Myofiber degeneration/regeneration is induced in the cachectic ApcMin/+ mouse.

Cachexia is characterized as an inflammatory state induced by the cancer environment, which is accompanied by the loss of muscle and fat mass. Well-investigated mechanisms of cachexia include the suppression of myofiber protein synthesis and the induction of the protein degradation. However, it is not well characterized whether chronic inflammation during cachexia induces myofiber degeneration, which contributes to muscle mass loss and decreased functional capacity. The purpose of this study was to determine whether Apc(Min/+) mice, which demonstrate a chronic systemic inflammatory state due to an intestinal tumor burden, undergo cachexia and whether the myofibers exhibit signs of degeneration and/or regeneration. Six-month-old female Apc(Min/+) body weight decreased 21% compared with C57BL/6 mice and was not the result of blunted growth. Apc(Min/+) gastrocnemius muscle was reduced 45%, and soleus mean fiber cross-sectional area decreased 24% vs. C57BL/6 mice. Soleus muscle morphology demonstrated pathology of myofibers undergoing degeneration and/or regeneration. These data demonstrate that the Apc(Min/+) mouse becomes cachectic by 6 mo of age and that skeletal muscle degeneration and regeneration may be related to the muscle loss.     

Interleukin-6 and cachexia in ApcMin/+ mice

The Apc(Min/+) mouse has a mutation in the Apc tumor suppressor gene and develops intestinal polyps, beginning at 4 wk of age. This mouse develops cachexia by 6 mo, characterized by significant loss of muscle and fat tissue. The purpose of the present study was to determine the role of circulating interleukin-6 (IL-6) and the polyp burden for the development of cachexia in Apc(Min/+) mice. At 26 wk of age, mice exhibiting severe cachectic symptoms had a 61% decrease in gastrocnemius muscle weight, complete loss of epididymal fat, a 10-fold increase in circulating IL-6 levels, and an 89% increase in intestinal polyps compared with mildly cachectic animals. Apc(Min/+)/IL-6(-/-) mice did not lose gastrocnemius muscle mass or epididymal fat pad mass while overall polyp number decreased by 32% compared with Apc(Min/+) mice. Plasmid-based IL-6 overexpression in Apc(Min/+)/IL-6(-/-) mice led to a decrease in gastrocnemius muscle mass and epididymal fat pad mass and increased intestinal polyp burden. IL-6 overexpression did not induce cachexia in non-tumor-bearing mice. These data demonstrate that IL-6 is necessary for the onset of adipose and skeletal muscle wasting in the Apc(Min/+) mouse and that circulating IL-6 can regulate Apc(Min/+) mouse tumor burden.     

The regulation of skeletal muscle protein turnover during the progression of cancer cachexia in the Apc(Min/+) mouse

Muscle wasting that occurs with cancer cachexia is caused by an imbalance in the rates of muscle protein synthesis and degradation. The Apc(Min/+) mouse is a model of colorectal cancer that develops cachexia that is dependent on circulating IL-6. However, the IL-6 regulation of muscle protein turnover during the initiation and progression of cachexia in the Apc(Min/+) mouse is not known. Cachexia progression was studied in Apc(Min/+) mice that were either weight stable (WS) or had initial (≤5%), intermediate (6-19%), or extreme (≥20%) body weight loss. The initiation of cachexia reduced %MPS 19% and a further ～50% with additional weight loss. Muscle IGF-1 mRNA expression and mTOR targets were suppressed with the progression of body weight loss, while muscle AMPK phosphorylation (Thr 172), AMPK activity, and raptor phosphorylation (Ser 792) were not increased with the initiation of weight loss, but were induced as cachexia progressed. ATP dependent protein degradation increased during the initiation and progression of cachexia. However, ATP independent protein degradation was not increased until cachexia had progressed beyond the initial phase. IL-6 receptor antibody administration prevented body weight loss and suppressed muscle protein degradation, without any effect on muscle %MPS or IGF-1 associated signaling. In summary, the %MPS reduction during the initiation of cachexia is associated with IGF-1/mTOR signaling repression, while muscle AMPK activation and activation of ATP independent protein degradation occur later in the progression of cachexia. IL-6 receptor antibody treatment blocked cachexia progression through the suppression of muscle protein degradation, while not rescuing the suppression of muscle protein synthesis. Attenuation of IL-6 signaling was effective in blocking the progression of cachexia, but not sufficient to reverse the process.     

Metabolic and physiologic characteristics of skeletal muscle determine its response to clenbuterol treatment

beta-Adrenoceptor agonists are reported to induce skeletal muscle hypertrophy and hence serve as valuable adjunct to the treatment of wasting disorders. In the present study, we attempted to find out whether metabolic and physiologic characteristics of fibres are important in determining skeletal muscle response to clenbuterol (an adrenergic receptor agonist) therapy, as proposed in the treatment of wasting disorders. The treatment of mice with clenbuterol (2 mg/kg body wt for 30 days) resulted in skeletal muscle hypertrophy, more common amongst fast-twitch glycolytic fibres/muscle, with increase in body mass and a parallel rise in muscle mass to body mass ratio. Measurement of fibre diameters in soleus (rich in slow-twitch oxidative fibres), ALD or anterior latissimus dorsi (with a predominance of fast-twitch glycolytic fibres) and gastrocnemius (a mixed-type of muscle) from clenbuterol-treated mice for 30 days revealed noticeable increase in the per cent population of narrow slow-twitch fibre and a corresponding decline in white-type or fast-twitch glycolytic fibres in gastrocnemius and ALD. As revealed by counting of muscle cells in soleus, narrow red fibres declined with corresponding increase in white-type glycolytic fibres population. A significant decline in the succinic dehydrogenase activity was observed, thereby suggesting abnormality in oxidative activity of skeletal muscles in response to clenbuterol therapy.     

Altered expression of skeletal muscle myosin isoforms in cancer cachexia

Cachexia is commonly seen in cancer and ischaracterized by severe muscle wasting, but little is known about theeffect of cancer cachexia on expression of contractile protein isoforms such as myosin. Other causes of muscle atrophy shift expression ofmyosin isoforms toward increased fast (type II) isoform expression. Weinjected mice with murine C-26 adenocarcinoma cells, a tumor cell linethat has been shown to cause muscle wasting. Mice were killed 21 daysafter tumor injection, and hindlimb muscles were removed. Myosin heavychain (MHC) and myosin light chain (MLC) content was determined inmuscle homogenates by SDS-PAGE. Body weight was significantly lower intumor-bearing (T) mice. There was a significant decrease in muscle massin all three muscles tested compared with control, with the largestdecrease occurring in the soleus. Although no type IIb MHC was detectedin the soleus samples from control mice, type IIb comprised 19% of thetotal MHC in T soleus. Type I MHC was significantly decreased in T vs. control soleus muscle. MHC isoform content was not significantly different from control in plantaris and gastrocnemius muscles. Thesedata are the first to show a change in myosin isoform expression accompanying muscle atrophy during cancer cachexia.

     

Fiber type-specific nitric oxide protects oxidative myofibers against cachectic stimuli

Oxidative skeletal muscles are more resistant than glycolytic muscles to cachexia caused by chronic heart failure and other chronic diseases. The molecular mechanism for the protection associated with oxidative phenotype remains elusive. We hypothesized that differences in reactive oxygen species (ROS) and nitric oxide (NO) determine the fiber type susceptibility. Here, we show that intraperitoneal injection of endotoxin (lipopolysaccharide, LPS) in mice resulted in higher level of ROS and greater expression of muscle-specific E3 ubiqitin ligases, muscle atrophy F-box (MAFbx)/atrogin-1 and muscle RING finger-1 (MuRF1), in glycolytic white vastus lateralis muscle than in oxidative soleus muscle. By contrast, NO production, inducible NO synthase (iNos) and antioxidant gene expression were greatly enhanced in oxidative, but not in glycolytic muscles, suggesting that NO mediates protection against muscle wasting. NO donors enhanced iNos and antioxidant gene expression and blocked cytokine/endotoxin-induced MAFbx/atrogin-1 expression in cultured myoblasts and in skeletal muscle in vivo. Our studies reveal a novel protective mechanism in oxidative myofibers mediated by enhanced iNos and antioxidant gene expression and suggest a significant value of enhanced NO signaling as a new therapeutic strategy for cachexia.     

A null mutation in skeletal muscle FAT/CD36 reveals its essential role in insulin- and AICAR-stimulated fatty acid metabolism

Fatty acid translocase (FAT)/CD36 is involved in regulating the uptake of long-chain fatty acids into muscle cells. However, the contribution of FAT/CD36 to fatty acid metabolism remains unknown. We examined the role of FAT/CD36 on fatty acid metabolism in perfused muscles (soleus and red and white gastrocnemius) of wild-type (WT) and FAT/CD36 null (KO) mice. In general, in muscles of KO mice, 1) insulin sensitivity and 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) sensitivity were normal, 2) key enzymes involved in fatty acid oxidation were altered minimally or not at all, and 3) except for an increase in soleus muscle FATP1 and FATP4, these fatty acid transporters were not altered in red and white gastrocnemius muscles, whereas plasma membrane-bound fatty acid binding protein was not altered in any muscle. In KO muscles perfused under basal conditions (i.e., no insulin, no AICAR), rates of hindquarter fatty acid oxidation were reduced by 26%. Similarly, in oxidative but not glycolytic muscles, the basal rates of triacylglycerol esterification were reduced by 40%. When muscles were perfused with insulin, the net increase in fatty acid esterification was threefold greater in the oxidative muscles of WT mice compared with the oxidative muscles in KO mice. With AICAR-stimulation, the net increase in fatty acid oxidation by hindquarter muscles was 3.7-fold greater in WT compared with KO mice. In conclusion, the present studies demonstrate that FAT/CD36 has a critical role in regulating fatty acid esterification and oxidation, particularly during stimulation with insulin or AICAR.     

Anatomic capillarization is maintained in relative excess of fiber oxidative capacity in some skeletal muscles of late middle-aged rats.

The anatomic size of the capillary-to-fiber (C/F) interface plays an important role in O(2) flux from blood to tissue by determining the surface area available for diffusion and is maintained in relative proportion to fiber mitochondrial volume across a wide range of muscle aerobic capacity. In the present study, we examined an estimate of the anatomic size of the C/F interface [the quotient of the individual C/F ratio and fiber perimeter, C/F perimeter exchange (CFPE) index] and fiber oxidative capacity in different skeletal muscles, or muscle regions, to test the hypothesis that capillarization would be maintained in relative excess of reduced fiber oxidative capacity in aged muscles. The right gastrocnemius, plantaris, and soleus muscles from young adult (8 mo old) and late middle-aged (28-30 mo old) Fischer 344 x Brown Norway F1 hybrid rats were excised for evaluation of flux through electron transport chain complexes I-III and/or morphometric estimation of capillarization. Muscle mass was lower in the gastrocnemius muscles of the older animals (2,076 +/- 32 vs. 1,825 +/- 47 mg in young adult vs. late middle-aged, respectively; mean +/- SE) but not the plantaris or soleus muscles. Fibers were smaller in the white region of gastrocnemius muscles but larger in the red region of gastrocnemius muscles of the older animals. There was no difference in the number of capillaries around a fiber, the individual C/F ratio, or the CFPE index between groups for any muscle/region, whereas flux through complexes I-III was reduced by 29-43% in late middle-aged animals. Thus the greater quotient of indexes of anatomic capillarity (individual C/F ratio or CFPE index) and fiber oxidative capacity in soleus and the white region of gastrocnemius muscles, but not in the red region of gastrocnemius muscles of the older animals, shows that anatomic capillarity is maintained in relative excess of oxidative capacity in some muscle regions in late middle-aged rats.     

Diminished AMPK signaling response to fasting in thioredoxin-interacting protein knockout mice

Thioredoxin-interacting protein (Txnip) knockout (TKO) mice exhibit impaired response to fasting. Herein, we showed that activation of adenine monophosphate-activated protein kinase and cellular AMP levels were diminished in the heart and soleus muscle but not in gastrocnemius muscle of fasting TKO mice. Similarly, glycogen content in fasted TKO mice was increased in oxidative muscles but was not different in glycolytic muscles. These data suggest Txnip deficiency has a higher impact on oxidative muscle than glycolytic muscles and provide new insights into the metabolic role of Txnip.     

Rat skeletal muscle triacylglycerol utilization during exhaustive swimming

The utilization of triacylglycerol in slow oxidative, fast oxidative-glycolytic, and fast glycolytic skeletal muscle fiber types was examined in rats subjected to a prolonged exhaustive swim. Significant reductions of intramuscular triacylglycerol occurred following 2 h and 40 min of swimming in all muscles containing a predominance of slow oxidative and fast oxidative-glycolytic fibers, which possess a high capacity for free fatty acid oxidation. Triacylglycerol content in the soleus decreased by 48%, and reductions of 41, 29, and 27% were measured in the red vastus lateralis, red gastrocnemius, and plantaris muscles, respectively. In the white vastus lateralis and white gastrocnemius muscles (fast glycolytic fibers) triacylglycerol concentrations were unaffected. In all muscles the variability of intramuscular triacylglycerol measurements between animals was 20-50% and the within animal variance (right vs. left hindlimb) was similar. Analytical repeatability was approximately 10% in all muscles and significantly less than the between- and within-animal variances. It was concluded that a real biological variation exists in the triacylglycerol content of all rat skeletal muscles and that intramuscular triacylglycerol is an important energy source during prolonged exercise of moderate intensity.     

Inhibition of intestinal polyposis with reduced angiogenesis in ApcMin/+ mice due to decreases in c-Myc expression

The c-myc oncogene plays an important role in tumorigenesis and is frequently deregulated in many human cancers, including gastrointestinal cancers. In humans, mutations of the adenomatous polyposis coli (Apc) tumor suppressor gene occur in most colorectal cancers. Mutation of Apc leads to stabilization of beta-catenin and increases in beta-catenin target gene expression (c-myc and cyclin D1), whose precise functional significance has not been examined using genetic approaches. Apc(Min/+) mice are a model of familial adenomatous polyposis and are heterozygous for an Apc truncation mutation. We have developed a model for examining the role of c-Myc in Apc-mediated tumorigenesis. We crossed c-myc(+/-) mice to Apc(Min/+) to generate Apc(Min/+) c-myc(+/-) animals. The compound Apc(Min/+) c-myc(+/-) mice were used to evaluate the effect of c-myc haploinsufficiency on the Apc(Min/+) phenotype. We observed a significant reduction in tumor numbers in the small intestine of Apc(Min/+) c-myc(+/-) mice compared with control Apc(Min/+) c-myc(+/+) mice. In addition, we observed one to three polyps per colon in Apc(Min/+) c-myc(+/+) mice, whereas only two lesions were observed in the colons of Apc(Min/+) mice that were haploinsufficient for c-myc. Moreover, reduction in c-myc levels resulted in a significant increase in the survival of these animals. Finally, we observed marked decreases in vascular endothelial growth factor, EphA2, and ephrin-B2 expression as well as marked decreases in angiogenesis in intestinal polyps in Apc(Min/+) c-myc(+/-) mice. This study shows that c-Myc is critical for Apc-dependent intestinal tumorigenesis in mice and provides a potential therapeutic target in the treatment of colorectal cancer.     

Effect of acute exercise on the content of free sphinganine and sphingosine in different skeletal muscle types of the rat.

It has previously been shown that prolonged exercise of moderate intensity reduces the content of ceramide in each type of skeletal muscle. This was accompanied by a reduction in the activity of neutral, Mg++-dependent sphingomyelinase (the major enzyme responsible for ceramide formation from sphingomyelin) in the soleus and red gastrocnemius, but not in the white gastrocnemius (A. Dobrzyń and J. Górski, Am. J. Physiol.: Endorcinol. Metab. 282: E277 - E285, 2002). No other data on regulation of ceramide metabolism in contracting muscles are available. The aim of the present study was to examine the content of sphinganine (a key precursor of ceramide on the de novo synthesis route) and the content of sphingosine (the main product of ceramide catabolism) in different skeletal muscle types after two kinds of acute exercise. The experiments were carried out on 30 male Wistar rats, 250 - 280 g of body weight. The rats were divided equally into three groups: 1 - control, 2 - run until exhaustion (1200 m/h, +10 degree incline), 3 - a group in which the sciatic nerve was stimulated 10 min with tetanic pulses (60 pulses/min). Samples were taken of the soleus and of the red and white section of the gastrocnemius. These muscles are composed mostly of the slow-twitch oxidative, fast-twitch oxidative-glycolytic and fast-twitch glycolytic fibers, respectively. Lipids were extracted with chloroform/methanol. Sphinganine and sphingosine were quantified by high-performance liquid chromatography. At rest, the content of sphinganine in the soleus was higher than in the red gastrocnemius (p < 0.05), and in the latter, it was higher than in the white gastrocnemius (p < 0.01). Prolonged exercise increased the content of sphinganine approximately 6-fold in each muscle. The resting content of sphingosine in the soleus and in the red gastrocnemius was similar--higher than in the white gastrocnemius (p < 0.001 and p < 0.01, respectively). The content of sphingosine increased over 3-fold in the soleus and nearly 2-fold in the red and white sections of the gastrocnemius. Stimulation of the sciatic nerve increased the content of both compounds approximately 2-fold in each muscle. We conclude that acute exercise increases both de novo synthesis and catabolism of ceramide in skeletal muscles. Accumulation of sphingosine in contracting muscles may contribute to the development of fatigue.     

Effect of endurance training on the phospholipid content of skeletal muscles in the rat

Only few data are available on the effect of training on phospholipid metabolism in skeletal muscles. The aim of the present study was to examine the effect of 6 weeks of endurance training on the content of particular phospholipid fractions and on the incorporation of blood-borne [14C]-palmitic acid into the phospholipids in different skeletal muscles (white and red sections of the gastrocnemius, the soleus and the diaphragm) of the rat. Lipids were extracted from the muscles and separated using thin-layer chromatography into the following fractions: sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, cardiolipin and neutral lipids (this fraction being composed mostly of triacylglycerols). It was found that training did not affect the content of any phospholipid fraction in soleus muscle. It increased the content of sphingomyelin in white gastrocnemius muscle, cardiolipin and phosphatidylethanolamine in red gastrocnemius muscle and phosphatidylinositol in white gastrocnemius muscle and diaphragm. The total phospholipid content in red gastrocnemius muscle of the trained group was higher than in the control group. Training reduced the specific activity of sphingomyelin and cardiolipin in all muscles, phosphatidylcholine in soleus, red, and white gastrocnemius muscles, phosphatidylserine in all muscles, phosphatidylinositol in all except the soleus muscle, and phosphatidylethanolamine in hindleg muscles, but not in the diaphragm compared to the corresponding values in the sedentary group. It was concluded that endurance training affects skeletal muscle phospholipid content and the rate of incorporation of the blood-borne [14C]palmitic acid into the phospholipid moieties.     

Differential structural features in soleus and gastrocnemius of carnitine-treated cancer cachectic rats

Muscle wasting is associated with chronic diseases and cancer. Elucidation of the biological mechanism involved in the process of muscle mass loss and cachexia may help identify therapeutic targets. We hypothesized that l -carnitine treatment may differentially revert muscle fiber atrophy and other structural alterations in slow- and fast-twitch limb muscles of rats bearing the Yoshida ascites hepatoma. In soleus and gastrocnemius of tumor-bearing rats (10⁸ AH-130 Yoshida ascites hepatoma cells inoculated intraperitoneally) with and without treatment with l -carnitine (1 g/kg body weight for 7 days, intragastric), food intake, body and muscle weights, fiber typing and morphometry, morphological features, redox balance, autophagy and proteolytic, and signaling markers were explored. Levels of carnitine palmitoyl transferase were also measured in all the study muscles. l - Carnitine treatment ameliorated the atrophy of both slow- and fast-twitch fibers (gastrocnemius particularly), muscle structural alterations (both muscles), and attenuated oxidative stress, proteolytic and signaling markers (gastrocnemius). Despite that carnitine palmitoyl transferase-1 levels increased in both muscle types in a similar fashion, l -carnitine ameliorated muscle atrophy and proteolysis in a muscle-specific manner in cancer-induced cachexia. These data reveal the need to study muscles of different fiber type composition and function to better understand whereby l -carnitine exerts its beneficial effects on the myofibers in muscle wasting processes. These findings also have potential clinical implications, since combinations of various exercise and muscle training modalities with l -carnitine should be specifically targeted for the muscle groups to be trained.     

Adaptation in synergistic muscles to soleus and plantaris muscle removal in the rat hindlimb.

Although the soleus muscle comprises only 6% of the ankle plantar flexor mass in the rat, a major role in stance and walking has been ascribed to it. The purpose of this study was to determine if removal of the soleus muscle would result in adaptations in the remaining gastrocnemius and plantaris muscles due to the new demands for force production imposed on them during stance or walking. A second purpose was to determine whether the mass or the fiber type of the muscle(s) removed was a more important determinant of compensatory adaptations. Male Sprague-Dawley rats underwent bilateral removal of soleus muscle, plantaris muscle, or both muscles. For comparison, compensatory hypertrophy was induced in soleus and plantaris muscles by gastrocnemius muscle ablation. After forty days, synergist muscles remaining intact were removed. Mass, and oxidative, glycolytic, and contractile enzyme activities were determined. Despite its role in stance and slow walking, removal of the soleus muscle did not elicit a measurable alteration in muscle mass, or in citrate synthase, lactate dehydrogenase, or myofibrillar ATPase activity in gastrocnemius or plantaris muscles. Similarly, removal of the plantaris muscle, or soleus and plantaris muscles, had no effect on the gastrocnemius muscle, suggesting that this muscle was able to easily meet the new demands placed on it. These results suggest that amount of muscle mass removed, rather than fiber type, is the most important stimulus for compensatory hypertrophy. They also suggest that slow-twitch motor units in the gastrocnemius muscle play an important role during stance and locomotion in the intact animal.     

Skeletal muscle fiber type comparison of pyruvate dehydrogenase phosphatase activity and isoform expression in fed and food-deprived rats

Fiber type specificity of pyruvate dehydrogenase (PDH) phosphatase (PDP) was determined in fed (CON) and 48-h food-deprived (FD) rats. PDP activity and isoform protein content were determined in soleus (slow-twitch oxidative), red gastrocnemius (RG; fast-twitch oxidative glycolytic), and white gastrocnemius (WG; fast-twitch glycolytic) muscles. When normalized for mitochondrial volume, there was no difference in PDP activity between muscle types or CON and FD. When expressed per gram wet tissue weight, PDP activity was higher in RG compared with soleus and WG in both CON and FD rats. PDP activities from CON muscles were 1.48 +/- 0.19, 2.68 +/- 0.65, and 1.20 +/- 0.33 nmol x min(-1) x g wet tissue wt(-1) in soleus, RG, and WG, respectively, and decreased in FD muscles (1.22 +/- 0.22, 2.00 +/- 0.57, and 0.84 +/- 0.18 nmol x min(-1) x g wet tissue wt(-1)). This correlated with increased PDP2 protein, however, only in RG, as PDP2 was not detectable in soleus or WG. PDP1 protein was not responsive to food deprivation in all fiber types. In conclusion, PDP activity and protein content were higher in fast-twitch oxidative glycolytic muscles from CON and FD rats, identifying a unique inter- and intramuscular distribution. FD induced a small but significant decrease in PDP activity that was partially due to decreases in PDP2 protein. As a result, coordinate changes to PDP activity opposite to those of the other regulatory enzyme, PDH kinase, during food deprivation would maximize the inactivation of skeletal muscle PDH and enhance carbohydrate conservation during periods of limited carbohydrate supply.     

Downregulation of ubiquitin-dependent proteolysis by eicosapentaenoic acid in acute starvation.

A number of acute wasting conditions are associated with an upregulation of the ubiquitin-proteasome system in skeletal muscle. Eicosapentaenoic acid (EPA) is effective in attenuating the increased protein catabolism in muscle in cancer cachexia, possibly due to inhibition of 15-hydroxyeicosatetraenoic acid (15-HETE) formation. To determine if a similar pathway is involved in other catabolic conditions, the effect of EPA on muscle protein degradation and activation of the ubiquitin-proteasome pathway has been determined during acute fasting in mice. When compared with a vehicle control group (olive oil) there was a significant decrease in proteolysis of the soleus muscles of mice treated with EPA after starvation for 24 h, together with an attenuation of the proteasome "chymotryptic-like" enzyme activity and the induction of the expression of the 20S proteasome alpha-subunits, the 19S regulator and p42, an ATPase subunit of the 19S regulator in gastrocnemius muscle, and the ubiquitin-conjugating enzyme E2(14k). The effect was not shown with the related (n-3) fatty acid docosahexaenoic acid (DHA) or with linoleic acid. However, 2,3,5-trimethyl-6-(3-pyridylmethyl)1,4-benzoquinone (CV-6504), an inhibitor of 5-, 12- and 15-lipoxygenases also attenuated muscle protein catabolism, proteasome "chymotryptic-like" enzyme activity and expression of proteasome 20S alpha-subunits in soleus muscles from acute fasted mice. These results suggest that protein catabolism in starvation and cancer cachexia is mediated through a common pathway, which is inhibited by EPA and is likely to involve a lipoxygenase metabolite as a signal transducer.     

Progressive changes in adherens junction structure during intestinal adenoma formation in Apc mutant mice

The C57BL/6J-Min/+ (Min/+) mouse bears a mutant Apc gene and therefore is an important in vivo model of intestinal tumorigenesis. Min/+ mice develop adenomas that exhibit loss of the wild-type Apc allele (Apc(Min/-)). Previously, we found that histologically normal enterocytes bearing a truncated Apc protein (Apc(Min/+)) migrated more slowly in vivo than enterocytes with either wild-type Apc (Apc(+/+)) or with heterozygous loss of Apc protein (Apc(1638N)). To study this phenotype further, we determined the effect of the Apc(Min) mutation upon cell-cell adhesion by examining the components of the adherens junction (AJ). We observed a reduced association between E-cadherin and beta-catenin in Apc(Min/+) enterocytes. Subcellular fractionation of proteins from Apc(+/+), Apc(Min/+), and Apc(Min/-) intestinal tissues revealed a cytoplasmic localization of intact E-cadherin only in Apc(Min/+), suggesting E-cadherin internalization in these enterocytes. beta-Catenin tyrosine phosphorylation was also increased in Apc(Min/+) enterocytes, consistent with its dissociation from E-cadherin. Furthermore, Apc(Min/+) enterocytes showed a decreased association between beta-catenin and receptor protein-tyrosine phosphatase beta/zeta (RPTPbeta/zeta), and Apc(Min/-) cells demonstrated an association between beta-catenin and receptor protein-tyrosine phosphatase gamma. In contrast to the Apc(Min/+) enterocytes, Apc(Min/-) adenomas displayed increased expression and association of E-cadherin, beta-catenin, and alpha-catenin relative to Apc(+/+) controls. These data show that Apc plays a role in regulating adherens junction structure and function in the intestine. In addition, discovery of these effects in initiated but histologically normal tissue (Apc(Min/+)) defines a pre-adenoma stage of tumorigenesis in the intestinal mucosa.     

Effect of streptozotocin-diabetes on the functioning of the sphingomyelin-signalling pathway in skeletal muscles of the rat.

AIMS/HYPOTHESIS: Ceramide is the main second messenger in the sphingomyelin-transmembrane signalling pathway. The compound is likely to play a role in the induction of insulin resistance. The aim of the present study was to examine the effect of streptozotocin diabetes on the content and composition of ceramides and sphingomyelins and the activity of neutral Mg (2+)-dependent sphingomyelinase and acid sphingomyelinase in different types of skeletal muscle of the rat. METHODS: The experiments were carried out on two groups of male Wistar rats weighing 250-280 g: controls and those treated with streptozotocin at a dose of 60 mg/kg. Determinations were performed on three types of skeletal muscle: the slow-twitch oxidative (soleus), fast-twitch oxidative-glycolytic (red section of the gastrocnemius) and fast-twitch glycolytic (white section of the same muscle). The content and composition of ceramide- and sphingomyelin-fatty acids were determined using gas-liquid chromatography. The activity of the enzymes was measured using N-[(14)CH (3)]-sphingomyelin as the substrate. RESULTS: Twelve different ceramides and sphingomyelins were identified and quantified in each muscle with regard to the fatty acid residue. The ratio of total content of ceramide-saturated fatty acids to the total content of ceramide-unsaturated fatty acids was more than two. In the case of sphingomyelin, the ratio was similar to ceramide in the soleus and much higher in both sections of the gastrocnemius. Treatment with streptozotocin increased the total content of ceramide-fatty acids by 78% (p < 0.001) in the soleus, 27.5% (p < 0.01) in the red and 36.9% (p < 0.001) in the white section of the gastrocnemius. Concomitantly, the total content of sphingomyelin-fatty acids decreased by 43.8%, 31.2%, 24.8% (p < 0.001 in each case) in the respective muscles. The activity of neutral Mg (2+)-dependent sphingomyelinase was elevated by 69.5%, 105.9% and 62.3% in the soleus and red and white gastrocnemius, respectively (p < 0.001 for each muscle). The activity of acid sphingomyelinase was stable in the soleus and white gastrocnemius and decreased by 15.7% (p < 0.01) in the red gastrocnemius. CONCLUSION/INTERPRETATION: The results obtained show that insulin deficiency results in elevation in the content of ceramide in skeletal muscles. This indicates that the hormone is involved in regulation of the activity of the sphingomyelin-signalling pathway in the muscles.     

Effect of exercise on biological pathways in ApcMin/+ mouse intestinal polyps

Many epidemiological studies have demonstrated that level of exercise is associated with reduced colorectal cancer risk. Treadmill training can decrease Apc(Min/+) mouse intestinal polyp number and size, but the mechanisms remain unclear. Understanding the molecular changes in the tumor following exercise training may provide insight on the mechanism by which exercise decreases Apc(Min/+) mouse polyp formation and growth. The purpose of this study was to determine if exercise can modulate Apc(Min/+) mouse intestinal polyp cellular signaling related to tumor formation and growth. Male Apc(Min/+) mice were randomly assigned to control (n = 20) or exercise (n = 20) treatment groups. Exercised mice ran on a treadmill at a moderate intensity (18 m/min, 60 min, 6 days/wk, 5% grade) for 9 wk. Polyps from Apc(Min/+) mice were used to quantify markers of polyp inflammation, apoptosis, and beta-catenin signaling. Exercise decreased the number of macrophages in polyps by 35%. Related to apoptosis, exercise decreased the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells by 73% in all polyps. Bax protein expression in polyps was decreased 43% by exercise. beta-Catenin phosphorylation was elevated 3.3-fold in polyps from exercised mice. Moderate-intensity exercise training alters cellular pathways in Apc(Min/+) mouse polyps, and these changes may be related to the exercise-induced reduction in polyp formation and growth.