Rudolf J. Schilder

The mTORC1 signaling repressors REDD1/2 are rapidly induced and activation of p70S6K1 by leucine is defective in skeletal muscle of an immobilized rat hindlimb

Limb immobilization, limb suspension, and bed rest cause substantial loss of skeletal muscle mass, a phenomenon termed disuse atrophy. To acquire new knowledge that will assist in the development of therapeutic strategies for minimizing disuse atrophy, the present study was undertaken with the aim of identifying molecular mechanisms that mediate control of protein synthesis and mechanistic target of rapamycin complex 1 (mTORC1) signaling. Male Sprague-Dawley rats were subjected to unilateral hindlimb immobilization for 1, 2, 3, or 7 days or served as nonimmobilized controls. Following an overnight fast, rats received either saline or L-leucine by oral gavage as a nutrient stimulus. Hindlimb skeletal muscles were extracted 30 min postgavage and analyzed for the rate of protein synthesis, mRNA expression, phosphorylation state of key proteins in the mTORC1 signaling pathway, and mTORC1 signaling repressors. In the basal state, mTORC1 signaling and protein synthesis were repressed within 24 h in the soleus of an immobilized compared with a nonimmobilized hindlimb. These responses were accompanied by a concomitant induction in expression of the mTORC1 repressors regulated in development and DNA damage responses (REDD) 1/2. The nutrient stimulus produced an elevation of similar magnitude in mTORC1 signaling in both the immobilized and nonimmobilized muscle. In contrast, phosphorylation of 70-kDa ribosomal protein S6 kinase 1 (p70S6K1) on Thr(229) and Thr(389) in response to the nutrient stimulus was severely blunted. Phosphorylation of Thr(229) by PDK1 is a prerequisite for phosphorylation of Thr(389) by mTORC1, suggesting that signaling through PDK1 is impaired in response to immobilization. In conclusion, the results show an immobilization-induced attenuation of mTORC1 signaling mediated by induction of REDD1/2 and defective p70S6K1 phosphorylation.

Metabolic syndrome and obesity in an insect

Dragonflies infected with noninvasive gregarine gut parasites (Microsporidia, Apicomplexa) have reduced flight-muscle performance, an inability to metabolize lipid in their muscles, twofold-elevated hemolymph carbohydrate concentrations, and they accumulate fat in their thorax in a manner analogous to mammalian obesity. Gregarine infection is associated with inappropriate responses of hemolymph carbohydrate concentration to insulin and with chronic activation in the flight muscles of p38 MAP kinase, a signaling molecule involved in immune and stress responses. Short-term exposure to gregarine excretory/secretory products caused elevated blood carbohydrate and p38 MAPK activation in healthy individuals. These characteristics comprise a set of symptoms and processes that are known in mammals as metabolic syndrome but which have not previously been described in other animal taxa. In addition to expanding the known taxonomic breadth of metabolic disease, these results indicate that insects may be useful experimental models for studying its underlying biology and mechanisms.

A hierarchical analysis of the scaling of force and power production by dragonfly flight motors

SUMMARY Maximum isometric force output by single muscles has long been known to be proportional to muscle mass0.67, i.e to muscle cross-sectional area. However, locomotion often requires a different muscle contraction regime than that used under isometric conditions. Moreover, lever mechanisms generally affect the force outputs of muscle–limb linkages, which is one reason why the scaling of net force output by intact musculoskeletal systems can differ from mass0.67. Indeed, several studies have demonstrated that force output by intact musculoskeletal systems and non-biological systems is proportional to motor mass1.0. Here we trace the mechanisms that cause dragonflies to achieve a change from muscle mass0.67 scaling of maximum force output by single flight muscles to mass1.0 scaling of dynamic force output by the intact dragonfly flight motor. In eight species of dragonflies, tetanic force output by the basalar muscle during isometric contraction scaled as muscle mass0.67. Mean force output by the basalar muscle under dynamic conditions (workloops) that simulated in vivo maximum musculoskeletal performance was proportional to muscle mass0.83, a significant increase in the scaling exponent over that of maximum isometric force output. The dynamic performance of the basalar muscle and the anatomy of its lever, consisting of the second moment of area of the forewing (d2) and the distance between the muscle apodeme and the wing fulcrum (d1), were used to analyze net force output by the integrated muscle-lever system (Find). The scaling of d2 conformed closely to the expected value from geometic similarity (proportional to muscle mass0.31), whereas d1 scaled as muscle mass0.54, a significant increase over the expected value from geometric similarity. Find scaled as muscle mass1.036, and this scaling exponent was not significantly different from unity or from the scaling exponent relating maximum load-lifting by flying dragonflies to their thorax mass. Thus, the combined effect of a change in the scaling of force output by the muscle during dynamic contraction compared to that during isometric contraction and the departure from geometric similarity of one of the two lever arm lengths provides an explanation for how mass1.0 scaling of force output by the intact musculoskeletal system is accomplished. We also show that maximum muscle mass-specific net work and power output available scale as mass0.43 and mass0.24, respectively.

Genetic variation in HIF signaling underlies quantitative variation in physiological and life-history traits within lowland butterfly populations.

Oxygen conductance to the tissues determines aerobic metabolic performance in most eukaryotes but has cost/benefit tradeoffs. Here we examine in lowland populations of a butterfly a genetic polymorphism affecting oxygen conductance via the hypoxia‐inducible factor (HIF) pathway, which senses intracellular oxygen and controls the development of oxygen delivery networks. Genetically distinct clades of Glanville fritillary (Melitaea cinxia) across a continental scale maintain, at intermediate frequencies, alleles in a metabolic enzyme (succinate dehydrogenase, SDH) that regulates HIF‐1α. One Sdhd allele was associated with reduced SDH activity rate, twofold greater cross‐sectional area of tracheoles in flight muscle, and better flight performance. Butterflies with less tracheal development had greater post‐flight hypoxia signaling, swollen & disrupted mitochondria, and accelerated aging of flight metabolic performance. Allelic associations with metabolic and aging phenotypes were replicated in samples from different clades. Experimentally elevated succinate in pupae increased the abundance of HIF‐1α and expression of genes responsive to HIF activation, including tracheal morphogenesis genes. These results indicate that the hypoxia inducible pathway, even in lowland populations, can be an important axis for genetic variation underlying intraspecific differences in oxygen delivery, physiological performance, and life history.

De novo transcriptome assembly from fat body and flight muscles transcripts to identify morph-specific gene expression profiles in Gryllus firmus.

Wing polymorphism is a powerful model for examining many aspects of adaptation. The wing dimorphic cricket species, Gryllus firmus, consists of a long-winged morph with functional flight muscles that is capable of flight, and two flightless morphs. One (obligately) flightless morph emerges as an adult with vestigial wings and vestigial flight muscles. The other (plastic) flightless morph emerges with fully-developed wings but later in adulthood histolyzes its flight muscles. Importantly both flightless morphs have substantially increased reproductive output relative to the flight-capable morph. Much is known about the physiological and biochemical differences between the morphs with respect to adaptations for flight versus reproduction. In contrast, little is known about the molecular genetic basis of these morph-specific adaptations. To address this issue, we assembled a de novo transcriptome of G. firmus using 141.5 million Illumina reads generated from flight muscles and fat body, two organs that play key roles in flight and reproduction. We used the resulting 34,411 transcripts as a reference transcriptome for differential gene expression analyses. A comparison of gene expression profiles from functional flight muscles in the flight-capable morph versus histolyzed flight muscles in the plastic flight incapable morph identified a suite of genes involved in respiration that were highly expressed in pink (functional) flight muscles and genes involved in proteolysis highly expressed in the white (histolyzed) flight muscles. A comparison of fat body transcripts from the obligately flightless versus the flight-capable morphs revealed differential expression of genes involved in triglyceride biosynthesis, lipid transport, immune function and reproduction. These data provide a valuable resource for future molecular genetics research in this and related species and provide insight on the role of gene expression in morph-specific adaptations for flight versus reproduction.

Body weight-dependent troponin T alternative splicing is evolutionarily conserved from insects to mammals and is partially impaired in skeletal muscle of obese rats

SUMMARY Do animals know at a physiological level how much they weigh, and, if so, do they make homeostatic adjustments in response to changes in body weight? Skeletal muscle is a likely tissue for such plasticity, as weight-bearing muscles receive mechanical feedback regarding body weight and consume ATP in order to generate forces sufficient to counteract gravity. Using rats, we examined how variation in body weight affected alternative splicing of fast skeletal muscle troponin T (Tnnt3), a component of the thin filament that regulates the actin–myosin interaction during contraction and modulates force output. In response to normal growth and experimental body weight increases, alternative splicing of Tnnt3 in rat gastrocnemius muscle was adjusted in a quantitative fashion. The response depended on weight per se, as externally attached loads had the same effect as an equal change in actual body weight. Examining the association between Tnnt3 alternative splicing and ATP consumption rate, we found that the Tnnt3 splice form profile had a significant association with nocturnal energy expenditure, independently of effects of weight. For a subset of the Tnnt3 splice forms, obese Zucker rats failed to make the same adjustments; that is, they did not show the same relationship between body weight and the relative abundance of five Tnnt3 β splice forms (i.e. Tnnt3 β2–β5 and β8), four of which showed significant effects on nocturnal energy expenditure in Sprague–Dawley rats. Heavier obese Zucker rats displayed certain splice form relative abundances (e.g. Tnnt3 β3) characteristic of much lighter, lean animals, resulting in a mismatch between body weight and muscle molecular composition. Consequently, we suggest that body weight-inappropriate skeletal muscle Tnnt3 expression in obesity is a candidate mechanism for muscle weakness and reduced mobility. Weight-dependent quantitative variation in Tnnt3 alternative splicing appears to be an evolutionarily conserved feature of skeletal muscle and provides a quantitative molecular marker to track how an animal perceives and responds to body weight.

Parasites, proteomics and performance: effects of gregarine gut parasites on dragonfly flight muscle composition and function

SUMMARY In previous work, we found that dragonflies infected with gregarine gut parasites have reduced muscle power output, loss of lipid oxidation in their flight muscles, and a suite of symptoms similar to mammalian metabolic syndrome. Here, we test the hypothesis that changes in muscle protein composition underlie the observed changes in contractile performance. We found that gregarine infection was associated with a 10-fold average reduction in abundance of a ∼155 kDa fragment of muscle myosin heavy chain (MHC;∼ 206 kDa intact size). Insect MHC gene sequences contain evolutionarily conserved amino acid motifs predicted for calpain cleavage, and we found that calpain digestion of purified dragonfly MHC produced a peptide of ∼155 kDa. Thus, gut parasites in dragonflies are associated with what appears to be a reduction in proteolytic degradation of MHC. MHC155 abundance showed a strong negative relationship to muscle power output in healthy dragonflies but either no relationship or a weakly positive relationship in infected dragonflies. Troponin T (TnT) protein isoform profiles were not significantly different between healthy and infected dragonflies but whereas TnT isoform profile was correlated with power output in healthy dragonflies, there was no such correlation in infected dragonflies. Multivariate analyses of power output based on MHC155 abundance and a principal component of TnT protein isoform abundances explained 98% of the variation in muscle power output in healthy dragonflies but only 29% when data from healthy and infected dragonflies were pooled. These results indicate that important, yet largely unexplored, functional relationships exist between (pathways regulating) myofibrillar protein expression and (post-translational) protein processing. Moreover, infection by protozoan parasites of the midgut is associated with changes in muscle protein composition (i.e. across body compartments) that, either alone or in combination with other unmeasured changes, alter muscle contractile performance.

The Biochemical Basis of Life History Adaptation: Molecular and Enzymological Causes of NADP+-Isocitrate Dehydrogenase Activity Differences Between Morphs of Gryllus firmus That Differ in Lipid Biosynthesis and Life History

Although whole-organism aspects of life-history physiology are well studied and molecular information (e.g., transcript abundance) on life-history variation is accumulating rapidly, much less information is available on the biochemical (enzymological) basis of life-history adaptation. The present study investigated the biochemical and molecular causes of specific activity differences of the lipogenic enzyme, NADP(+)-isocitrate dehydrogenase, between genetic lines of the wing-polymorphic cricket, Gryllus firmus, which differ in lipid biosynthesis and life history. With one exception, variation among 21 Nadp(+)-Idh genomic sequences, which spanned the entire coding sequence of the gene, was restricted to a few synonymous substitutions within and among replicate flight-capable or flightless lines. No NADP(+)-IDH electromorph variation was observed among individuals within or among lines as determined by polyacrylamide gel electrophoresis. Nor did any NADP(+)-IDH kinetic or stability parameter, such as K(M) for substrate or cofactor, k(cat), or thermal denaturation, differ between flight-capable and flightless lines. By contrast, line differences in NADP(+)-IDH-specific activity strongly covaried with transcript abundance and enzyme protein concentration. These results demonstrate that NADP(+)-IDH-specific activity differences between artificially selected lines of G. firmus are due primarily, if not exclusively, to genetic variation in regulators of NADP(+)-IDH gene expression, with no observed contribution from altered catalytic efficiency of the enzyme due to changes in amino acid sequence or posttranslational modification. Kinetic analyses indicate that in vitro differences in enzyme-specific activity between flight-capable and flightless lines likely occur in vivo. This study constitutes the most comprehensive analysis to date of the biochemical and molecular causes of naturally occurring genetic variation in enzyme activity that covaries strongly with life history.

Cell-autonomous regulation of fast troponin T pre-mRNA alternative splicing in response to mechanical stretch

How mechanochemical signals induced by the amount of weight borne by the skeletal musculature are translated into modifications to muscle sarcomeres is poorly understood. Our laboratory recently demonstrated that, in response to experimentally induced increases in the weight load borne by a rat, alternative splicing of the fast skeletal muscle troponin T (Tnnt3) pre-mRNA in gastrocnemius was adjusted in a correlated fashion with the amount of added weight. (Schilder RJ, Kimball SR, Marden JH, Jefferson LS. J Exp Biol 214: 1523-1532, 2011). Thus muscle load is perceived quantitatively by the body, and mechanisms that sense it appear to control processes that generate muscle sarcomere composition plasticity, such as alternative pre-mRNA splicing. Here we demonstrate how mechanical stretch (see earlier comment) of C2C12 muscle cells in culture results in changes to Tnnt3 pre-mRNA alternative splicing that are qualitatively similar to those observed in response to added weight in rats. Moreover, inhibition of Akt signaling, but not that of ERK1/2, prevents the stretch-induced effect on Tnnt3 pre-mRNA alternative splicing. These findings suggest that effects of muscle load on Tnnt3 pre-mRNA alternative splicing are controlled by a cell-autonomous mechanism, rather than systemically. They also indicate that, in addition to its regulatory role in protein synthesis and muscle mass plasticity, Akt signaling may regulate muscle sarcomere composition by modulating alternative splicing events in response to load. Manipulation of Tnnt3 pre-mRNA alternative splicing by mechanical stretch of cells in culture provides a model to investigate the biology of weight sensing by skeletal muscles and facilitates identification of mechanisms through which skeletal muscles match their performance and experienced load.

Role of Precursor mRNA Splicing in Nutrient-Induced Alterations in Gene Expression and Metabolism

Precursor mRNA (pre-mRNA) splicing is a critical step in gene expression that results in the removal of intronic sequences from immature mRNA, leading to the production of mature mRNA that can be translated into protein. Alternative pre-mRNA splicing is the process whereby alternative exons and/or introns are selectively included or excluded, generating mature mRNAs that encode proteins that may differ in function. The resulting alterations in the pattern of protein isoform expression can result in changes in protein-protein interaction, subcellular localization, and flux through metabolic pathways. Although basic mechanisms of pre-mRNA splicing of introns and exons are reasonably well characterized, how these mechanisms are regulated remains poorly understood. The goal of this review is to highlight selected recent advances in our understanding of the regulation of pre-mRNA splicing by nutrients and modulation of nutrient metabolism that result from changes in pre-mRNA splicing.