High-fat, high-fructose, high-cholesterol feeding causes severe NASH and cecal microbiota dysbiosis in juvenile Ossabaw swine.

Pediatric obesity and nonalcoholic steatohepatitis (NASH) are on the rise in industrialized countries, yet our ability to mechanistically examine this relationship is limited by the lack of a suitable higher animal models. Here, we examined the effects of high-fat, high-fructose corn syrup, high-cholesterol Western-style diet (WD)-induced obesity on NASH and cecal microbiota dysbiosis in juvenile Ossabaw swine. Juvenile female Ossabaw swine (5 wk old) were fed WD (43.0% fat; 17.8% high-fructose corn syrup; 2% cholesterol) or low-fat diet (CON/lean; 10.5% fat) for 16 wk ( n = 6 each) or 36 wk ( n = 4 each). WD-fed pigs developed obesity, dyslipidemia, and systemic insulin resistance compared with CON pigs. In addition, obese WD-fed pigs developed severe NASH, with hepatic steatosis, hepatocyte ballooning, inflammatory cell infiltration, and fibrosis after 16 wk, with further exacerbation of histological inflammation and fibrosis after 36 wk of WD feeding. WD feeding also resulted in robust cecal microbiota changes including increased relative abundances of families and genera in Proteobacteria ( P < 0.05) (i.e., Enterobacteriaceae, Succinivibrionaceae, and Succinivibrio) and LPS-containing Desulfovibrionaceae and Desulfovibrio and a greater ( P < 0.05) predicted microbial metabolic function for LPS biosynthesis, LPS biosynthesis proteins, and peptidoglycan synthesis compared with CON-fed pigs. Overall, juvenile Ossabaw swine fed a high-fat, high-fructose, high-cholesterol diet develop obesity and severe microbiota dysbiosis with a proinflammatory signature and a NASH phenotype directly relevant to the pediatric/adolescent and young adult population.

Muscle adaptation to exercise: New Saltin's paradigms.

The title assigned for my lecture at the Saltin Symposium was "Muscle adaptation to exercise: new paradigms." The title's topic made me remember that some of Saltin's paradigms for his development of a novel exercise model were either originated by him or modified by him from existing information. Therefore, I deemed it would be instructive for future generations to consider one facet of his 54-year career--human exercise models. I arbitrarily selected to share five examples of new paradigm models initiated by Saltin. They are: bed rest; arms vs legs; one leg vs the other leg; myokine communication from skeletal muscle to other organs/tissues; and 42-day cross-country skiing expedition. I arbitrarily selected the above as examples of novel approaches that he used to the study humans during maximal endurance exercise. Noteworthy though is that Saltin's lifetime demeanor, itself, is a model for other scientists. In final analysis, the world is richer due to his passion to study humans to advance medical science by uncovering mechanisms as to how the human body is constructed to perform endurance types of exercise at maximal intensities and durations.

Basic concepts about genes, inactivity and aging.

Remarkably, 80-year-old humans who have partaken in lifelong aerobic or strength training have maximal aerobic capacities or muscle strengths comparable with that of sedentary individuals aged 50 or 55-year-old, respectively. Such delays in functional aging are clinically significant because lower aerobic and lower strength capacities increase the risk of premature death. In this short review, we speculate that the lack of daily physical activity induces evolutionarily selected mechanisms to use or lose, one of which is related to nutritional status.

Lack of adequate appreciation of physical exercise's complexities can pre-empt appropriate design and interpretation in scientific discovery.

Two major issues are presented. First, a challenge is made by us that a misunderstanding of physiology has led to incomplete or wrong functional designations of genes in some cases. Normal physiological processes are dynamic, integrated and periodic, and, therefore, it is difficult to define normal physiological function by looking at a single time point or single process in a non-stressed subject. The ability of the organism to successfully respond to homeostatic disruptions defines normal physiology. Genes were selected for survival and to appropriately respond to stresses, such as physical activity. Omitting gene functions by restricting them to non-stressful conditions could lead to less than optimal primary preventions, treatments and cures for diseases. Physical exercise, as a stressor, should be used to better demonstrate the complete functional classifications of some genes. Second, the challenge from others of an 'exercise pill' as a mimetic of natural physical activity will be shown to be lacking a scientific basis. The concept of an 'exercise pill'/'exercise mimetic' demonstrates an inadequate appreciation of the complexities in integrating cell, tissue, organ and systems during both acute disruptions in homeostasis by a single bout of exercise, and longer-term chronic adaptations to different types of exercise such as resistance and endurance. It is our opinion that those promoting drugs targeting a single or few molecules should not redefine the term 'exercise' and exercise concepts in an attempt to sensationalize findings. Additionally, the scientific criteria that the authors demand to be met to legitimately use the terms 'exercise pill' and 'exercise mimetic' are presented.

Linking performance and chronic disease risk: indices of physical performance are surrogates for health.

Recent studies have identified a remarkable association between indices of athletic performance and optimal health of the general public. Both high aerobic capacity and high skeletal muscle strength are associated with lower mortality. Furthermore, higher aerobic capacity and often higher skeletal muscle strength are associated with a lower prevalence of most chronic diseases. Also, maintenance of aerobic capacity and skeletal muscle strength by lifelong physical activity delays the biological ageing in most organ systems, therefore delaying premature death. These facts raise the question whether associations between high aerobic capacity and muscle strength are causally or associatively related to either metabolic health or elite performance. If a causal relationship was noted at the molecular level, it would have major public health implications. In this review, evidence is presented for the assertion that research on elite athletes and chronic disease prevention by exercise is actually addressing the same biochemical, physiological and genomic phenomena.

Primary rat muscle progenitor cells have decreased proliferation and myotube formation during passages.

Adult skeletal muscle contains populations of satellite cells and muscle-derived stem cells that are capable of forming multinucleate myotubes. The purpose of this study was to determine the phenotype of cells isolated from a common satellite cell isolation and passaging procedure from whole skeletal muscle. To ascertain the characteristics of the cellular phenotype, the myogenic markers MyoD and desmin, the satellite-cell-specific marker Pax7, and the haemopoietic stem cell markers CD34 and CD45 were examined by immunohistochemical analysis. Immediately after isolation, > 90% myogenic marker-positive cells were positive for desmin, MyoD and Pax7. In contrast, approximately 10% of the isolated cells expressed only CD34 or CD45. After three passages, the percentage of cells that were positive for the myogenic markers desmin, MyoD and Pax7 was reduced to approximately 55%, while the population of CD34- or CD45-positive cells increased to approximately 30% after the third passage. Immunohistochemical detection of bromodeoxyuridine demonstrated that the number of proliferating cells decreased progressively after each passaging. Finally, after the third passage the percentage of nuclei in myotubes decreased from 46.7% to 12.5%. Since passaging of muscle progenitor cells is common practice, the results of the current report suggest that characterization of cell heterogeneity needs to be made frequently.

Cytochrome c mRNA levels decrease in senescent rat heart.

The concentration of mitochondria decreases in the heart as rodents age from maturity to senescence. The reason for this change is not known. One purpose of the present study was to determine if cytochrome c mRNA, representative of proteins of the inner mitochondrial membrane, decreased in the hearts of Fischer 344 rats as they aged from 12 to 24 months. Twenty-two percent less cytochrome c mRNA existed per given quantity of extracted RNA from the heart in 24-month-old rats as compared with the 12-month-old group. No change in the quantities of cardiac alpha-actin mRNA, Ca2+/calmodulin protein kinase II mRNA or 18S rRNA was noted between 12- and 24-month-old hearts. Thus, the decrease in cytochrome c mRNA suggests that decreases in mRNAs for proteins of the inner mitochondrial membrane could play some role in the diminished concentration of mitochondria that exists in the senescent heart.

Long-term insulin-like growth factor-I expression in skeletal muscles attenuates the enhanced in vitro proliferation ability of the resident satellite cells in transgenic mice.

Insulin-like growth factor-I (IGF-I) overexpression for 1-month in mouse skeletal muscle increases satellite cell proliferation potential. However, it is unknown whether this beneficial enhancement by IGF-I expression would persist over a longer-term duration in aged mice. This is an important issue to address if a prolonged course of IGF-I is to be used clinically in muscle-wasting conditions where satellite cells may become limiting. Using the IGF-I transgenic (IGF-I Tg) mouse that selectively expresses the IGF-I transgene in striated muscles, we found that 18-months of continuous IGF-I overexpression led to a loss in the enhanced in vitro proliferative capacity of satellite cells from Tg skeletal muscles. Also 18-month-old IGF-I Tg satellite cells lost the enhanced BrdU incorporation, greater pRb and Akt phosphorylations, and decreased p27(Kip1) levels initially observed in cells from 1-month-old IGF-I Tg mice. The levels of those biochemical markers reverted to similar values seen in the 18-months WT littermates. These findings, therefore, suggest that there is no further beneficial effect on enhancing satellite cell proliferation ability with persistent long-term expression of IGF-I in skeletal muscles of these transgenic mice.

Strength and aerobic training attenuate muscle wasting and improve resistance to the development of disability with aging.

By the age of 50 yrs old, humans become aware that they are losing muscle strength (mass) and endurance (mitochondria). A frequent symptom of neuromuscular disorders is muscle weakness (Walton, 1988). We define the aging-associated muscle wasting as a progressive neuromuscular syndrome that will lower the quality of life in the elderly by (1) decreasing the ability to lift loads (progressing to difficulty arising from a chair), and (2) decreasing endurance (leading to an inability to perform the activities of daily living, which increases health care costs). Campion (1994) states that the most successful outcome would be for the very elderly to take control of the last stage of their life and make it worth living. To obtain this goal, prevention of muscle wasting is an absolute requirement. Muscle mass and motor unit number, activation, and synchronization are highly related to strength; both decrease with aging (Rodgers and Evans, 1993). Resistance-training is the best way to increase muscle mass, neural coordination, and strength. Mitochondrial concentration is highly related to endurance capacity in young and old (Holloszy and Coyle, 1984). Both muscle contractile and mitochondrial protein decrease with aging in sedentary humans (reviewed by Rodgers and Evans, 1993). Endurance training, which is the best exercise to increase/maintain mitochondrial concentration with aging, has generally resulted in relatively small functional benefits to nursing home patients (Fiatarone et al., 1994).(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochrome c protein synthesis rate in rat skeletal muscle.

Endurance training is associated with increases in mitochondrial density, of which cytochrome c protein is an index. Increases in the synthesis rates of cytochrome c protein in skeletal muscle during endurance training have been inferred (Biochem. Biophys. Res. Commun. 66: 173, 1975; J. Biol. Chem. 252: 416, 1977). One purpose of the present study was to test these indirect approximations with direct measurements of the synthesis rates of cytochrome c protein in skeletal muscles postexercise. No change in the fractional synthesis rate of cytochrome c was detected in the red quadriceps muscle of rats either 2-7 h after a 104-min run on a motor-driven treadmill or 17-22 h after the final bout of 4 days of running 100 min/day. If the 16% increase in cytochrome c protein concentration in the red quadriceps muscle on the 5th day of training is used to calculate the nanomoles of cytochrome c synthesized per gram of wet muscle weight, the normalized rate of cytochrome c protein synthesis is increased 29% on the 5th day of training. The observation of no significant alteration in cytochrome c mRNA in the red quadriceps muscle of rats during the 1st wk of training implies that the initial increase in the synthesis rate of cytochrome c protein normalized per unit of muscle mass during treadmill training is likely to occur at a translational or posttranslational step. These results suggest that the control of increased cytochrome c expression in skeletal muscle during exercise training involves a complex mechanism.

Perspectives on molecular and cellular exercise physiology.

A challenge to applied physiologists is to continue to apply new methods to their field. The purpose of this review is to speculate how recent advances in the fields of molecular and cell biology might be applied to exercise physiology to provide greater insights into the mechanisms underlying adaptations to a single bout of exercise and to repeated bouts of exercise (training). The review is organized to consider the actions occurring outside the cell, through the cellular membrane and cytoplasm to the nucleus. These topics are as follows: blood-borne signals, polypeptide growth factors, membrane receptors (insulin receptor, adrenergic receptors, acetylcholine receptors, phosphorylation of receptors, and mutant receptors), transduction through the plasmalemma (glucose transporters and G proteins), second messengers (phosphatidylinositol phosphates and calcium), cis sequences required for muscle-specific gene expression, transcriptional regulation by physiological signals, and gene expression. Exercise physiology is a challenging discipline that integrates molecules, cell-to-cell interaction, tissues, and the whole organism during the physiological stress of exercise by the live, unanesthetized animal.

Atrophy of the soleus muscle by hindlimb unweighting.

The unweighting model is a unique whole animal model that will permit the future delineation of the mechanism(s) by which gravity maintains contractile mass in postural (slow-twitch) skeletal muscle. Since the origination of the model of rodent hindlimb unweighting almost one decade ago, about half of the 59 refereed articles in which this model was utilized have been published in the Journal of Applied Physiology. Thus the purpose of this review is to provide, for those researchers with an interest in the hindlimb unweighting model, a summation of the data derived from this model to data and hopefully to stimulate research interest in aspects of the model for which data are lacking. The stress response of the animal to hindlimb unweighting is transient, minimal in magnitude, and somewhat variable. After 1 wk of unweighting, the animal exhibits no chronic signs of stress. The atrophy of the soleus muscle, a predominantly slow-twitch muscle, is emphasized because unweighting preferentially affects it compared with other calf muscles, which are mainly fast-twitch muscles. The review considers the following information about the unweighted soleus muscle: electromyogram activity, amount and type of protein lost, capillarization, oxidative capacity, glycolytic enzyme activities, fiber cross section, contractile properties, glucose uptake, sensitivity to insulin, protein synthesis and degradation rates, glucocorticoid receptor numbers, responses of specific mRNAs, and changes in metabolite concentrations.

Skeletal muscle enlargement with weight-lifting exercise by rats.

A rat model of weight lifting that produces skeletal muscle enlargement utilizing regimens of resistance training similar to those employed in human training programs is described. The model consists of electrically stimulating the lower leg muscles to contract against a weighted pulley bar. Animals were subjected to training protocols employing low-frequency repetitions with high training loads within a training session. Initial maximum loads of between 200 and 800 g were progressively increased during the 16 wk of training. Work done at the end of the training period increased to an average value 66% higher than that performed at the start of training. The gastrocnemius wet weight and protein content increased (P less than 0.001) by 18 and 17%, respectively, in the stimulated loaded leg in all but one training protocol, a program in which rats were exercised more frequently. RNA content, but not concentration, was increased in the trained gastrocnemius muscle from each protocol, resulting in muscle enlargement. These data indicate that the basic model presented here provides a suitable vehicle for future studies into the biochemical events that may cause skeletal muscle enlargement during resistance training but, based on limited data, suggests that an increased frequency of training days may hinder muscle enlargement in this model.

Cytochrome c mRNA and alpha-actin mRNA in muscles of rats fed beta-GPA.

A diet of 1% beta-guanidinopropionic acid (beta-GPA) fed to rats for weeks results in decreased muscle adenosine triphosphate and creatine phosphate concentrations (J. Biol. Chem. 249: 1060-1063, 1974), increased activities of selected mitochondrial enzymes (Biochem. J. 232: 125-131, 1985), and atrophied type IIb fibers (Lab. Invest. 33: 151-158, 1975). The hypothesis of the present study was that chronic beta-GPA feeding would increase cytochrome c mRNA in muscle and would decrease alpha-skeletal actin mRNA in type IIb muscle. Data collected supported, in part, the hypothesis. After 22 days of a 1% beta-GPA diet, cytochrome c mRNA was increased 60-67% in muscles with inherently low cytochrome c mRNA but was not altered in muscles with higher cytochrome c mRNA levels. alpha-Skeletal actin mRNA was unchanged in muscles with low and high cytochrome c mRNA after 22 days of 1% beta-GPA. After 66 days of beta-GPA feeding, both cytochrome c mRNA and alpha-skeletal actin mRNA were decreased 18 and 26%, respectively, per unit of total RNA, in white quadriceps muscle. At the same time muscles composed of predominantly type II fibers atrophied 22%, whereas type I muscle size was unaltered. These data suggest that high-energy phosphate levels could play some role in adaptive changes in muscle composition.

Protein metabolism in rat gastrocnemius muscle after stimulated chronic concentric exercise.

Previous results by use of a model of resistance exercise consisting of nonvoluntary electrical contraction of rat skeletal muscle have shown that significant gastrocnemius muscle enlargement was produced after 16 wk of chronic concentric resistance training with progressively increased weights but not after the same training program without weights (J. Appl. Physiol. 65: 950-954, 1988). In the present study we examined whether this differential effect on muscle mass between high- and low-resistance exercise is mediated through differential actions on muscle protein synthesis rates. In addition, we determined whether accumulation of specific mRNA quantities had a primary role in the protein synthesis response to this type of exercise. The data revealed that as little as 8 min of total contractile duration increased gastrocnemius protein synthesis rates by nearly 50%. Contrary to our hypothesis, post-exercise protein synthesis rates do not appear to be differentially regulated by the resistance imposed on the muscle during exercise but rather by the number of repetitions performed during the acute bout. This observation, the failure of high-frequency chronic training to produce gastrocnemius enlargement, and the relatively minor effects on mRNA levels collectively suggest that translational and posttranslational mechanisms, including protein degradation, may be the principal processes by which gastrocnemius protein expression is regulated in this model of stimulated concentric exercise.

Protein metabolism in rat tibialis anterior muscle after stimulated chronic eccentric exercise.

In another study (J. Appl. Physiol. 69: 1709-1717, 1990) we reported that gastrocnemius (GAST) muscle enlargement failed to occur after 10 wk of 192 contractions performed every 3rd or 4th day. This result was surprising because increased protein synthesis rates were determined after an initial acute exercise bout with the same paradigms. In the same set of animals, tibialis anterior (TA) muscles were enlarged 16-30% compared with sedentary control muscles after the same chronic training regimen. This indicated that the regulation of protein expression may be different between the GAST and TA muscles. The present experiment attempted to define and explain these differences by comparing changes in various indexes of protein metabolism in TA with the same parameters determined in the accompanying study for the GAST. As in the GAST, results showed that TA protein synthesis rates are increased by acute exercise and principally regulated by translational and possibly posttranslational mechanisms. The differential response in muscle mass between the GAST and TA muscles after training may be due, in part, to greater relative resistances imposed on the TA than on the GAST that result in a more-prolonged effect on protein synthesis rates, with lower numbers of stimulated contractions required to stimulate increases in protein synthesis. Data also revealed that although as little as 1 min of total contractile duration (24 repetitions) increased TA protein synthesis rate by 30%, 8 min of total contractile duration (192 repetitions) further increased TA protein synthesis rates to only 45% above control.

Skeletal muscle adaptation to exercise: a century of progress.

Skeletal muscle physiology and biochemistry is an established field with Nobel Prize-winning scientists, dating back to the 1920s. Not until the mid to late 1960s did there appear a major focus on physiological and biochemical training adaptations in skeletal muscle. The study of adaptations to exercise training reveals a wide range of integrative approaches, from the systemic to the molecular level. Advances in our understanding of training adaptations have come in waves caused by the introduction of new experimental approaches. Research has revealed that exercise can be effective at preventing and/or treating some of the most common chronic diseases of the latter half of the 20th century. Endurance-trained muscle is more effective at clearing plasma triglyceride, glucose, and free fatty acids. However, at the present time, most of the mechanisms underlying the adaptation of human skeletal muscle to exercise still remain to be discovered. Little is known about the regulatory factors (e.g., trans-acting proteins or signaling pathways) directly modulating the expression of exercise-responsive genes. Because so many potential physiological and biochemical signals change during exercise, it will be an important challenge in the next century to move beyond "correlational studies" and to identify responsible mechanisms. Skeletal muscle metabolic adaptations may prove to be a critical component to preventing diseases such as coronary heart disease, type 2 diabetes, and obesity. Therefore, training studies have had an impact on setting the stage for a potential "preventive medicine reformation" in a society needing a return to a naturally active lifestyle of our ancestors.

Cytochrome c promoter activity in soleus and white vastus lateralis muscles in rats.

Cytochrome c protein and mRNA are 300 and 100% higher, respectively, in the soleus muscle (predominantly slow-twitch oxidative) than the white vastus lateralis (predominately fast-twitch glycolytic) muscle (W. W. Winder, K. M. Baldwin, and J. O. Holloszy. Eur. J. Biochem. 47: 461-467, 1974; M. M. Lai and F. W. Booth. J. Appl. Physiol. 69: 843-848, 1990). However, the mechanisms controlling these differences in cytochrome c mRNA are largely unknown. The present study employed direct plasmid injection techniques to determine whether the proximal promoter (-726 to +610) of the rat somatic cytochrome c gene was more active in the soleus than in white vastus lateralis muscles in rats. No difference between the soleus and white vastus lateralis muscles for the activities of the -726, -631, -489, -326, -215, -159 and -149 cytochrome c promoters was noted. The results of this study suggest that additional elements (outside of -726 to +610) in the cytochrome c gene may be required, or posttranscriptional regulation may account, for the higher cytochrome c mRNA in the slow-twitch oxidative muscle.

Expression of acetylcholine receptor mRNAs in atrophying and nonatrophying skeletal muscles of old rats.

We examined the age-related association in skeletal muscle between atrophy and expression of mRNAs encoding both the gamma-subunit of the nicotinic acetylcholine receptor (AChR), and myogenin, a transcription factor that upregulates expression of the gamma-subunit promoter. Gastrocnemius and biceps brachii muscles were collected from young (2-mo-old), adult (18-mo-old), and old (31-mo-old) Fischer 344/Brown Norway F1 generation cross male rats. In the gastrocnemius muscles of old vs. young and adult rats, lower muscle mass was accompanied by significantly elevated AChR gamma-subunit and myogenin mRNA levels. In contrast, the biceps brachii muscle exhibited neither atrophy nor as drastic a change in AChR gamma-subunit and myogenin mRNA levels with age. Expression of the AChR epsilon-subunit mRNA did not change with age in either gastrocnemius or biceps brachii muscles. Thus changes in skeletal muscle AChR gamma-subunit and myogenin mRNA levels may be more related to atrophy than to chronological age in old rats.

Serum response factor mRNA induction in the hypertrophying chicken patagialis muscle.

Gene expression in the stretched chicken patagialis (Pat) muscle has not been extensively examined. This study's purpose was to determine the Pat muscle's expression pattern of serum response factor (SRF), skeletal alpha-actin, and MyoD mRNAs after 3 days (onset of stretch), 6 days (end of first week of rapid growth), and 14 days (slowed rate of stretch-induced growth) of stretch. SRF mRNA demonstrated two species (B1 and B2), with B2 being more prevalent in the predominantly fast-twitch Pat muscle, compared with the slow-tonic muscle. Stretch overload increased B1 and B2 SRF mRNA concentrations, and the increase in B1 SRF mRNA concentration was greater at day 6 compared with days 3 or 14. MyoD mRNA concentration was greater in 3-day-stretched Pat muscles, compared with days 6 or 14. Skeletal alpha-actin mRNA concentration was not changed during the study. Gel mobility shift assays demonstrated that SRF binding with serum response element 1 of the skeletal alpha-actin promoter had no altered binding patterns from 6-day-stretched Pat nuclear extracts. It appears that SRF and MyoD mRNAs are induced in the stretch-overloaded Pat muscle but at different time points.