Skeletal Muscle AMP-activated Protein Kinase Is Essential for the Metabolic Response to Exercise in Vivo

AMP-activated protein kinase (AMPK) has been postulated as a super-metabolic regulator, thought to exert numerous effects on skeletal muscle function, metabolism, and enzymatic signaling. Despite these assertions, little is known regarding the direct role(s) of AMPK in vivo, and results obtained in vitro or in situ are conflicting. Using a chronically catheterized mouse model (carotid artery and jugular vein), we show that AMPK regulates skeletal muscle metabolism in vivo at several levels, with the result that a deficit in AMPK activity markedly impairs exercise tolerance. Compared with wild-type littermates at the same relative exercise capacity, vascular glucose delivery and skeletal muscle glucose uptake were impaired; skeletal muscle ATP degradation was accelerated, and arterial lactate concentrations were increased in mice expressing a kinase-dead AMPKα2 subunit (α2-KD) in skeletal muscle. Nitric-oxide synthase (NOS) activity was significantly impaired at rest and in response to exercise in α2-KD mice; expression of neuronal NOS (NOSμ) was also reduced. Moreover, complex I and IV activities of the electron transport chain were impaired 32 ± 8 and 50 ± 7%, respectively, in skeletal muscle of α2-KD mice (p < 0.05 versus wild type), indicative of impaired mitochondrial function. Thus, AMPK regulates neuronal NOSμ expression, NOS activity, and mitochondrial function in skeletal muscle. In addition, these results clarify the role of AMPK in the control of muscle glucose uptake during exercise. Collectively, these findings demonstrate that AMPK is central to substrate metabolism in vivo, which has important implications for exercise tolerance in health and certain disease states characterized by impaired AMPK activation in skeletal muscle.The ubiquitously expressed serine/threonine AMP-activated protein kinase (AMPK)² is an αβγ heterotrimer postulated to play a key role in the response to energetic stress (1, 2), because of its sensitivity to increased cellular AMP levels (3). Pharmacological activation of AMPK (primarily via the AMP analogue ZMP) increases catabolic processes such as GLUT4 translocation (4, 5), glucose uptake (6, 7), long chain fatty acid (LCFA) uptake (8), and substrate oxidation (6). Concomitantly, pharmacological activation of AMPK inhibits anabolic processes, and in skeletal muscle genetic reduction of the catalytic AMPKα2 subunit eliminates these pharmacological effects (9–12). Thus, AMPK has been proposed to act as a metabolic master switch (2, 13, 14). Physiologically, exercise at intensities sufficient to increase free cytosolic AMP (AMP_free) levels is a potent stimulus of AMPK, preferentially activating AMPKα2 in skeletal muscle (15–17). The metabolic profile of skeletal muscle during moderate to high intensity exercise is remarkably similar to skeletal muscle in which AMPK has been pharmacologically activated (i.e. increases in catabolic processes). This is consistent with the hypothesis that AMPK activation is required for the metabolic response to increased cellular stress. Given this, it is surprising that the direct role(s) of skeletal muscle AMPK during exercise under physiological in vivo conditions is unknown.A number of studies have tried to attribute causality to the AMPK and metabolic responses to exercise using transgenic models. In mouse models in which AMPKα2 protein expression and/or activity has been impaired, contractions performed in isolated skeletal muscle in vitro, ex vivo, or in situ have demonstrated that skeletal muscle glucose uptake (MGU) is normal (9, 10), partially impaired (11, 18), or ablated (19). Furthermore, ex vivo skeletal muscle LCFA uptake and oxidation in response to contraction appears to be AMPK-independent (20, 21). A key limitation of these studies is that the experimental models were not physiological. Under in vivo conditions, mice expressing a kinase-dead (18) or inactive (22) AMPKα2 subunit in cardiac and skeletal muscle have impaired voluntary and maximal physical activity, respectively, indicative of a physiological role for AMPK during exercise. In this context, obese non-diabetic and diabetic individuals have impaired skeletal muscle AMPK activation during moderate intensity exercise (23) as well as during the post-exercise period (24), yet the contribution of this impairment to the disease state is unclear. Thus, in vivo studies are essential to define the role of AMPK in skeletal muscle during exercise.Physical exercise of a moderate intensity is an effective adjunct treatment for chronic metabolic diseases such as obesity and type 2 diabetes (25). Given the importance of elucidating the molecular mechanism(s) regulating skeletal muscle substrate metabolism during exercise and the putative role of AMPK as a critical mediator in this process, we tested the hypothesis that AMPKα2 is functionally linked to substrate metabolism in vivo.     

Hyperlipidemic chicken as a model of non-alcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH) is part of the spectrum of non-alcoholic fatty liver disease (NAFLD), currently the most common cause of abnormal liver tests. Given the difficulty of studying all the factors involved in it in human populations, studies in animal models might provide crucial insights in the pathogenesis of steatohepatitis. Several physiological features predispose birds to fat deposition in the liver. The present study was conceived to explore the possibilities of the chicken fed a cholesterol and fat enriched diet as a model for steatohepatitis. We used two different diets: a standard growing mash (control group) and a standard growing mash enriched with 2% cholesterol and 20% palm oil (hyperlipidemic group). We investigated the effect of feeding a cholesterol and fat enriched diet, on plasma lipid levels, liver enzymes and hepatic histopathology. Semiquantitative and quantitative assessment by image analysis was performed to determine changes in lipid deposits and inflammatory infiltration. Statistically significant increases were observed in all plasma lipid parameters, liver macroscopic features, fat deposits and cell-ballooning of hepatocytes between control and hyperlipidemic animals. Significant differences were also observed in the inflammatory infiltration parameters (number of foci, density, area and maximal diameter). Results show that diet-induced hypercholesterolemia and hypertriglyceridemia are associated with severe impairment of liver histology (fat accumulation, inflammation and cell-ballooning), reproducing histological features of human NAFLD. This model, which is easy and reproducible, offers economic and technical advantages. Furthermore, the reversibility of the pathologic changes makes it suitable for drug intervention studies of steatohepatitis.     

TGF-β1 induces an age-dependent inflammation of nerve ganglia and fibroplasia in the prostate gland stroma of a novel transgenic mouse

TGF-β1 is overexpressed in wound repair and in most proliferative disorders including benign prostatic hyperplasia and prostate cancer. The stromal microenvironment at these sites is reactive and typified by altered phenotype, matrix deposition, inflammatory responses, and alterations in nerve density and biology. TGF-β1 is known to modulate several stromal responses; however there are few transgenic models to study its integrated biology. To address the actions of TGF-β1 in prostate disorders, we targeted expression of an epitope tagged and constitutively active TGF-β1 via the enhanced probasin promoter to the murine prostate gland epithelium. Transgenic mice developed age-dependent lesions leading to severe, yet focal attenuation of epithelium, and a discontinuous basal lamina. These changes were associated with elevated fibroplasia and frequency of collagenous micronodules in collapsed acini, along with an induced inflammation in nerve ganglia and small vessels. Elevated recruitment of CD115+ myeloid cells but not mature macrophages was observed in nerve ganglia, also in an age-dependent manner. Similar phenotypic changes were observed using a human prostate epithelium tissue recombination xenograft model, where epithelial cells engineered to overexpress TGF-β1 induced fibrosis and altered matrix deposition concurrent with inflammation in the stromal compartment. Together, these data suggest that elevated TGF-β1 expression induces a fibroplasia stromal response associated with breach of epithelial wall structure and inflammatory involvement of nerve ganglia and vessels. The novel findings of ganglia and vessel inflammation associated with formation of collagenous micronodules in collapsed acini is important as each of these are observed in human prostate carcinoma and may play a role in disease progression.     

Genetic mapping of a major codominant QTL associated with β-carotene accumulation in watermelon

The common flesh color of commercially grown watermelon is red due to the accumulation of lycopene. However, natural variation in carotenoid composition that exists among heirloom and exotic accessions results in a wide spectrum of flesh colors. We previously identified a unique orange flesh watermelon accession (NY0016) that accumulates mainly β-carotene and no lycopene. We hypothesized this unique accession could serve as a viable source for increasing provitamin A content in watermelon. Here we characterize the mode of inheritance and genetic architecture of this trait. Analysis of testcrosses of NY0016 with yellow and red fruited lines indicated a codominant mode of action as F₁ fruits exhibited a combination of carotenoid profiles from both parents. We combined visual color phenotyping with genotyping-by-sequencing of an F_2:3 population from a cross of NY0016 by a yellow fruited line, to map a major locus on chromosome 1, associated with β-carotene accumulation in watermelon fruit. The QTL interval is approximately 20 cM on the genetic map and 2.4 Mb on the watermelon genome. Trait-linked marker was developed and used for validation of the QTL effect in segregating populations across different genetic backgrounds. This study is a step toward identification of a major gene involved in carotenoid biosynthesis and accumulation in watermelon. The codominant inheritance of β-carotene provides opportunities to develop, through marker-assisted breeding, β-carotene-enriched red watermelon hybrids.     

Interplay among cyclic diguanylate, HapR, and the general stress response regulator (RpoS) in the regulation of Vibrio cholerae hemagglutinin/protease

Vibrio cholerae secretes the Zn-dependent metalloprotease hemagglutinin (HA)/protease (mucinase), which is encoded by hapA and displays a broad range of potential pathogenic activities. Expression of HA/protease has a stringent requirement for the quorum-sensing regulator HapR and the general stress response regulator RpoS. Here we report that the second messenger cyclic diguanylic acid (c-di-GMP) regulates the production of HA/protease in a negative manner. Overexpression of a diguanylate cyclase to increase the cellular c-di-GMP pool resulted in diminished expression of HA/protease and its positive regulator, HapR. The effect of c-di-GMP on HapR was independent of LuxO but was abolished by deletion of the c-di-GMP binding protein VpsT, the LuxR-type regulator VqmA, or a single-base mutation in the hapR promoter that prevents autorepression. Though expression of HapR had a positive effect on RpoS biosynthesis, direct manipulation of the c-di-GMP pool at a high cell density did not significantly impact RpoS expression in the wild-type genetic background. In contrast, increasing the c-di-GMP pool severely inhibited RpoS expression in a ΔhapR mutant that is locked in a regulatory state mimicking low cell density. Based on the above findings, we propose a model for the interplay between HapR, RpoS, and c-di-GMP in the regulation of HA/protease expression.     

Superparasitism strategies by a native and an exotic parasitoid species attacking the Mexican fruit fly,Anastrepha ludens (Diptera: Tephritidae)

Superparasitism refers to the action of parasitoids ovipositing eggs in hosts that are already parasitized; this inevitably results in the elimination of supernumerary larvae in solitary parasitoids. Here, we investigated superparasitism performed by two species of solitary parasitoids on the larvae of Anastrepha ludens (Loew; Diptera: Tephritidae): a native species, Doryctobracon crawfordi (Viereck; Hymenoptera: Braconidae), and an exotic species, Diachasmimorpha tryoni (Cameron; Hymenoptera: Braconidae). Tests were conducted under laboratory conditions evaluating the behaviour of females acting alone (self-superparasitism) or in groups (conspecific superparasitism). Parasitism strategies were different between these two species. In D. crawfordi, the number of first instar larvae found in each dissected host pupa was never greater than two, regardless of the number of oviposition scars observed per pupa. In contrast, there was a positive correlation between the number of oviposition scars and the number of first instar larvae in D. tryoni. The survival and fecundity of D. crawfordi females emerging from pupae with one scar was higher than in females emerging from pupae with more scars. In D. tryoni, the number of oviposition scars did not show deleterious effects on life history traits and was positively correlated with the proportion of emerging females. An understanding of the superparasitism strategy adopted by parasitoid species could be of great interest to augmentative biological control programmes because the mass rearing of natural enemies could be negatively or positively affected by this condition.     

Patterns of selection on Plasmodium falciparum erythrocyte‐binding antigens after the colonization of the New World

Pathogens, which have recently colonized a new host species or new populations of the same host, are interesting models for understanding how populations may evolve in response to novel environments. During its colonization of South America from Africa, Plasmodium falciparum, the main agent of malaria, has been exposed to new conditions in distinctive new human populations (Amerindian and populations of mixed origins) that likely exerted new selective pressures on the parasite's genome. Among the genes that might have experienced strong selective pressures in response to these environmental changes, the eba genes (erythrocyte‐binding antigens genes), which are involved in the invasion of the human red blood cells, constitute good candidates. In this study, we analysed, in South America, the polymorphism of three eba genes (eba‐140, eba‐175, eba‐181) and compared it to the polymorphism observed in African populations. The aim was to determine whether these genes faced selective pressures in South America distinct from what they experienced in Africa. Patterns of genetic variability of these genes were compared to the patterns observed at two housekeeping genes (adsl and serca) and 272 SNPs to separate adaptive effects from demographic effects. We show that, conversely to Africa, eba‐140 seemed to be under stronger diversifying selection in South America than eba‐175. In contrast, eba‐181 did not show any sign of departure from neutrality. These changes in the patterns of selection on the eba genes could be the consequence of changes in the host immune response, the host receptor polymorphisms and/or the ability of the parasite to silence or express differentially its invasion proteins.     

Track analysis and conservation priorities in the cloud forests of Hidalgo,Mexico

Abstract . A track analysis based on the distributional patterns of 967 species of vascular plant taxa (gymnosperms, angiosperms and pteridophytes) was performed to assess conservation priorities for cloud forests in the state of Hidalgo, Mexico, ranged in the municipalities of Chapulhuacán, Eloxochitlán, Molocotlán, Pisaflores, Tenango de Doria, Tlahuelompa and Tlanchinol, as well as five floristically equivalent areas in the states of Veracruz (Teocelo and Helechales), Tamaulipas (Gómez Farías), Morelos‐México (Ocuilan) and Oaxaca (Huautla de Jiménez). In order to detect generalized tracks we employed a new parsimony method, where clades (considered equivalent to generalized tracks) are defined forbidding homoplasy and acting like a compatibility algorithm. Several generalized tracks were found connecting these areas. Cloud forests of Chapulhuacán were connected according to three different generalized tracks and thus have a higher value, qualifying as a priority area for the conservation of cloud forests in the state of Hidalgo.     

A novel founder mutation in the RNASEL gene, 471delAAAG,is associated with prostate cancer in Ashkenazi Jews

HPC1/RNASEL was recently identified as a candidate gene for hereditary prostate cancer. We identified a novel founder frameshift mutation in RNASEL, 471delAAAG, in Ashkenazi Jews. The mutation frequency in the Ashkenazi population, estimated on the basis of the frequency in 150 healthy young women, was 4% (95% confidence interval [CI] 1.9%-8.4%). Among Ashkenazi Jews, the mutation frequency was higher in patients with prostate cancer (PRCA) than in elderly male control individuals (6.9% vs. 2.4%; odds ratio = 3.0; 95% CI 0.6-15.3; P=.17). 471delAAAG was not detected in the 134 non-Ashkenazi patients with PRCA and control individuals tested. The median age at PRCA diagnosis did not differ significantly between the Ashkenazi carriers and noncarriers included in our study. However, carriers received diagnoses at a significantly earlier age, compared with patients with PRCA who were registered in the Israeli National Cancer Registry (65 vs. 74.4 years, respectively; P<.001). When we examined two brothers with PRCA, we found a heterozygous 471delAAAG mutation in one and a homozygous mutation in the other. Loss of heterozygosity was demonstrated in the tumor of the heterozygous sib. Taken together, these data suggest that the 471delAAAG null mutation is associated with PRCA in Ashkenazi men. However, additional studies are required to determine whether this mutation confers increased risk for PRCA in this population.