Considerable information exists on the physiological correlates of life history adaptation, while molecular data on this topic are rapidly accumulating. However, much less is known about the enzymological basis of life history adaptation in outbred populations. In the present study, we compared developmental profiles of fat body specific activity, kinetic constants of homogeneously purified and unpurified enzyme, and fat body enzyme concentration of the pentose-shunt enzyme, 6-phosphogluconate dehydrogenase (6PGDH, E.C.22.214.171.124) between the dispersing [long-winged, LW(f)] and flightless [short-winged, SW] genotypes of the cricket Gryllus firmus. Neither kcat nor the Michaelis constant for 6-phosphogluconate differed between 6PGDH from LW(f) versus SW morphs for either homogeneously purified or unpurified enzyme. Purified enzyme from the LW(f) morph exhibited reduced KM for NADP(+), but this was not observed for multiple KM(NADP+) estimates for unpurified enzyme. A polyclonal antibody was generated against 6PGDH which was used to develop a chemiluminescence assay to quantify 6PGDH concentration in fat body homogenates. Elevated enzyme concentration accounted for all of the elevated 6PGDH specific activity in the LW(f) morph during the juvenile and adult stages. Finally, activity of another pentose-shunt enzyme, glucose-6-phosphate dehydrogenase, strongly covaried with 6PGDH activity suggesting that variation in 6PGDH activity gives rise to variation in pentose shunt flux. This is one of the first life-history studies and one of the few studies of intraspecific enzyme adaptation to identify the relative importance of evolutionary change in enzyme concentration vs. kinetic constants to adaptive variation in enzyme activity in an outbred population.
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.
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.
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 ? splice forms (i.e. Tnnt3 ?2-?5 and ?8), 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 ?3) 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.
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.
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.
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-1?. 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-1? 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.
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