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.

Non-passaged muscle precursor cells from 32-month old rat skeletal muscle have delayed proliferation and differentiation.

OBJECTIVES: The systemic environment and satellite cell dysfunction have been proposed as important contributors in the development of sarcopenia and impaired skeletal muscle regrowth with ageing. In the present study, we investigated effects of serum age on proliferation of muscle precursor cells (MPCs) isolated from skeletal muscles of young and old rats. MATERIALS AND METHODS: We examined proliferation and subsequent differentiation of non-passaged MPCs isolated from skeletal muscles of 1-, 3- and 32-month old rats over a 72-h time course, using a serum cross-over design. RESULTS AND CONCLUSIONS: We found no effect of serum age on MPC proliferation, but we did discover that MPCs isolated from skeletal muscle of 32-month old rats had delayed onset of, and exit from proliferation, compared to MPCs isolated from skeletal muscle of 1-month old rats. Delayed proliferation of MPCs from 32-month old rats was associated with delayed p38 MAPK phosphorylation, and MyoD and p21(Cip1) protein expression. We also demonstrate that MPCs from 32-month old rats exhibited lower levels of muscle creatine kinase mRNA compared to 1-month old rats, but elevated levels of myogenin mRNA, when stimulated to differentiate after 36 h proliferation. These findings suggest that delayed entry and exit of the cell cycle observed in MPCs from 32-month old rats may compromise their ability to respond to differentiation stimuli and subsequently impair myogenic potential of 32-month old skeletal muscle, in this model.

The future: genes, physical activity and health.

The assigned title for the Lindhard presentation was to examine the future of genes, physical activity and health. The current review is a summary of this presentation. Caution is expressed that technology is improving so rapidly that a future view is limited to a few years as opposed to the 100 years passing since Lindhard's achievements. The near futuristic opportunities and challenges for four major topic topics are reviewed here. Concerns are expressed over current usage of the terms 'control' group and 'non-responders' in exercise research. Our view is that 'control' needs to be differentiated between its usage for treatments of exercise to restore natural functions in individuals with less than healthy levels of physical activity and the inherited genome's expectation for physical activity levels to maintain normal function. For the second discussed topic, it is proposed that the term 'non-responders' should be replaced by the term 'low sensitivity' as there may be no such human who is a non-responder to every exercise adaptation. The third futuristic topic is exercise prescription as envisioned for individualized medicine. However, numerous limitations and challenges exist to truly optimal exercise medicine at the level of one individual. Finally, preventative physical activity medicine is discussed. Physical activity as a therapy now exists to prevent most of the chronic diseases. The future needs to understand the molecular basis for how the body becomes dysfunctional when its level of physical activity does not match the norm of physical activity that selected our inherited genome.

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.

p21(Cip1) expression is increased in ambient oxygen, compared to estimated physiological (5%) levels in rat muscle precursor cell culture.

OBJECTIVE: While it is common practice to culture cells in the presence of ambient oxygen (approximately 21% O2), O2 level observed in the physiological environment is often much lower. Previous efforts to culture a variety of different stem cells, including muscle precursor cells (MPC), under O2 conditions that better mimic in vivo conditions have resulted in enhanced proliferation. In the present study, we hypothesized that 20% O2 in culture represents a sufficient stimulus to cause increased expression of two key negative regulators of the cell-cycle Cip/Kip family of cyclin-dependent kinase inhibitors, p21(Cip1) and p27(Kip1), in MPCs. MATERIALS AND METHODS: MPCs were isolated from Fischer 344 x Brown Norway F(1) hybrid male rats and O2 was adjusted in culture using a tri-gas incubator. RESULTS: 5-Bromo-2'-deoxyuridine incorporation, cell number and nuclear proliferating cell nuclear antigen expression were all decreased after 48 h culture in 20% O2, compared to 5% O2. Twenty per cent O2 had no effect on either p27(Kip1) promoter activity or protein expression. Although p21(Cip1) promoter activity remained unchanged between 5% and 20% O2, there were significant increases in both p21(Cip1) mRNA and protein expression. Furthermore, 20% O2 caused an increase in p21(Cip1) mRNA stability and p53 transcription factor activity. CONCLUSION: These findings are considered important because they reveal p21(Cip1) as a critical regulatory protein that needs to be considered when interpreting proliferation data from MPCs studied in culture. In addition, O2-dependent regulation of MPC proliferation is relevant to conditions, including sarcopenia, heart failure, cancer and muscular dystrophy, where increased oxidative stress exists.

Changes in signalling molecule levels in 10-day hindlimb immobilized rat muscles.

AIM: Hindlimb immobilization produces similar percentage decreases in muscle mass in the predominantly type I soleus and type II vastus lateralis muscles. Consequently we hypothesized that the percentage changes in potential regulatory molecules for atrophy would be similar in the two muscle fibre types. METHODS: Therefore, the purpose of the current study was to measure phosphorylated p38 MAPK and JNK, as well as the protein levels of p53, growth arrest and DNA damage-inducible 45 (GADD45), and full-length poly(ADP-ribose) polymerase (PARP) to determine whether their changes in expression in the soleus and vastus lateralis muscles were similar at 10th day of hindlimb immobilization in young rats. RESULTS: Unexpectedly, preferential increases in phosphorylation of p38 MAPK and JNK and in the protein levels of p53, GADD45, as well as decreases in full-length PARP occurred in the soleus muscle, while only p38 phosphorylation increased in the white portion of the vastus lateralis muscle at 10th day of hindlimb immobilization. CONCLUSION: Taken together, these results are interpreted to suggest that some of regulatory processes or kinetics in the atrophy of type I and II muscle fibres during limb immobilization may differ at the 10th day of limb immobilization.

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.

Molecular regulation of individual skeletal muscle fibre types.

The purpose of this review is to present current understanding of cellular and molecular regulation of fibre type expression in skeletal muscle. Published literature seems to conclusively suggest that muscle fibre type expression is regulated by multiple signalling pathways and transcription factors rather than a single 'master' switch or signalling pathway. While the current nomenclature for fibre types is convenient for communication, based upon the evolution of this nomenclature, the prediction that fibre type classifications may change in the future to incorporate post-genomic information is made. It is predicted that future fibre type classifications could be based upon the contractile-activity-induced changes in a common regulatory factor(s) within a subpopulation of genes whose expressions are altered to modify and maintain the new muscle fibre phenotype.

Regenerated mdx mouse skeletal muscle shows differential mRNA expression.

Despite over 3,000 articles published on dystrophin in the last 15 years, the reasons underlying the progression of the human disease, differential muscle involvement, and disparate phenotypes in different species are not understood. The present experiment employed a screen of 12,488 mRNAs in 16-wk-old mouse mdx muscle at a time when the skeletal muscle is avoiding severe dystrophic pathophysiology, despite the absence of a functional dystrophin protein. A number of transcripts whose levels differed between the mdx and human Duchenne muscular dystrophy were noted. A fourfold decrease in myostatin mRNA in the mdx muscle was noted. Differential upregulation of actin-related protein 2/3 (subunit 4), beta-thymosin, calponin, mast cell chymase, and guanidinoacetate methyltransferase mRNA in the more benign mdx was also observed. Transcripts for oxidative and glycolytic enzymes in mdx muscle were not downregulated. These discrepancies could provide candidates for salvage pathways that maintain skeletal muscle integrity in the absence of a functional dystrophin protein in mdx skeletal muscle.

Recovery of neuromuscular junction morphology following 16 days of spaceflight.

It has previously been established that spaceflight elicits alterations in the morphology of the neuromuscular system that includes expansion of the neuromuscular junction (NMJ) and myofiber atrophy. The purpose of this study was to determine the capacity of the neuromuscular system to recover from spaceflight-induced modifications upon return to normal gravity. Soleus muscles were obtained from rats participating in the 16-day Neurolab space shuttle mission at 1 day and 14 days after returning to Earth: solei were also taken at the same time points from ground-based control rats. Cytofluorescent techniques, coupled with confocal microscopy, were used to assess NMJ morphology. Histochemistry, in conjunction with phase contrast microscopy, was employed to examine myofiber size and type. Results indicate that 1 day after landing both pre- and postsynaptic stained areas of the NMJ were significantly (P < or = 0.05) larger in the spaceflight group than in controls. Moreover, significant myofiber atrophy was demonstrated in animals subjected to 0 gravity. By 14 days following return to the Earth, however, NMJ stained areas and muscle fiber size were no longer different from control values at that same interval. These results suggest that the neuromuscular system possesses a robust capacity to recover from spaceflight-induced perturbations upon return to normal gravitational influences.

Time course of the MAPK and PI3-kinase response within 24 h of skeletal muscle overload.

Knowledge of the molecular mechanisms by which skeletal muscle hypertrophies in response to increased mechanical loading may lead to the discovery of novel treatment strategies for muscle wasting and frailty. To gain insight into potential early signaling mechanisms associated with skeletal muscle hypertrophy, the temporal pattern of mitogen-activated protein kinase (MAPK) phosphorylation and phosphatidylinositol 3-kinase (PI3-kinase) activity during the first 24 h of muscle overload was determined in the rat slow-twitch soleus and fast-twitch plantaris muscles after ablation of the gastrocnemius muscle. p38alpha MAPK phosphorylation was elevated for the entire 24-h overload period in both muscles. In contrast, Erk 2 and p54 JNK phosphorylation were transiently increased by overload, returning to the levels of sham-operated controls by 24 h. PI3-kinase activity was increased by muscle overload only at 12 h of overload and only in the plantaris muscle. In summary, sustained elevation of p38alpha MAPK phosphorylation occurred early in response to muscle overload, identifying this pathway as a potential candidate for mediating early hypertrophic signals in response to skeletal muscle overload.

Culture in low levels of oxygen enhances in vitro proliferation potential of satellite cells from old skeletal muscles.

The proliferation ability of satellite cells (considered the 'stem cells' of mature myofibers) declines with increasing age when cultured under standard cell culture conditions of 21% oxygen. However, actual oxygen levels in the intact myofiber in vivo are an order of magnitude lower. No studies to date have addressed the issue of whether culturing satellite cells from old muscles under more 'physiologic' conditions would enhance their proliferation and/or differentiation ability. Therefore, we analyzed satellite cells derived from 31-month-old rats in standard cultures with 21% O2 and in lowered (approximately 3%) O2. Under the lowered O2 conditions, we noted a remarkable increase in the percentage of large-sized colonies, activation of cell cycle progression factors, phosphorylation of Akt, and downregulation of the cell cycle inhibitor p27Kip1. These data suggest that lower O2 levels provide a milieu that stimulates proliferation by allowing continued cell cycle progression, to result ultimately in the enhanced in vitro replicative life span of the old satellite cells. Such a method therefore provides an improved means for the ex vivo generation of progenitor satellite cell populations for potential therapeutic stem cell transplantation.

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.

Differential global gene expression in red and white skeletal muscle.

The differences in gene expression among the fiber types of skeletal muscle have long fascinated scientists, but for the most part, previous experiments have only reported differences of one or two genes at a time. The evolving technology of global mRNA expression analysis was employed to determine the potential differential expression of approximately 3,000 mRNAs between the white quad (white muscle) and the red soleus muscle (mixed red muscle) of female ICR mice (30-35 g). Microarray analysis identified 49 mRNA sequences that were differentially expressed between white and mixed red skeletal muscle, including newly identified differential expressions between muscle types. For example, the current findings increase the number of known, differentially expressed mRNAs for transcription factors/coregulators by nine and signaling proteins by three. The expanding knowledge of the diversity of mRNA expression between white and mixed red muscle suggests that there could be quite a complex regulation of phenotype between muscles of different fiber types.

Selected Contribution: Skeletal muscle focal adhesion kinase, paxillin, and serum response factor are loading dependent.

This investigation examined the effect of mechanical loading state on focal adhesion kinase (FAK), paxillin, and serum response factor (SRF) in rat skeletal muscle. We found that FAK concentration and tyrosine phosphorylation, paxillin concentration, and SRF concentration are all lower in the lesser load-bearing fast-twitch plantaris and gastrocnemius muscles compared with the greater load-bearing slow-twitch soleus muscle. Of these three muscles, 7 days of mechanical unloading via tail suspension elicited a decrease in FAK tyrosine phosphorylation only in the soleus muscle and decreases in FAK and paxillin concentrations only in the plantaris and gastrocnemius muscles. Unloading decreased SRF concentration in all three muscles. Mechanical overloading (via bilateral gastrocnemius ablation) for 1 or 8 days increased FAK and paxillin concentrations in the soleus and plantaris muscles. Additionally, whereas FAK tyrosine phosphorylation and SRF concentration were increased by < or =1 day of overloading in the soleus muscle, these increases did not occur until somewhere between 1 and 8 days of overloading in the plantaris muscle. These data indicate that, in the skeletal muscles of rats, the focal adhesion complex proteins FAK and paxillin and the transcription factor SRF are generally modulated in association with the mechanical loading state of the muscle. However, the somewhat different patterns of adaptation of these proteins to altered loading in slow- vs. fast-twitch skeletal muscles indicate that the mechanisms and time course of adaptation may partly depend on the prior loading state of the muscle.

ANG II is required for optimal overload-induced skeletal muscle hypertrophy.

ANG II mediates the hypertrophic response of overloaded cardiac muscle, likely via the ANG II type 1 (AT(1)) receptor. To examine the potential role of ANG II in overload-induced skeletal muscle hypertrophy, plantaris and/or soleus muscle overload was produced in female Sprague-Dawley rats (225-250 g) by the bilateral surgical ablation of either the synergistic gastrocnemius muscle (experiment 1) or both the gastrocnemius and plantaris muscles (experiment 2). In experiment 1 (n = 10/group), inhibiting endogenous ANG II production by oral administration of an angiotensin-converting enzyme (ACE) inhibitor during a 28-day overloading protocol attenuated plantaris and soleus muscle hypertrophy by 57 and 96%, respectively (as measured by total muscle protein content). ACE inhibition had no effect on nonoverloaded (sham-operated) muscles. With the use of new animals (experiment 2; n = 8/group), locally perfusing overloaded soleus muscles with exogenous ANG II (via osmotic pump) rescued the lost hypertrophic response in ACE-inhibited animals by 71%. Furthermore, orally administering an AT(1) receptor antagonist instead of an ACE inhibitor produced a 48% attenuation of overload-induced hypertrophy that could not be rescued by ANG II perfusion. Thus ANG II may be necessary for optimal overload-induced skeletal muscle hypertrophy, acting at least in part via an AT(1) receptor-dependent pathway.