ter titin has potential as a facile, non-invasive and translational biomarker for DMD.

Proteome-wide muscle protein fractional synthesis rates predict muscle mass gain in response to a selective androgen receptor modulator in rats.

Biomarkers of muscle protein synthesis rate could provide early data demonstrating anabolic efficacy for treating muscle-wasting conditions. Androgenic therapies have been shown to increase muscle mass primarily by increasing the rate of muscle protein synthesis. We hypothesized that the synthesis rate of large numbers of individual muscle proteins could serve as early response biomarkers and potentially treatment-specific signaling for predicting the effect of anabolic treatments on muscle mass. Utilizing selective androgen receptor modulator (SARM) treatment in the ovariectomized (OVX) rat, we applied an unbiased, dynamic proteomics approach to measure the fractional synthesis rates (FSR) of 167-201 individual skeletal muscle proteins in triceps, EDL, and soleus. OVX rats treated with a SARM molecule (GSK212A at 0.1, 0.3, or 1 mg/kg) for 10 or 28 days showed significant, dose-related increases in body weight, lean body mass, and individual triceps but not EDL or soleus weights. Thirty-four out of the 94 proteins measured from the triceps of all rats exhibited a significant, dose-related increase in FSR after 10 days of SARM treatment. For several cytoplasmic proteins, including carbonic anhydrase 3, creatine kinase M-type (CK-M), pyruvate kinase, and aldolase-A, a change in 10-day FSR was strongly correlated (r(2) = 0.90-0.99) to the 28-day change in lean body mass and triceps weight gains, suggesting a noninvasive measurement of SARM effects. In summary, FSR of multiple muscle proteins measured by dynamics of moderate- to high-abundance proteins provides early biomarkers of the anabolic response of skeletal muscle to SARM.

Genetic Ablation of CD38 Protects against Western Diet-Induced Exercise Intolerance and Metabolic Inflexibility.

Nicotinamide adenine dinucleotide (NAD+) is a key cofactor required for essential metabolic oxidation-reduction reactions. It also regulates various cellular activities, including gene expression, signaling, DNA repair and calcium homeostasis. Intracellular NAD+ levels are tightly regulated and often respond rapidly to nutritional and environmental changes. Numerous studies indicate that elevating NAD+ may be therapeutically beneficial in the context of numerous diseases. However, the role of NAD+ on skeletal muscle exercise performance is poorly understood. CD38, a multi-functional membrane receptor and enzyme, consumes NAD+ to generate products such as cyclic-ADP-ribose. CD38 knockout mice show elevated tissue and blood NAD+ level. Chronic feeding of high-fat, high-sucrose diet to wild type mice leads to exercise intolerance and reduced metabolic flexibility. Loss of CD38 by genetic mutation protects mice from diet-induced metabolic deficit. These animal model results suggest that elevation of tissue NAD+ through genetic ablation of CD38 can profoundly alter energy homeostasis in animals that are maintained on a calorically-excessive Western diet.

Total body skeletal muscle mass: estimation by creatine (methyl-d3) dilution in humans.

Current methods for clinical estimation of total body skeletal muscle mass have significant limitations. We tested the hypothesis that creatine (methyl-d3) dilution (D3-creatine) measured by enrichment of urine D3-creatinine reveals total body creatine pool size, providing an accurate estimate of total body skeletal muscle mass. Healthy subjects with different muscle masses [n = 35: 20 men (19-30 yr, 70-84 yr), 15 postmenopausal women (51-62 yr, 70-84 yr)] were housed for 5 days. Optimal tracer dose was explored with single oral doses of 30, 60, or 100 mg D3-creatine given on day 1. Serial plasma samples were collected for D3-creatine pharmacokinetics. All urine was collected through day 5. Creatine and creatinine (deuterated and unlabeled) were measured by liquid chromatography mass spectrometry. Total body creatine pool size and muscle mass were calculated from D3-creatinine enrichment in urine. Muscle mass was also measured by magnetic resonance imaging (MRI), dual-energy x-ray absorptiometry (DXA), and traditional 24-h urine creatinine. D3-creatine was rapidly absorbed and cleared with variable urinary excretion. Isotopic steady-state of D3-creatinine enrichment in the urine was achieved by 30.7 ± 11.2 h. Mean steady-state enrichment in urine provided muscle mass estimates that correlated well with MRI estimates for all subjects (r = 0.868, P < 0.0001), with less bias compared with lean body mass assessment by DXA, which overestimated muscle mass compared with MRI. The dilution of an oral D3-creatine dose determined by urine D3-creatinine enrichment provides an estimate of total body muscle mass strongly correlated with estimates from serial MRI with less bias than total lean body mass assessment by DXA.

Post-transcriptional regulation of ITGB6 protein levels in damaged skeletal muscle.

We have identified integrin beta 6 (Itgb6) as a transcript highly enriched in skeletal muscle. This finding is unexpected because Itgb6 is typically associated with epithelial expression domains in normal tissue. Further we find that ITGB6 protein expression in muscle is post-transcriptionally regulated. Uninjured muscle expresses Itgb6 RNA but no ITGB6 protein is detectable. Muscle injury induces ITGB6 protein accumulation rapidly post-injury in myofibers adjacent to the site of injury. As regeneration of the injured muscle tissue progresses ITGB6 protein is found in newly formed fibers up to at least 15 days post-injury.

Longitudinal changes in total body creatine pool size and skeletal muscle mass using the D3-creatine dilution method.

BACKGROUND: We recently validated in cross-sectional studies a new method to determine total body creatine pool size and skeletal muscle mass based on D3-creatine dilution from an oral dose and detection of urinary creatinine enrichment by isotope ratio mass spectrometry (IRMS). Routine clinical use of the method in aging and disease will require repeated application of the method, with a more widely available technology than IRMS, to enable determination of change in skeletal muscle mass in longitudinal studies. We therefore adapted the method to liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology, and sought to establish proof of concept for the repeated application of the method in a longitudinal study. Because the turnover of creatine is slow, it was also critical to determine the impact of background enrichment from an initial dose of oral D3-creatine on subsequent, longitudinal measurements of change in muscle mass. METHODS: Rats were given an oral tracer dose of D3-creatine (1.0 mg/kg body weight) at 10 and 17 weeks of age. LC-MS/MS was used to determine urinary D3-creatine, and urinary D3-creatinine enrichment, at time intervals after D3-creatine administration. Total body creatine pool size was calculated from urinary D3-creatinine enrichment at isotopic steady state 72 h after administration of D3-creatine tracer. RESULTS: At 10 weeks of age, rat lean body mass (LBM) measured by quantitative magnetic resonance correlated with creatine pool size (r = 0.92, P = 0.0002). Over the next 7 weeks, the decline in urinary D3-creatinine enrichment was slow and linear, with a rate constant of 2.73 ± 0.06 %/day. Subtracting background urinary D3-creatinine enrichment from the elevated enrichment following a second dose of D3-creatine at 17 weeks permitted repeat calculations of creatine pool size. As at 10 weeks, 17-week LBM correlated with creatine pool size (r = 0.98, P <0.0001). In addition, the change in creatine pool size was correlated with the change in LBM during the 7 weeks of rat growth between measurements (r = 0.96, P <0.0001). CONCLUSION: The LC-MS/MS-based D3-creatine dilution method can be applied repeatedly to measure total body creatine skeletal muscle mass change in longitudinal study.

Total-body creatine pool size and skeletal muscle mass determination by creatine-(methyl-D3) dilution in rats.

There is currently no direct, facile method to determine total-body skeletal muscle mass for the diagnosis and treatment of skeletal muscle wasting conditions such as sarcopenia, cachexia, and disuse. We tested in rats the hypothesis that the enrichment of creatinine-(methyl-d(3)) (D(3)-creatinine) in urine after a defined oral tracer dose of D(3)-creatine can be used to determine creatine pool size and skeletal muscle mass. We determined 1) an oral tracer dose of D(3)-creatine that was completely bioavailable with minimal urinary spillage and sufficient enrichment in the body creatine pool for detection of D(3)-creatine in muscle and D(3)-creatinine in urine, and 2) the time to isotopic steady state. We used cross-sectional studies to compare total creatine pool size determined by the D(3)-creatine dilution method to lean body mass determined by independent methods. The tracer dose of D(3)-creatine (<1 mg/rat) was >99% bioavailable with 0.2-1.2% urinary spillage. Isotopic steady state was achieved within 24-48 h. Creatine pool size calculated from urinary D(3)-creatinine enrichment at 72 h significantly increased with muscle accrual in rat growth, significantly decreased with dexamethasone-induced skeletal muscle atrophy, was correlated with lean body mass (r = 0.9590; P < 0.0001), and corresponded to predicted total muscle mass. Total-body creatine pool size and skeletal muscle mass can thus be accurately and precisely determined by an orally delivered dose of D(3)-creatine followed by the measurement of D(3)-creatinine enrichment in a single urine sample and is promising as a noninvasive tool for the clinical determination of skeletal muscle mass.

Effectiveness of the PPARγ agonist, GW570, in liver fibrosis.

INTRODUCTION: The peroxisome proliferator-activated receptors (PPARs) are well established to be important in modulating the fibrogenic response to liver injury. PPARγ plays a role in hepatic fibrosis, presumably by virtue of its expression in hepatic stellate cells, which are key effectors of fibrosis. In this study, we evaluated whether the potent nonthiozolidinedione PPARγ agonist, GW570, had effects on isolated stellate cells and hepatic fibrosis in vivo. METHODS: Liver fibrosis and stellate cell activation were induced in vivo by either bile duct ligation (BDL) or administration of carbon tetrachloride (CCl(4)). Primary cultures of stellate cells isolated from normal rats were exposed to GW570. The PPARγ agonist was also given to male Sprague-Dawley rats before or during injury to test its ability to ameliorate fibrosis. Fibrosis biomarkers including total collagen, hydroxyproline, collagen I α1 and smooth muscle α actin were measured. RESULTS: GW570 had potent effects on isolated stellate cells, both simulating PPARγ mediated gene transcription, as well as inhibiting collagen I α1 mRNA and protein expression and smooth muscle α actin protein abundance, consistent with suppression of stellate cell activation. In BDL liver injury, a daily dose of 10 mg/kg per day of GW570 inhibited collagen I α1 mRNA, while concentrations of 1 also inhibited fibrosis as measured by hydroxyproline and total collagen content. Lower doses of GW570 (0.1-1.0 mg/kg per day) did not significantly abrogate whole liver collagen or hydroxyproline content in this model. In a CCl(4) model, 0.1-1.0 mg/kg per day GW570 reduced expression of smooth muscle α actin, but did not affect whole liver collagen or hydroxyproline content. Finally, we found that GW570 had anti-inflammatory effects on Kupffer cells as well as in vivo during CCl(4) injury. CONCLUSION: PPARγ receptor agonism with the nonthiozolidinedione, GW570, inhibited stellate cell activation in vitro and in vivo, and abrogated the fibrogenic response to injury in a dose responsive fashion.

The identification of pyrazolo[1,5-a]pyridines as potent p38 kinase inhibitors.

A series of pyrazolo[1,5-a]pyridine derivatives was designed and synthesized as novel potent p38 kinase inhibitors. Our approaches towards improving in vitro metabolism and in vivo pharmacokinetic properties of the series are described.

RU486 did not exacerbate cytokine release in mice challenged with LPS nor in db/db mice.

BACKGROUND: Glucocorticoids down-regulate cytokine synthesis and suppress inflammatory responses. The glucocorticoid receptor (GR) antagonist RU486 may exacerbate the inflammatory response, and concerns over this exacerbation have limited the development and clinical use of GR antagonists in the treatment of diabetes and depression. We investigated the effects of RU486 on serum cytokines in db/db mice and on lipopolysaccharide (LPS)-induced circulating TNFalpha levels in both normal AKR mice and diet-induced obese (DIO) C57BL/6 mice. RESULTS: Chronic treatment of db/db mice with RU486 dose-dependently decreased blood glucose, increased serum corticosterone and ACTH, but did not affect serum MCP-1 and IL-6 levels. LPS dose-dependently increased serum TNFalpha in both AKR and C57BL/6 DIO mice, along with increased circulating corticosterone and ACTH. Pretreatment of the mice with RU486 dose-dependently suppressed the LPS induced increases in serum TNFalpha and further increased serum corticosterone. CONCLUSION: RU486 at doses that were efficacious in lowering blood glucose did not exacerbate cytokine release in these three mouse models. RU486 actually suppressed the lower dose LPS-mediated TNFalpha release, possibly due to the increased release of glucocorticoids.

Effects of the cFMS kinase inhibitor 5-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine (GW2580) in normal and arthritic rats.

The cFMS (cellular homolog of the V-FMS oncogene product of the Susan McDonough strain of feline sarcoma virus) (Proc Natl Acad Sci U S A 83:3331-3335, 1986) kinase inhibitor 5-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine (GW2580) inhibits colony-stimulating factor (CSF)-1-induced monocyte growth and bone degradation in vitro and inhibits CSF-1 signaling through cFMS kinase in 4-day models in mice (Proc Natl Acad Sci U S A 102:16078, 2005). In the present study, the kinase selectivity of GW2580 was further characterized, and the effects of chronic treatment were evaluated in normal and arthritic rats. GW2580 selectively inhibited cFMS kinase compared with 186 other kinases in vitro and completely inhibited CSF-1-induced growth of rat monocytes, with an IC(50) value of 0.2 microM. GW2580 dosed orally at 25 and 75 mg/kg 1 and 5 h before the injection of lipopolysaccharide inhibited tumor necrosis factor-alpha production by 60 to 85%, indicating a duration of action of at least 5 h. In a 21-day adjuvant arthritis model, GW2580 dosed twice a day (b.i.d.) from days 0 to 21, 7 to 21, or 14 to 21 inhibited joint connective tissue and bone destruction as assessed by radiology, histology and bone mineral content measurements. In contrast, GW2580 did not affect ankle swelling in the adjuvant model nor did it affect ankle swelling in a model where local arthritis is reactivated by peptidoglycan polysaccharide polymers. GW2580 administered to normal rats for 21 days showed no effects on tissue histology and only modest changes in serum clinical chemistry and blood hematology. In conclusion, GW2580 was effective in preserving joint integrity in the adjuvant arthritis model while showing minimal effects in normal rats.

PPARgamma agonists prevent TGFbeta1/Smad3-signaling in human hepatic stellate cells.

PPARgamma agonists inhibit liver fibrosis, but the mechanisms involved are uncertain. We hypothesized that PPARgamma agonists inhibit transforming growth factor (TGF)beta1-activation of TGFbeta receptor (TGFbetaR)-1 signaling in quiescent stellate cells, thereby abrogating Smad3-dependent induction of extracellular matrix (ECM) genes, such as PAI-1 and collagen-1alphaI. To test this, human HSC were cultured to induce a quiescent phenotype, characterized by lipid accumulation and PPARgamma expression and transcriptional activity. These adipocytic HSC were then treated with TGFbeta1+/-a TGFbetaR-1 kinase inhibitor (SB431542) or a PPARgamma agonist (GW7845). TGFbeta1 caused dose- and time-dependent increases in Smad3 phosphorylation, followed by induction of collagen and PAI-1 expression. Like the TGFbetaR-1 kinase inhibitor, the PPARgamma agonist caused dose-dependent inhibition of all of these responses without effecting HSC proliferation or viability. Thus, the anti-fibrotic actions of PPARgamma agonists reflect their ability to inhibit TGFbeta1-TGFbetaR1 signaling that initiates ECM gene expression in quiescent HSC.

Regulation of peroxisome proliferator-activated receptor-gamma in liver fibrosis.

The peroxisome proliferator-activated receptors (PPARs) impart diverse cellular effects in biological systems. Because stellate cell activation during liver injury is associated with declining PPARgamma expression, we hypothesized that its expression is critical in stellate cell-mediated fibrogenesis. We therefore modulated its expression during liver injury in vivo. PPARgamma was depleted in rat livers by using an adenovirus-Cre recombinase system. PPARgamma was overexpressed by using an additional adenoviral vector (AdPPARgamma). Bile duct ligation was utilized to induce stellate cell activation and liver fibrosis in vivo; phenotypic effects (collagen I, smooth muscle alpha-actin, hydroxyproline content, etc.) were measured. PPARgamma mRNA levels decreased fivefold and PPARgamma protein was undetectable in stellate cells after culture-induced activation. During activation in vivo, collagen accumulation, assessed histomorphometrically and by hydroxyproline content, was significantly increased after PPARgamma depletion compared with controls (1.28 +/- 0.14 vs. 1.89 +/- 0.21 mg/g liver tissue, P < 0.03). In isolated stellate cells, AdPPARgamma overexpression resulted in significantly increased adiponectin mRNA expression and decreased collagen I and smooth muscle alpha-actin mRNA expression compared with controls. During in vivo fibrogenesis, rat livers exposed to AdPPARgamma had significantly less fibrosis than controls. Collagen I and smooth muscle alpha-actin mRNA expression were significantly reduced in AdPPARgamma-infected rats compared with controls (P < 0.05, n = 10). PPARgamma-deficient mice exhibited enhanced fibrogenesis after liver injury, whereas PPARgamma receptor overexpression in vivo attenuated stellate cell activation and fibrosis. The data highlight a critical role for PPARgamma during in vivo fibrogenesis and emphasize the importance of the PPARgamma pathway in stellate cells during liver injury.

Changes of skeletal muscle adiponectin content in diet-induced insulin resistant rats.

The current study examined the relationship between skeletal muscle levels of adiponectin and parameters of insulin sensitivity. A high fat/sucrose diet (HFD) for 20 weeks resulted in significant increases in body weight, serum insulin, triglycerides (TG), and free fatty acids (FFA) (all p < 0.01). Interestingly, this diet leads to a slight increase in serum adiponectin, but significant decreases in gastrocnemius muscle and white adipose adiponectin (all p < 0.05). HFD for 4 weeks also resulted in a significant decrease in muscle adiponectin, which correlated with serum insulin, TG, and FFA (all p < 0.05). Treatment of the 4-week HFD rats with a PPARgamma agonist GI262570 ameliorated the diet-induced hyperinsulinemia and dyslipidemia, and effectively restored muscle adiponectin (all p < 0.05). This study demonstrated that HFD-induced hyperinsulinemia and dyslipidemia appeared without changes in serum adiponectin, but were associated with decreased tissue adiponectin. This provides the first evidence for a connection between tissue adiponectin and diet-induced hyperinsulinemia and dyslipidemia.

PPARgamma agonists diminish serum VEGF elevation in diet-induced insulin resistant SD rats and ZDF rats.

We investigated the effect of peroxisome proliferator-activated receptor gamma (PPARgamma) agonists on serum vascular endothelial growth factor (VEGF) in diet-induced insulin resistant SD rats and ZDF rats. SD rats fed a high fat/sucrose diet showed increases in serum insulin and VEGF (both p < 0.01). Treatment with a PPARgamma agonist GI262570 normalized the diet-elevated insulin and VEGF (both p < 0.01). There was a positive correlation between serum insulin and VEGF (p < 0.05) in SD rats. ZDF rats had higher serum glucose, insulin, and VEGF than Zucker lean rats (all p < 0.01). Treatment of ZDF rats with PPARgamma agonist pioglitazone decreased serum glucose and VEGF (both p <0.01). There was a positive correlation between glucose and VEGF in ZDF rats (p < 0.05). In 3T3-L1 adipocytes, GI262570 did not affect insulin-stimulated VEGF secretion. These studies demonstrated that hyperinsulinemia in SD rats and hyperglycemia in ZDF rats were associated with increased serum VEGF; PPARgamma agonists normalized serum insulin, glucose, and VEGF, but did not affect VEGF secretion in vitro.

GI262570, a peroxisome proliferator-activated receptor {gamma} agonist, changes electrolytes and water reabsorption from the distal nephron in rats.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) agonists have been shown to have significant therapeutic benefits such as desirable glycemic control in type 2 diabetic patients; however, these agents may cause fluid retention in susceptible individuals. Since PPARgamma is expressed selectively in distal nephron epithelium, we studied the mechanism of PPARgamma agonist-induced fluid retention using male Sprague-Dawley rats treated with either vehicle or GI262570 (farglitazar), a potent PPARgamma agonist. GI262570 (20 mg/kg/day) induced a plasma volume expansion. The plasma volume expansion was accompanied by a small but significant decrease in plasma potassium concentration. Small but significant increases in plasma sodium and chloride concentrations were also observed. These changes in serum electrolytes suggested an activation of the renal mineralocorticoid response system; however, GI262570-treated rats had lower plasma levels of aldosterone compared with vehicle-treated controls. mRNA levels for a group of genes involved in distal nephron sodium and water absorption are changed in the kidney medulla with GI262570 treatment. In addition, due to a possible rebound effect on epithelial sodium channel (ENaC) activity, a low dose of amiloride did not prevent GI262570-induced fluid retention. On the contrary, the rebound effect after amiloride treatment potentiated GI262570-induced plasma volume expansion. This is at least partially due to a synergistic effect of GI262570 and the rebound from amiloride treatment on ENaCalpha expression. In summary, our current data suggest that GI262570 can increase water and sodium reabsorption in distal nephron by stimulating the ENaC and Na,K-ATPase system. This may be an important mechanism for PPARgamma agonist-induced fluid retention.

Serum adiponectin as a biomarker for in vivo PPARgamma activation and PPARgamma agonist-induced efficacy on insulin sensitization/lipid lowering in rats.

BACKGROUND: PPARgamma agonists ameliorate insulin resistance and dyslipidemia in type 2 diabetic patients. Adiponectin possesses insulin sensitizing properties, and predicts insulin sensitivity of both glucose and lipid metabolism. In diet-induced insulin resistant rats and ZDF rats, the current studies determined the correlation between PPARgamma agonist-upregulated fatty acid binding protein(FABP3) mRNA in adipose tissue and PPARgamma agonist-elevated serum adiponectin, and the correlation between PPARgamma agonist-elevated serum adiponectin and PPARgamma agonist-mediated efficacy in insulin sensitization and lipid lowering. RESULTS: Parallel groups of SD rats were fed a high fat/sucrose (HF) diet for 4 weeks. These rats were orally treated for the later 2 weeks with vehicle, either PPARgamma agonist GI262570 (0.2-100 mg/kg, Q.D.), or GW347845 (3 mg/kg, B.I.D). Rats on HF diet showed significant increases in postprandial serum triglycerides, free fatty acids (FFA), insulin, and area under curve (AUC) of serum insulin during an oral glucose tolerance test, but showed no change in serum glucose, adiponectin, and glucose AUC. Treatment with GI262570 dose-dependently upregulated adipose FABP3 mRNA, and increased serum adiponectin. There was a position correlation between adipose FABP3 mRNA and serum adiponectin (r = 0.7350, p < 0.01). GI262570 dose-dependently decreased the diet-induced elevations in triglycerides, FFA, insulin, and insulin AUC. Treatment with GW347845 had similar effects on serum adiponectin and the diet-induced elevations. There were negative correlations for adiponectin versus triglycerides, FFA, insulin, and insulin AUC (For GI262570, r = -0.7486, -0.4581, -0.4379, and -0.3258 respectively, all p < 0.05. For GW347845, r = -0.6370, -0.6877, -0.5512, and -0.3812 respectively, all p < 0.05). In ZDF rats treated with PPARgamma agonists pioglitazone (3-30 mg/kg, B.I.D.) or GW347845 (3 mg/kg, B.I.D.), there were also negative correlations for serum adiponectin versus glucose, triglycerides, FFA (for pioglitazone, r = -0.7005, -0.8603, and -0.9288 respectively; for GW347845, r = -0.9721, -0.8483, and -0.9453 respectively, all p < 0.01). CONCLUSIONS: This study demonstrated that (a) PPARgamma agonists improved insulin sensitivity and ameliorated dyslipidemia in HF fed rats and ZDF rats, which were correlated with serum adiponectin; (b) Serum adiponectin was positively correlated with adipose FABP3 mRNA in GI262570-treated rats. These data suggest that serum adiponectin can serve as a biomarker for both in vivo PPARgamma activation and PPARgamma agonist-induced efficacy on insulin resistance and dyslipidemia in rats.

CD14 mediates the innate immune responses to arthritopathogenic peptidoglycan-polysaccharide complexes of Gram-positive bacterial cell walls.

Bacterial infections play an important role in the multifactorial etiology of rheumatoid arthritis. The arthropathic properties of Gram-positive bacteria have been associated with peptidoglycan-polysaccharide complexes (PG-PS), which are major structural components of bacterial cell walls. There is little agreement as to the identity of cellular receptors that mediate innate immune responses to PG-PS. A glycosylphosphatidylinositol-linked cell surface protein, CD14, the lipopolysaccharide receptor, has been proposed as a PG-PS receptor, but contradictory data have been reported. Here, we examined the inflammatory and pathogenic responses to PG-PS in CD14 knockout mice in order to examine the role for CD14 in PG-PS-induced signaling. We found that PG-PS-induced responses in vitro, including transient increase in intracellular calcium, activation of nuclear factor-kappaB, and secretion of the cytokines tumor necrosis factor-alpha and interleukin-6, were all strongly inhibited in CD14 knockout macrophages. In vivo, the incidence and severity of PG-PS induced acute polyarthritis were significantly reduced in CD14 knockout mice as compared with their wild-type counterparts. Consistent with these findings, CD14 knockout mice had significantly inhibited inflammatory cell infiltration and synovial hyperplasia, and reduced expression of inflammatory cytokines in PG-PS arthritic joints. These results support an essential role for CD14 in the innate immune responses to PG-PS and indicate an important role for CD14 in PG-PS induced arthropathy.

Phosphorylation of p70s6 kinase is implicated in androgen-induced levator ani muscle anabolism in castrated rats.

Androgens are known to increase muscle mass, strength and muscle protein synthesis. However, the molecular mechanisms by which androgens regulate skeletal muscle development remain poorly understood. The ribosomal protein kinase p70(s6k) is a regulator of ribosome biogenesis and plays an important role in the regulation of growth-related protein synthesis. The phosphorylation of p70(s6k) has been implicated in load-induced skeletal muscle hypertrophy. In the current study, we determined the effect of DHT on the phosphorylation of p70(s6k) in the androgen-sensitive levator ani muscle of castrated rats. DHT induced a rapid increase in the phosphorylation of p70(s6k), which was detectable within 6 h after a single injection. Interestingly, DHT-induced phosphorylation of p70(s6k) occurred only in androgen-sensitive muscles, but not prostate and seminal vesicle. Co-administration of flutamide, an AR antagonist, inhibited DHT-induced p70(s6k) phosphorylation. While serum IGF-I levels were not changed by DHT treatment, IGF-I gene expression levels increased and the mRNA levels of IGFBP3 and IGFBP5 were suppressed in the LA muscle after DHT replacement in castrated rats. These results suggest that the phosphorylation of p70(s6k), likely via the IGF-I pathway, may play an important role in androgen-induced skeletal muscle hypertrophy.