Potential involvement of dietary advanced glycation end products in impairment of skeletal muscle growth and muscle contractile function in mice.

Diets enriched with advanced glycation end products (AGE) have recently been related to muscle dysfunction processes. However, it remains unclear whether long-term exposure to an AGE-enriched diet impacts physiological characteristics of skeletal muscles. Therefore, we explored the differences in skeletal muscle mass, contractile function and molecular responses between mice receiving a diet high in AGE (H-AGE) and low in AGE (L-AGE) for 16 weeks. There were no significant differences between L-AGE and H-AGE mice with regard to body weight, food intake or epididymal fat pad weight. However, extensor digitorum longus (EDL) and plantaris (PLA) muscle weights in H-AGE mice were lower compared with L-AGE mice. Higher levels of N ε -(carboxymethyl)-l-lysine, a marker for AGE, in EDL muscles of H-AGE mice were observed compared with L-AGE mice. H-AGE mice showed lower muscle strength and endurance in vivo and lower muscle force production of PLA muscle in vitro. mRNA expression levels of myogenic factors including myogenic factor 5 and myogenic differentiation in EDL muscle were lower in H-AGE mice compared with L-AGE mice. The phosphorylation status of 70-kDa ribosomal protein S6 kinase Thr389, an indicator of protein synthesis signalling, was lower in EDL muscle of H-AGE mice than that of L-AGE mice. These findings suggest that long-term exposure to an AGE-enriched diet impairs skeletal muscle growth and muscle contractile function, and that these muscle dysfunctions may be attributed to the inhibition of myogenic potential and protein synthesis.

Salicylate acutely stimulates 5'-AMP-activated protein kinase and insulin-independent glucose transport in rat skeletal muscles.

Salicylate (SAL) has been recently implicated in the antidiabetic effect in humans. We assessed whether 5'-AMP-activated protein kinase (AMPK) in skeletal muscle is involved in the effect of SAL on glucose homeostasis. Rat fast-twitch epitrochlearis and slow-twitch soleus muscles were incubated in buffer containing SAL. Intracellular concentrations of SAL increased rapidly (<5 min) in both skeletal muscles, and the Thr(172) phosphorylation of the α subunit of AMPK increased in a dose- and time-dependent manner. SAL increased both AMPKα1 and AMPKα2 activities. These increases in enzyme activity were accompanied by an increase in the activity of 3-O-methyl-D-glucose transport, and decreases in ATP, phosphocreatine, and glycogen contents. SAL did not change the phosphorylation of insulin receptor signaling including insulin receptor substrate 1, Akt, and p70 ribosomal protein S6 kinase. These results suggest that SAL may be transported into skeletal muscle and may stimulate AMPK and glucose transport via energy deprivation in multiple muscle types. Skeletal muscle AMPK might be part of the mechanism responsible for the metabolic improvement induced by SAL.

The Effects of Caffeine on Metabolomic Responses to Muscle Contraction in Rat Skeletal Muscle.

Exercise has beneficial effects on our health by stimulating metabolic activation of skeletal muscle contraction. Caffeine is a powerful metabolic stimulant in the skeletal muscle that has ergogenic effects, including enhanced muscle power output and endurance capacity. In the present study, we aim to characterize the metabolic signatures of contracting muscles with or without caffeine stimulation using liquid chromatography-mass spectrometry and capillary electrophoresis coupled to mass spectrometry. Isolated rat epitrochlearis muscle was incubated in the presence or absence or of 3 mM caffeine for 30 min. Electrical stimulation (ES) was used to induce tetanic contractions during the final 10 min of incubation. Principal component analysis and hierarchical clustering analysis detected 184 distinct metabolites across three experimental groups-basal, ES, and ES with caffeine (ES + C). Significance Analysis of Microarray identified a total of 50 metabolites with significant changes in expression, and 23 metabolites significantly changed between the ES and ES + C groups. Changes were observed in metabolite levels of various metabolic pathways, including the pentose phosphate, nucleotide synthesis, ß-oxidation, tricarboxylic acid cycle, and amino acid metabolism. In particular, D-ribose 5-phosphate, IMP, O-acetylcarnitine, butyrylcarnitine, L-leucine, L-valine, and L-aspartate levels were higher in the ES + C group than in the ES group. These metabolic alterations induced by caffeine suggest that caffeine accelerates contraction-induced metabolic activations, thereby contributing to muscle endurance performance and exercise benefits to our health.

The Protective Effect of Brazilian Propolis against Glycation Stress in Mouse Skeletal Muscle.

We investigated the protective effect of Brazilian propolis, a natural resinous substance produced by honeybees, against glycation stress in mouse skeletal muscles. Mice were divided into four groups: (1) Normal diet + drinking water, (2) Brazilian propolis (0.1%)-containing diet + drinking water, (3) normal diet + methylglyoxal (MGO) (0.1%)-containing drinking water, and (4) Brazilian propolis (0.1%)-containing diet + MGO (0.1%)-containing drinking water. MGO treatment for 20 weeks reduced the weight of the extensor digitorum longus (EDL) muscle and tended to be in the soleus muscle. Ingestion of Brazilian propolis showed no effect on this change in EDL muscles but tended to increase the weight of the soleus muscles regardless of MGO treatment. In EDL muscles, Brazilian propolis ingestion suppressed the accumulation of MGO-derived advanced glycation end products (AGEs) in MGO-treated mice. The activity of glyoxalase 1 was not affected by MGO, but was enhanced by Brazilian propolis in EDL muscles. MGO treatment increased mRNA expression of inflammation-related molecules, interleukin (IL)-1ß, IL-6, and toll-like receptor 4 (TLR4). Brazilian propolis ingestion suppressed these increases. MGO and/or propolis exerted no effect on the accumulation of AGEs, glyoxalase 1 activity, and inflammatory responses in soleus muscles. These results suggest that Brazilian propolis exerts a protective effect against glycation stress by inhibiting the accumulation of AGEs, promoting MGO detoxification, and reducing proinflammatory responses in the skeletal muscle. However, these anti-glycation effects does not lead to prevent glycation-induced muscle mass reduction.

Negative regulation of urokinase-type plasminogen activator production through FGF-2-mediated activation of phosphoinositide 3-kinase.

Activation of phosphoinositide 3-kinase (PI3-kinase) is involved in many cellular responses. FGF-2 is one of the potent inducers of urokinase-type plasminogen activator (uPA) production in endothelial cells. However, little is known about the molecular mechanisms underlying FGF-2-mediated uPA production. Here we examined the signal transduction pathways involved in the regulation of uPA production by FGF-2-treatment. FGF-2 potently upregulated uPA production in murine brain capillary endothelial cells (IBE cells), as well as porcine aortic endothelial (PAE) cells and L6 myoblasts ectopically expressing FGFR1. PI3-kinase inhibitors, wortmannin and LY294002, both enhanced FGF-2-dependent uPA production by these cells. Stable expression of activated mutant p110alpha catalytic subunit of PI3-kinase into IBE cells decreased FGF-2-mediated uPA production, suggesting that PI3-kinase exhibited the negative regulatory effect on uPA production. No increase in FGF-2-induced PI3-kinase activity was observed in proteins immunoprecipitated by anti-phosphotyrosine antibody. Although stable expression of deleted mutant p85alpha regulatory subunit, which lacks association with p110 catalytic subunit, in IBE cells showed no dominant negative effect, transient expression of dominant negative Ras inhibited FGF-2-mediated PI3-kinase activation. These results suggest that only activated Ras contributed the FGF-2-mediated PI3-kinase activation. In cells stably expressing mutant p85alpha subunit, FGF-2 efficiently induced uPA production. Taken together, activation of PI3-kinase by FGF-2 is Ras-dependent and results in down-regulation of uPA production.

[A case of renal pelvic squamous cell carcinoma accompanied with humoral hypercalcemia of malignancy (HHM)].

A 78-year-old male was urgently admitted to our hospital because of consciousness disturbance. Laboratory data showed marked hypercalcemia (17.0 mg/dl), hypophosphatemia, low intact PTH level, high PTH relating peptide (PTHrP) level, normal osteocalcin and normal 1-25(OH)2D level. Computed tomography revealed a right renal tumor with extracapsular extension. Bone scintigram appeared normal. We performed right nephrectomy under the diagnosis of right renal tumor. Pathological diagnosis was poorly differentiated squamous cell carcinoma (SCC) of the right pelvis. Immunohistochemical study of the resected specimen for PTHrP was positive. Therefore, we diagnosed it as renal pelvic SCC with humoral hypercalcemia of malignancy (HHM). After nephrectomy, serum calcium returned to normal, but 5 months after nephrectomy, local recurrence appeared and serum calcium was re-elevated. She died 7 months after nephrectomy.

Caffeine and contraction synergistically stimulate 5'-AMP-activated protein kinase and insulin-independent glucose transport in rat skeletal muscle.

5'-Adenosine monophosphate-activated protein kinase (AMPK) has been identified as a key mediator of contraction-stimulated insulin-independent glucose transport in skeletal muscle. Caffeine acutely stimulates AMPK in resting skeletal muscle, but it is unknown whether caffeine affects AMPK in contracting muscle. Isolated rat epitrochlearis muscle was preincubated and then incubated in the absence or presence of 3 mmol/L caffeine for 30 or 120 min. Electrical stimulation (ES) was used to evoke tetanic contractions during the last 10 min of the incubation period. The combination of caffeine plus contraction had additive effects on AMPKα Thr(172) phosphorylation, α-isoform-specific AMPK activity, and 3-O-methylglucose (3MG) transport. In contrast, caffeine inhibited basal and contraction-stimulated Akt Ser(473) phosphorylation. Caffeine significantly delayed muscle fatigue during contraction, and the combination of caffeine and contraction additively decreased ATP and phosphocreatine contents. Caffeine did not affect resting tension. Next, rats were given an intraperitoneal injection of caffeine (60 mg/kg body weight) or saline, and the extensor digitorum longus muscle was dissected 15 min later. ES of the sciatic nerve was performed to evoke tetanic contractions for 5 min before dissection. Similar to the findings from isolated muscles incubated in vitro, the combination of caffeine plus contraction in vivo had additive effects on AMPK phosphorylation, AMPK activity, and 3MG transport. Caffeine also inhibited basal and contraction-stimulated Akt phosphorylation in vivo. These findings suggest that caffeine and contraction synergistically stimulate AMPK activity and insulin-independent glucose transport, at least in part by decreasing muscle fatigue and thereby promoting energy consumption during contraction.

Evidence for organic cation transporter-mediated metformin transport and 5'-adenosine monophosphate-activated protein kinase activation in rat skeletal muscles.

OBJECTIVE: 5'-Adenosine monophosphate-activated protein kinase (AMPK) is a key molecule of metabolic enhancement in skeletal muscle. We investigated whether metformin (MET) acts directly on skeletal muscle, is transported into skeletal muscle via organic cation transporters (OCTs), and activates AMPK. MATERIALS/METHODS: Isolated rat epitrochlearis and soleus muscles were incubated in vitro either in the absence or in the presence of MET. The activation status of AMPK, the intracellular energy status, and glucose and MET transport activity were then evaluated. The effect of cimetidine, which is an OCT inhibitor, on AMPK activation was also examined. RESULTS: MET (10 mmol/L, ≥60 min) increased the phosphorylation of Thr¹7² at the catalytic α subunit of AMPK in both muscles. AMPK activity assays showed that both AMPKα1 and AMPKα2 activity increased significantly. The AMPK activation was associated with energy deprivation, which was estimated from the ATP, phosphocreatine (PCr), and glycogen content, and with increased rates of 3-O-methyl-D-glucose (3MG) transport. MET did not change the basal phosphorylation status of insulin receptor signaling molecules. MET was transported into the cytoplasm in a time-dependent manner, and cimetidine suppressed MET-induced AMPK phosphorylation and 3MG transport. CONCLUSION: These results suggest that MET is acutely transported into skeletal muscle by OCTs, and stimulates AMPKα1 and α2 activity in both fast- and slow-twitch muscle types, at least in part by reducing the energy state.

Heat stress acutely activates insulin-independent glucose transport and 5'-AMP-activated protein kinase prior to an increase in HSP72 protein in rat skeletal muscle.

Heat stress (HS) stimulates heat shock protein (HSP) 72 mRNA expression, and the period after an increase in HSP72 protein is characterized by enhanced glucose metabolism in skeletal muscle. We have hypothesized that, prior to an increase in the level of HSP72 protein, HS activates glucose metabolism by acutely stimulating 5'-AMP-activated protein kinase (AMPK). Rat epitrochlearis muscle was isolated and incubated either with or without HS (42°C) for 10 and 30 min. HS for 30 min led to an increase in the level of Hspa1a and Hspa1b mRNA but did not change the amount of HSP72 protein. However, HS for both 10 and 30 min led to a significant increase in the rate of 3-O-methyl-d-glucose (3MG) transport, and the stimulatory effect of 3MG transport was completely blocked by cytochalasin B. HS-stimulated 3MG transport was also inhibited by dorsomorphin but not by wortmannin. HS led to a decrease in the concentration of ATP, phosphocreatine, and glycogen, to an increase in the level of phosphorylation of AMPKα Thr(172), and to an increase in the activity of both AMPKα1 and AMPKα2. HS did not affect the phosphorylation status of insulin receptor signaling or Ca(2+)/calmodulin-dependent protein kinase II. These results suggest that HS acts as a rapid stimulator of insulin-independent glucose transport, at least in part by stimulating AMPK via decreased energy status. Although further research is warranted, heat treatment of skeletal muscle might be a promising method to promote glucose metabolism acutely.

The selective tyrosine kinase inhibitor, STI571, inhibits growth of anaplastic thyroid cancer cells.

To establish a molecular targeting therapy for anaplastic thyroid carcinomas, we studied the effect of the specific tyrosine kinase inhibitor, STI571, on anaplastic thyroid cancer cell lines highly expressing c-ABL ARO (mutated p53) and FRO (undetectable p53). These lines showed marked inhibition of cell growth after treatment with STI571. In contrast, the growth of papillary thyroid cancer cell lines that harbor wild-type p53 and have low levels of c-ABL was not affected by STI571. Fluorescent-activated cell sorting analysis revealed that STI571 treatment increased the fraction of FRO and ARO cells in S and G(2)/M phases, respectively, indicating induction of S and G(2)/M transition arrest. These changes were accompanied by inhibition of c-ABL phosphorylation/activation and increased expression of p21(cip1) in FRO and p27(kip1) in both FRO and ARO cells. Treatment with STI571 also led to reduction of cyclin A, B1, and CDC2 levels. The growth of FRO cells implanted into immunocompromised mice was significantly inhibited by STI571. Taken together, these results suggest that selective suppression of c-ABL activity by STI571 may represent a potential anticancer strategy for p53-mutated undifferentiated thyroid carcinomas.

[A case of prostate cancer with invasion to the middle ureter].

A 69-year-old male was diagnosed as having prostate cancer with ureter invasion. His serum prostate specific antigen level was not so high (19.6 ng/ml) before treatment, but clinical stage was D2, poorly differentiated adenocarcinoma of prostate. He was treated with chemoendocrine therapy. Prostate cancer with continuous invasion to the middle ureter is rare. To the best of our knowledge, this is the 6th case report of such an unusual invasion of prostate cancer.

[Feasibility and change in the level of blood paclitaxel concentration after paclitaxel therapy for a hemodialysis patient with cisplatin resistant metastatic transitional cell cancer].

The patient was a 54-year-old woman with recurrent urinary tract cancer. A CT of the lung showed multiple nodules after bil-nephroureterectomy, cystectomy and combination chemotherapy with cisplatin (CDDP) and epirubicin (EPI). As second line chemotherapy for the patient, TXL was administered. She was hemodialized after operation, and there are few reports about paclitaxel (TXL) therapy for hemodialysis (HD) patients. Peak blood TXL concentration, about 1,200 ng/ml, was achieved 6 hours after the administration of TXL. The blood TXL concentration was 41 ng/ml 24 h after the administration of TXL. However, the TXL therapy was not repeated because of unacceptable neurological side effects. An almost 40% shrinkage in the size of the metastatic lung nodule was obtained after 1 cycle of treatment.

Pu-erh tea hot-water extract activates Akt and induces insulin-independent glucose transport in rat skeletal muscle.

Skeletal muscle is a major organ that is important for whole-body glucose metabolism. We found that when isolated rat epitrochlearis muscle was incubated with a Pu-erh tea hot-water extract (PTE) for 30 min, the rate of 3-O-methyl-D-glucose (3MG) transport increased in the absence of insulin. This activation was associated with an increase in Ser(473) phosphorylation of Akt, a signaling intermediary leading to insulin-dependent glucose transport, but not Tyr(458) phosphorylation of phosphoinositide 3-kinase p85, an upstream molecule of Akt. PTE-stimulated 3MG transport was also not accompanied by Thr(172) phosphorylation of the catalytic α-subunit of 5'-AMP-activated protein kinase (AMPK). Gallic acid, a water-soluble ingredient in Pu-erh tea, stimulated Akt phosphorylation, but not AMPK phosphorylation. These results suggest that Pu-erh tea potentially promotes skeletal muscle glucose transport at least in part by activating Akt.

Coffee polyphenol caffeic acid but not chlorogenic acid increases 5'AMP-activated protein kinase and insulin-independent glucose transport in rat skeletal muscle.

Chlorogenic acid is an ester of caffeic and quinic acids, and is one of the most widely consumed polyphenols because it is abundant in foods, especially coffee. We explored whether chlorogenic acid and its metabolite, caffeic acid, act directly on skeletal muscle to stimulate 5'-adenosine monophosphate-activated protein kinase (AMPK). Incubation of rat epitrochlearis muscles with Krebs buffer containing caffeic acid (≥0.1 mM, ≥30 min) but not chlorogenic acid increased the phosphorylation of AMPKα Thr(172), an essential step for kinase activation, and acetyl CoA carboxylase Ser(79), a downstream target of AMPK, in a dose- and time-dependent manner. Analysis of isoform-specific AMPK activity revealed that AMPKα2 activity increased significantly, whereas AMPKα1 activity did not change. This enzyme activation was associated with a reduction in phosphocreatine content and an increased rate of 3-O-methyl-d-glucose transport activity in the absence of insulin. These results suggest that caffeic acid but not chlorogenic acid acutely stimulates skeletal muscle AMPK activity and insulin-independent glucose transport with a reduction of the intracellular energy status.

Alterations of the base excision repair gene MUTYH in sporadic colorectal cancer.

The base excision repair gene MUTYH encodes glycosylase which removes adenine residues mispaired with 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OHG). Biallelic germline mutations of the MUTYH gene are known to cause multiple colorectal adenomas including polyposis and cancer, mostly due to G:C➝T:A transversions in proto-oncogenes or tumor suppressor genes. The risk of colorectal cancer (CRC) in monoallelic mutation carriers of MUTYH is estimated to be higher in comparison with non-carriers. To investigate the possible role in sporadic CRC, we examined alterations of the MUTYH gene including somatic mutations and allelic loss in 101 cases of sporadic CRC, together with the KRAS mutation in some cases. MUTYH mutations in cancer DNA were detected in 3 cases, while mutations were also found in DNA samples from normal tissues, indicating that all were germline mutations. Allelic loss at the MUTYH locus was found in 10 of 51 (20.0%) CRC cases and KRAS mutations were found in 33 of the 101 (32.7%) samples. There was no significant difference in the rate of G:C➝T:A transversion in KRAS between cases with allelic loss (1 of 10, 10.0%) and without allelic loss (9 of 41, 22.0%). Investigation of quantitative allelic imbalance at SNP rs3219489 of MUTYH showed that CRC cases with C allele dominance (minor type corresponding to His) were more frequently detected with G:C➝T:A transversions than in those with G allele dominance (major type corresponding to Gln). In conclusion, somatic alterations of MUTYH in sporadic CRC were rare, similar to other DNA repair genes. However, it is possible that unknown mutations of regions not analyzed in this study and epigenetic changes of the promoter region of MUTYH may contribute to the disease.

Caffeine modulates phosphorylation of insulin receptor substrate-1 and impairs insulin signal transduction in rat skeletal muscle.

Caffeine decreases insulin sensitivity and insulin-stimulated glucose transport in skeletal muscle; however, the precise mechanism responsible for this deleterious effect is not understood fully. We investigated the effects of incubation with caffeine on insulin signaling in rat epitrochlearis muscle. Caffeine (≥1 mM, ≥15 min) suppressed insulin-stimulated insulin receptor substrate (IRS)-1 Tyr(612) phosphorylation in a dose- and time-dependent manner. These responses were associated with inhibition of the insulin-stimulated phosphorylation of phosphatidylinositol 3-kinase (PI3K) Tyr(458), Akt Ser(473), and glycogen synthase kinase-3ß Ser(9) and with inhibition of insulin-stimulated 3-O-methyl-d-glucose (3MG) transport but not with inhibition of the phosphorylation of insulin receptor-ß Tyr(1158/62/63). Furthermore, caffeine enhanced phosphorylation of IRS-1 Ser(307) and an IRS-1 Ser(307) kinase, inhibitor-κB kinase (IKK)-α/ß Ser(176/180). Blockade of IKK/IRS-1 Ser(307) by caffeic acid ameliorated the caffeine-induced downregulation of IRS-1 Tyr(612) phosphorylation and 3MG transport. Caffeine also increased the phosphorylation of IRS-1 Ser(789) and an IRS-1 Ser(789) kinase, 5'-AMP-activated protein kinase (AMPK). However, inhibition of IRS-1 Ser(789) and AMPK phosphorylation by dantrolene did not rescue the caffeine-induced downregulation of IRS-1 Tyr(612) phosphorylation or 3MG transport. In addition, caffeine suppressed the phosphorylation of insulin-stimulated IRS-1 Ser(636/639) and upstream kinases, including the mammalian target of rapamycin and p70S6 kinase. Intravenous injection of caffeine at a physiological dose (5 mg/kg) in rats inhibited the phosphorylation of insulin-stimulated IRS-1 Tyr(612) and Akt Ser(473) in epitrochlearis muscle. Our results indicate that caffeine inhibits insulin signaling partly through the IKK/IRS-1 Ser(307) pathway, via a Ca(2+)- and AMPK-independent mechanism in skeletal muscle.

Role of c-Fyn in FGF-2-mediated tube-like structure formation by murine brain capillary endothelial cells.

Tube formation of endothelial cells is an important step of angiogenesis. However, little is known about the molecular mechanisms underlying growth factor-mediated tube formation by endothelial cells. FGF-2 stimulates tube formation by a murine brain capillary endothelial cell line, IBE cells, when cultured on collagen gels (differentiation-associated culture condition), whereas cells proliferate and migrate without forming tube on fibronectin-coated surface (proliferation/migration-associated condition). To elucidate FGF-2-mediated signal transduction pathways leading to tube formation by endothelial cells, we focused on the contribution of Src family kinases. Src family kinase inhibitor PP2 attenuated FGF-2-induced tube formation. Stable expression of kinase-inactive c-Src in IBE cells demonstrated no dominant negative effect on FGF-2-induced tube formation. In vitro kinase assay revealed that c-Fyn was activated by FGF-2 only in cells cultured on collagen gels. Three independent cell lines, expressing kinase-inactive c-Fyn, all exhibited attenuation of FGF-2-mediated tube formation. However, FGF-2-mediated proliferation or migration was not clearly perturbed in these cells. These results show the first time that c-Fyn plays a pivotal role in tube formation by endothelial cells.

Combined adrenal adenoma and myelolipoma in a patient with Cushing's syndrome: case report and review of the literature.

Myelolipoma is an uncommon benign tumor of unknown etiology and adrenal myelolipoma is rarely associated with endocrine disorders. We report a 67-year-old woman with Cushing's syndrome due to left adrenal adenoma associated with myelolipoma. The patient underwent laparoscopic left adrenalectomy and pathological examination revealed an adrenocortical adenoma associated with myelolipoma. To the best of our knowledge, 25 cases of endocrine dysfunction associated with myelolipoma have been reported in the English and Japanese literature. We review and discuss the pathogenesis of adrenal myelolipoma.