Keith Baar

Resveratrol ameliorates aging-related metabolic phenotypes by inhibiting cAMP phosphodiesterases

Resveratrol, a polyphenol in red wine, has been reported as a calorie restriction mimetic with potential antiaging and antidiabetogenic properties. It is widely consumed as a nutritional supplement, but its mechanism of action remains a mystery. Here, we report that the metabolic effects of resveratrol result from competitive inhibition of cAMP-degrading phosphodiesterases, leading to elevated cAMP levels. The resulting activation of Epac1, a cAMP effector protein, increases intracellular Ca(2+) levels and activates the CamKKβ-AMPK pathway via phospholipase C and the ryanodine receptor Ca(2+)-release channel. As a consequence, resveratrol increases NAD(+) and the activity of Sirt1. Inhibiting PDE4 with rolipram reproduces all of the metabolic benefits of resveratrol, including prevention of diet-induced obesity and an increase in mitochondrial function, physical stamina, and glucose tolerance in mice. Therefore, administration of PDE4 inhibitors may also protect against and ameliorate the symptoms of metabolic diseases associated with aging.

Adaptations of skeletal muscle to exercise: rapid increase in the transcriptional coactivator PGC-1

Endurance exercise induces increases in mitochondria and the GLUT4 isoform of the glucose transporter in muscle. Although little is known about the mechanisms underlying these adaptations, new information has accumulated regarding how mitochondrial biogenesis and GLUT4 expression are regulated. This includes the findings that the transcriptional coactivator PGC‐1 promotes mitochondrial biogenesis and that NRF‐1 and NRF‐2 act as transcriptional activators of genes encoding mitochondrial enzymes. We tested the hypothesis that increases in PGC‐1, NRF‐1, and NRF‐2 are involved in the initial adaptive response of muscle to exercise. Five daily bouts of swimming induced increases in mitochondrial enzymes and GLUT4 in skeletal muscle in rats. One exercise bout resulted in ~ twofold increases in full‐length muscle PGC‐1 mRNA and PGC‐1 protein, which were evident 18 h after exercise. A smaller form of PGC‐1 increased after exercise. The exercise induced increases in muscle NRF‐1 and NRF‐2 that were evident 12 to 18 h after one exercise bout. These findings suggest that increases in PGC‐1, NRF‐1, and NRF‐2 represent key regulatory components of the stimulation of mitochondrial biogenesis by exercise and that PGC‐1 mediates the coordinated increases in GLUT4 and mitochondria.—Baar, K., Wende, A. R., Jones, T. E., Marison, M., Nolte, L. A., Chen, M., Kelly, D. P., Holloszy, J. O. Adaptations of skeletal muscle to exercise: rapid increase in the transcriptional coactivator PGC‐1. FASEB J. 16, 1879–1886 (2002)

Myogenic gene expression signature establishes that brown and white adipocytes originate from distinct cell lineages

Attainment of a brown adipocyte cell phenotype in white adipocytes, with their abundant mitochondria and increased energy expenditure potential, is a legitimate strategy for combating obesity. The unique transcriptional regulators of the primary brown adipocyte phenotype are unknown, limiting our ability to promote brown adipogenesis over white. In the present work, we used microarray analysis strategies to study primary preadipocytes, and we made the striking discovery that brown preadipocytes demonstrate a myogenic transcriptional signature, whereas both brown and white primary preadipocytes demonstrate signatures distinct from those found in immortalized adipogenic models. We found a plausible SIRT1-related transcriptional signature during brown adipocyte differentiation that may contribute to silencing the myogenic signature. In contrast to brown preadipocytes or skeletal muscle cells, white preadipocytes express Tcf21, a transcription factor that has been shown to suppress myogenesis and nuclear receptor activity. In addition, we identified a number of developmental genes that are differentially expressed between brown and white preadipocytes and that have recently been implicated in human obesity. The interlinkage between the myocyte and the brown preadipocyte confirms the distinct origin for brown versus white adipose tissue and also represents a plausible explanation as to why brown adipocytes ultimately specialize in lipid catabolism rather than storage, much like oxidative skeletal muscle tissue.

Is irisin a human exercise gene

Arising from P. Boström et al. 481, 463–468 (2012)10.1038/nature10777Boström et al. report that exercise training induces the expression of the FNDC5 gene in human muscle, producing irisin, which can convert white fat into brown fat, so enhancing metabolic uncoupling and hence caloric expenditure, and propose that this is a new health promoting hormone. This assertion is based on experimental evidence that exogenous FNDC5 induces uncoupling protein 1 (UCP1) expression in white subcutaneous adipocytes; overexpression of FNDC5 in liver (elevating systemic irisin) prevents diet-induced weight gain and metabolic dysfunction and stimulates oxygen consumption in mice; and FNDC5 mRNA expression levels double after exercise training in eight human skeletal muscle samples. However, the UCP1 induction was lower than observed during Brite formation or the level associated with an improved diabetes profile in humans. Here we demonstrate that muscle FNDC5 induction occurs only in a minority of subjects—whereas all types of exercise training programmes, in the vast majority of people, yield some gain in cardiovascular or metabolic health, in our analysis of ∼200 subjects muscle FNDC5 was increased only in highly active elderly subjects, whereas FNDC5 expression was unrelated to metabolic status, which casts doubt over the general relevance of skeletal muscle FNDC5 to human health.

Supplementation of a suboptimal protein dose with leucine or essential amino acids: effects on myofibrillar protein synthesis at rest and following resistance exercise in men

•  Essential amino acids (EAAs) stimulate increased rates of myofibrillar protein synthesis (MPS). •  Leucine is a key regulator of MPS in rodents; however, its importance relative to the other EAAs is not clear. •  About 20 g of protein maximally stimulates MPS after resistance exercise in young men, but we do not know if smaller doses can be made better by adding certain amino acids. •  We report that a suboptimal dose of whey protein (6.25 g) supplemented with either leucine or a mixture of EAAs without leucine stimulates MPS similar to 25 g of whey protein under resting conditions; however, only 25 g of whey sustains exercise‐induced rates of MPS. •  Adding leucine or a mixture of EAAs without leucine to a suboptimal dose of whey is as effective as 25 g whey at stimulating fed rates of MPS; however, 25 g of whey is better suited to increase resistance exercise‐induced muscle anabolism.

Engineering of Functional Tendon

Surgical tendon repair is limited by the availability of viable tissue for transplantation. Because of its relatively avascular nature, tendon is a prime candidate for engineered tissue replacement. To address this problem, cells isolated from rat Achilles tendon were grown to confluence in culture and allowed to self-assemble into a cylinder between two anchor points. The resulting scaffold-free tissue was composed of aligned, small-diameter collagen fibrils, a large number of cells, and an excess of noncollagenous extracellular matrix; all characteristics of embryonic tendon. The stress-strain response of the constructs also resembles the nonlinear behavior of immature tendons, and the ultimate tensile strength is approximately equal to that of embryonic chick tendon, roughly 2 MPa. These physical and mechanical properties indicate that these constructs are the first viable tendons engineered in vitro, without the aid of artificial scaffolding.

Sirt1 enhances skeletal muscle insulin sensitivity in mice during caloric restriction

Skeletal muscle insulin resistance is a key component of the etiology of type 2 diabetes. Caloric restriction (CR) enhances the sensitivity of skeletal muscle to insulin. However, the molecular signals within skeletal muscle linking CR to improved insulin action remain largely unknown. Recently, the mammalian ortholog of Sir2, sirtuin 1 (Sirt1), has been identified as a potential transducer of perturbations in cellular energy flux into subsequent metabolic adaptations, including modulation of skeletal muscle insulin action. Here, we have demonstrated that CR increases Sirt1 deacetylase activity in skeletal muscle in mice, in parallel with enhanced insulin-stimulated phosphoinositide 3-kinase (PI3K) signaling and glucose uptake. These adaptations in skeletal muscle insulin action were completely abrogated in mice lacking Sirt1 deacetylase activity. Mechanistically, Sirt1 was found to be required for the deacetylation and inactivation of the transcription factor Stat3 during CR, which resulted in decreased gene and protein expression of the p55α/p50α subunits of PI3K, thereby promoting more efficient PI3K signaling during insulin stimulation. Thus, these data demonstrate that Sirt1 is an integral signaling node in skeletal muscle linking CR to improved insulin action, primarily via modulation of PI3K signaling.

Sirtuin 1 (SIRT1) Deacetylase Activity Is Not Required for Mitochondrial Biogenesis or Peroxisome Proliferator-activated Receptor-γ Coactivator-1α (PGC-1α) Deacetylation following Endurance Exercise

The protein deacetylase, sirtuin 1 (SIRT1), is a proposed master regulator of exercise-induced mitochondrial biogenesis in skeletal muscle, primarily via its ability to deacetylate and activate peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). To investigate regulation of mitochondrial biogenesis by SIRT1 in vivo, we generated mice lacking SIRT1 deacetylase activity in skeletal muscle (mKO). We hypothesized that deacetylation of PGC-1α and mitochondrial biogenesis in sedentary mice and after endurance exercise would be impaired in mKO mice. Skeletal muscle contractile characteristics were determined in extensor digitorum longus muscle ex vivo. Mitochondrial biogenesis was assessed after 20 days of voluntary wheel running by measuring electron transport chain protein content, enzyme activity, and mitochondrial DNA expression. PGC-1α expression, nuclear localization, acetylation, and interacting protein association were determined following an acute bout of treadmill exercise (AEX) using co-immunoprecipitation and immunoblotting. Contrary to our hypothesis, skeletal muscle endurance, electron transport chain activity, and voluntary wheel running-induced mitochondrial biogenesis were not impaired in mKO versus wild-type (WT) mice. Moreover, PGC-1α expression, nuclear translocation, activity, and deacetylation after AEX were similar in mKO versus WT mice. Alternatively, we made the novel observation that deacetylation of PGC-1α after AEX occurs in parallel with reduced nuclear abundance of the acetyltransferase, general control of amino-acid synthesis 5 (GCN5), as well as reduced association between GCN5 and nuclear PGC-1α. These findings demonstrate that SIRT1 deacetylase activity is not required for exercise-induced deacetylation of PGC-1α or mitochondrial biogenesis in skeletal muscle and suggest that changes in GCN5 acetyltransferase activity may be an important regulator of PGC-1α activity after exercise.

Involvement of PPARγ co-activator-1, nuclear respiratory factors 1 and 2, and PPARα in the adaptive response to endurance exercise

Endurance exercise training induces an increase in the respiratory capacity of muscle, resulting in an increased capacity to generate ATP as well as improved efficiency of muscle contraction. Such adaptations are largely the result of a coordinated genetic response that increases mitochondrial proteins, fatty acid oxidation enzymes and the exercise- and insulin-stimulated glucose transporter GLUT4, and shifts the contractile and regulatory proteins to their more efficient isoforms. In recent years a number of the transcriptional regulators involved in this genetic response have been identified and these factors can be classified into two different groups. The first group comprises transcription factors such as nuclear respiratory factors (NRF) 1 and 2 and PPARα that bind DNA in a sequence-specific manner. The second group, referred to as transcriptional co-activators, alter transcription without directly binding to DNA. The PPARγ co-activator (PGC) family of proteins have been identified as the central family of transcriptional co-activators for induction of mitochondrial biogenesis. PGC-1α is activated by exercise, and is sufficient to produce the endurance phenotype through direct interactions with NRF-1 and PPARα, and potentially NRF-2. Furthering the understanding of the activation of PGC proteins following exercise has implications beyond improving athletic performance, including the possibility of providing targets for the treatment of frailty in the elderly, obesity and diseases such as mitochondrial myopathies and diabetes.

Self-organization of rat cardiac cells into contractile 3-D cardiac tissue

The mammalian heart is not known to regenerate following injury. Therefore, there is great interest in developing viable tissue‐based models for cardiac assist. Recent years have brought numerous advances in the development of scaffold‐based models of cardiac tissue, but a self‐organizing model has yet to be described. Here, we report the development of an in vitro cardiac tissue without scaffolding materials in the contractile region. Using an optimal concentration of the adhesion molecule laminin, a confluent layer of neonatal rat cardiomyogenic cells can be induced to self‐organize into a cylindrical construct, resembling a papillary muscle, which we have termed a cardioid. Like endogenous heart tissue, cardioids contract spontaneously and can be electrically paced between 1 and 5 Hz indefinitely without fatigue. These engineered cardiac tissues also show an increased rate of spontaneous contraction (chronotropy), increased rate of relaxation (lusitropy), and increased force production (inotropy) in response to epinephrine. Cardioids have a developmental protein phenotype that expresses both α‐ and β‐tropomyosin, very low levels of SERCA2a, and very little of the mature isoform of cardiac troponin T.