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A correction to this paper has been published: https://doi.org/10.1007/s10592-021-01376-9

Subtelomeric transcription and chromatin can have a significant impact on telomere repeat maintenance and chromosome stability. We have previously found that tumor suppressor protein p53 (TP53) can bind to retrotransposon-like elements in a majority of human subtelomeres to regulate TERRA transcription and telomeric histone acetylation in response to DNA damage. TP53 also prevents the accumulation of γH2AX DNA-damage signaling at telomeres. We now show that the inherited TP53 polymorphism Pro47Ser (hereafter S47), which is enriched in populations of African descent, is associated with elevated marks of telomere dysfunction. We found that human and mouse cells carrying the S47 variant show increased γH2AX DNA-damage signals at telomeres, as well as reduced TERRA transcription and subtelomeric histone acetylation in response to DNA damage stress. Cell-lines containing inducible genes for P47 or S47 versions of p53, as well mouse embryo fibroblasts (MEFs) reconstituted with human p53, showed elevated telomere-induced DNA damage foci and metaphase telomere signal loss in cells with S47. Human lymphoblastoid cell lines (LCLs) derived from individuals homozygous for S47, show increased accumulation of subtelomeric γH2AX and unstable telomere repeats in response to DNA damage relative to age matched LCLs homozygous for P47. Furthermore, LCLs with S47 had reduced replicative lifespan. These studies indicate that the naturally occurring S47 variant of p53 can affect telomeric chromatin, telomere repeat stability, and replicative capacity. We discuss the potential evolutionary significance of the S47 variant to African populations with respect to telomere regulation and the implications for inherited health disparities.

ispartof: CONSERVATION GENETICS vol:22 issue:4 pages:533-536 status: published

Interspecific introgression is considered a potential threat to endangered taxa. One example where this has had a major impact on conservation policy is the lesser white-fronted goose (LWfG). After a dramatic decline in Sweden, captive breeding birds were released between 1981–1999 with the aim to reinforce the population. However, the detection of greater white-fronted goose (GWfG) mitochondrial DNA in the LWfG breeding stock led to the release program being dismantled, even though the presence of GWfG introgression in the actual wild Swedish LWfG population was never documented. To examine this, we sequenced the complete genomes of 21 LWfG birds from the Swedish, Russian and Norwegian populations, and compared these with genomes from other goose species, including the GWfG. We found no evidence of interspecific introgression into the wild Swedish LWfG population in either nuclear genomic or mitochondrial data. Moreover, Swedish LWfG birds are genetically distinct from the Russian and Norwegian populations and display comparatively low genomic diversity and high levels of inbreeding. Our findings highlight the utility of genomic approaches in providing scientific evidence that can help improve conservation management as well as policies for breeding and reinforcement programmes.

Objective: The aim of this in vitro study is to compare the antimicrobial efficacy of four different endodontic sealers against Enterococcus faecalis. Material and Method: Four different endodontic sealers, namely, resin based (AH Plus), zinc oxide-eugenol based (Tubliseal), calcium hydroxide based (Sealapex), and mineral trioxide aggregate (MTA Fillapex) based were tested for their antimicrobial efficacy against E. faecalis using agar diffusion method. Four wells were made by the removal of agar at equidistant points and filled with freshly mixed respective root canal sealers and were inoculated with E. faecalis. All the three plates were incubated for a period of 72 h at 37°C under aerobic conditions. The diameter of inhibition zones was measured at 24, 48, and 72 h time intervals. Data obtained were statistically analyzed using one-way analysis of variance and unpaired t-test. Results: All the tested sealers showed some bacterial growth inhibition of E. faecalis. Their efficacy in descending order of antibacterial activity was as follows: Sealapex > AH Plus > Tubliseal > MTA Fillapex. The efficacy of the root canal sealers decreased marginally with increase in their duration of action. Conclusion: Antimicrobial efficacy of calcium hydroxide-based sealer was highest followed by resin-based sealer and was the least with MTA based sealer.

Human herpesvirus 7 (HHV-7) is a betaherpesvirus, and is phylogenetically related to both HHV-6A and HHV-6B. The presence of telomeric repeat sequences at both ends of its genome should make it equally likely to integrate into the human telomere as HHV-6. However, numerous studies have failed to detect germline integration of HHV-7, suggesting an important difference between the HHV-6A/-6B and HHV-7 genomes. In search of possible germline integrated HHV-7, we developed a sensitive and quantitative real-time PCR assay and discovered that primers designed against some parts of the HHV-7 genome can frequently miss HHV-7 positive clinical samples even though they work efficiently in cell-culture-derived HHV-7 positive materials. Using a primer pair against the U90 ORF of HHV-7, we identified a possible case of germline integration of HHV-7 with one copy of viral genome per cell in both peripheral blood cells and hair follicles. Chromosomal integration of HHV-7 in these individuals was confirmed by fluorescence in situ hybridization analysis. Germline integration of HHV-7 was further confirmed by detection of ~2.6 copies of HHV-7 in the hair follicles of one of the parents. Our results shed light on the complex nature of the HHV-7 genome in human-derived materials in comparison to cell-culture-derived materials and show the need for stringent criteria in the selection of primers for epidemiological HHV-7 studies.

Human herpesvirus 6A and 6B frequently acquires latency. HHV-6 activation has been associated with various human diseases. Germ line inheritance of chromosomally integrated HHV-6 makes viral DNA-based analysis difficult for determination of early stages of viral activation. We characterized early stages of HHV-6 activation using high throughput transcriptomics studies and applied the results to understand virus activation under clinical conditions. Using a latent HHV-6A cell culture model in U2OS cells, we identified an early stage of viral reactivation, which we define as transactivation that is marked by transcription of several viral small non-coding RNAs (sncRNAs) in the absence of detectable increase in viral replication and proteome. Using deep sequencing approaches, we detected previously known as well as a new viral sncRNAs that characterized viral transactivation and differentiated it from latency. Here we show changes in human transcriptome upon viral transactivation that reflect multiple alterations in mitochondria-associated pathways, which was supported by observation of increased mitochondrial fragmentation in virus reactivated cells. Furthermore, we present here a unique clinical case of DIHS/DRESS associated death where HHV-6 sncRNA-U14 was abundantly detected throughout the body of the patient in the presence of low viral DNA. In this study, we have identified a unique and early stage of viral activation that is characterized by abundant transcription of viral sncRNAs, which can serve as an ideal biomarker under clinical conditions., Virology: a biomarker for HHV-6 reactivation The human herpesvirus 6 (HHV-6) expresses high levels of small non-coding RNA (sncRNA) molecules early in its reactivation from latency. Bhupesh Prusty from the University of Würzburg, Germany, and colleagues developed a laboratory system for studying HHV-6 infections in a human bone cancer cell line. They reawakened the virus with a drug stimulus and detected several sncRNAs but few other viral RNAs that might promote replication or protein production. They term this unique stage of the viral life cycle ‘transactivation’, and show that it alters both host and viral physiology. The authors also describe a teenage girl with high sncRNA levels in her blood who fell ill after an acne drug spurred the reactivation of a dormant HHV-6 infection. They thus argue that sncRNAs could serve as an early diagnostic indicator of HHV-6 reactivation.

Chlamydia trachomatis infections have been associated with ovarian cancer by several epidemiological studies. Here, we show that C. trachomatis-infected primary human ovarian epithelial cells display elevated oxidative DNA damage. Base excision repair, an important cellular mechanism to repair oxidative DNA lesions, was impaired in infected primary ovarian and in several other types of cells. Polymerase β was downregulated in infected cells associated with upregulation of microRNA-499a (miR-499a). Stabilising polymerase β by inhibiting miR-499a significantly improved repair. Moreover, downregulation of tumour suppressor p53 also resulted in attenuated repair in these cells. Thus, our data show that downregulation of polymerase β by direct inhibition through miR-499a and downregulation of p53 debilitate the host-cell base excision repair during C. trachomatis infection.

The scarce heath (Coenonympha hero) is an internationally threatened butterfly in Western Europe, where it occurs primarily on hay fields and abandoned arable land in a small-scale agricultural landscape of south-central Scandinavia. Due to afforestation, this habitat is becoming increasingly fragmented in Sweden, and it can be expected that the scarce heath will decline abruptly when threshold conditions for metapopulation persistence are no longer met. We used stepwise polychotomous logistic regression to compare habitat characteristics and isolation measures for patches that harbour large, small or no populations, respectively, in an area of south-western Sweden. We found that patch area, distance to the nearest large population and amount of Galium spp. explained a significant part of the variation in relative abundance among patches. Distance to nearest large population resulted in a better model to predict occupancy than both distance to the nearest inhabited patch and connectivity, which suggests that primarily large populations act as sources for small satellite populations. Today, sites of three of the eight larger populations in the study area have been planted with spruce or pine and will disappear within 20 years. We argue that the disappearance of these patches may very well lead to rapid extinction of the whole metapopulation system.

We have created a novel quaternary ammonium silane, K21 through sol-gel chemistry, using an ethoxylated version of an organosilane quaternary ammonium compound and TetraEthyl Ortho Silicate (TEOS) as precursors. Previous studies using the precursor molecule quaternary ammonium compounds (QACs) and a methacryloxy version of K21, primarily designed for use in dental healthcare, have shown inhibited growth properties against several types of gram-positive and gram-negative bacteria including Escherichia coli , Streptococcus mutans, Actinomyces naeslundii and Candida albicans etc . Here we tested the effect of K21 on HSV-1, HHV-6A and HHV-7 in in vitro cell culture infection models. Our results show growth inhibitory effect of K21 on HSV-1, HHV-6A and HHV-7 infection.

Dispersal between habitat patches may be important for the long-term persistence of populations. We conducted a mark–release–recapture study and analysed the dispersal pattern in the scarce heath butterfly inhabiting a network of suitable habitat patches using stepwise logistic regression (SLR) and the Virtual Migration (VM) model. We also analysed the influence of different types of matrices. We found that the majority of the recaptured butterflies remained within the patch where they were originally caught. However, dispersal between patches did occur and both the SLR analysis and the VM model indicated that the migration pattern was significantly associated with patch area and its level of isolation. The SLR model also showed that there was a positive association between immigration rate and tree density, supporting earlier observations that this species prefers semi-open habitat. We discuss the use of SLR versus the VM model to analyse recapture data in dispersal studies. This system is not at equilibrium, as a number of the most important patches in the network are continuously being lost due to afforestation and a number of populations are facing deterministic extinction. This increases the risk of a chain reaction of local extinctions, which may cause a collapse of the whole system.

Allozyme variability was examined in relation to population size among sympatric moor frogs (Rana arvalis) and pool frogs (R. lessonae) at a pond in east-central Sweden (60 degrees N). Average hete ...

Two aspects of patch geometry—area and isolation—currently dominate the field of metapopulation dynamics. Under this area-and-isolation paradigm, models commonly assume that the probability of local extinction decreases as patch area increases and that the probability of colonization increases as patch connectivity increases. Environmental variables other than patch area and isolation are assumed to have relatively little effect on metapopulation dynamics. Our work on a metapopulation of the butterfly Speyeria nokomis apacheana highlights the need for a broader view of metapopulation dynamics. In this system, neither occupancy nor turnover patterns were best modeled as functions of patch area or isolation. Instead, other measures of habitat quality explained the most variance in occupancy and turnover. Our study also revealed temporal variation in the factors associated with occupancy and turnover. This variation can cause the results of analyses to vary with the temporal scale of analysis. For example, factors associated with turnover in this system differed among single-year and multiple-year analyses. We emphasize that factors other than patch geometry may drive extinction and colonization processes in metapopulations, especially in systems that experience substantial natural and anthropogenic environmental variability. Resumen: Dos aspectos de la geometria de parches —area y aislamiento—dominan actualmente el campo de la dinamica metapoblacional. Bajo este paradigma de “area y aislamiento”, los modelos comunmente suponen que la probabilidad de una extincion local disminuye si el area del parche aumenta y la probabilidad de colonizacion aumenta si la conectividad del parche aumenta. Se asume que otras variables ambientales diferentes al area y al aislamiento del parche tienen relativamente poco efecto en la dinamica metapoblacional. Nuestro trabajo en una metapoblacion de la mariposa Speyeria nokomis apacheana resalta la necesidad de una vision mas amplia de las dinamicas metapoblacionales. En este sistema, no se modelaron bien ni la ocupacion ni los patrones de rendimiento como funciones del area o aislamiento del parche. Por lo contrario, otras medidas de calidad del habitat explicaron la mayor parte de la variacion en cuanto a ocupacion y rendimiento. Nuestro estudio tambien revelo una variacion temporal en los factores asociados con la ocupacion y el rendimiento. Esta variacion puede hacer que los resultados de los analisis varien con la escala temporal del analisis. Por ejemplo, los factores asociados con el rendimiento en este sistema discreparon en los analisis entre anos individuales y entre anos multiples. Senalamos que factores diferentes a la geometria del parche pueden conducir a procesos de extincion y colonizacion en metapoblaciones, especialmente en sistemas que experimentan una variabilidad ambiental natural y antropogenica substancial.

Patterns and levels of genetic diversity mayhave significant influence on the long termpersistence of local populations and revealingsuch information is important in protectingrare species. In this study we investigated thegenetic pattern in five microsatellite lociwithin five Swedish populations of the rareorchid species Gymnadenia odoratissima. Thegeographic distribution of G. odoratissima isrestricted to Europe and in Scandinavia it isonly found in three provinces in southernSweden; Ostergotland,Vastergotland and on the island ofGotland.Compared with the more widespread congener G.conopsea our results indicate lower levels ofgenetic variation within and higher degrees ofgenetic differentiation among populations ofG. odoratissima (HEL = 0.6–0.8 in G. conopseaand 0.3–0.7 in G. odoratissima; FST over allpopulations = 0.06 in G. conopsea and 0.19 inG. odoratissima). Also, we found a cleardistinction among mainland and islandpopulations of G. odoratissima wherepopulations on the island of Gotland seem toexhibit higher levels of gene flow andintragenetic variation, probably as a result ofa larger number of existing populations.Future conservation of this species shouldfocus on facilitation on colonisation events,especially on the mainland, and preservation ofthe genetically more variable Gotlandpopulations.

Insulin resistance can be induced in vivo by intravenous infusion of glucosamine or in cells by incubation with glucosamine. However, a publication (Hresko, R. C., et al. (1998) J. Biol. Chem. 273, 20658-20668) suggests a trivial explanation of glucosamine-induced insulin resistance whereby intracellular ATP pools are depleted presumably due to the phosphorylation of glucosamine to glucosamine 6-phosphate, a hexosamine pathway intermediate. The reduced ATP level impaired insulin receptor (IR) autophosphorylation and tyrosine kinase activity toward substrates. The present work describes the development and comparison of two methods for inducing insulin resistance, by treating 3T3-L1 adipocytes overnight using either 25 mM glucose/5 nM insulin or 2 mM glucosamine. Under these conditions basal glucose transport rates were comparable with controls. Insulin-stimulated 2-deoxyglucose uptake, however, was reduced by approximately 45% in response to both high glucose/insulin and glucosamine treatment, relative to control cells. The total relative amounts of the insulin-responsive glucose transporter, Glut4, remained constant under both treatment conditions. The relative phosphotyrosine (Tyr(P)) contents of the insulin receptor and its substrate 1 (IRS-1) were assessed in whole cell homogenates. With both methods to induce insulin resistance, IR/IRS-1 Tyr(P) levels were virtually indistinguishable from those in control cells. Insulin-stimulated phosphorylation of Akt on Ser(473) was not impaired in insulin-resistant cells. Furthermore, the relative Tyr(P) content of the PDGF receptor was comparable in high glucose/insulin- or glucosamine-treated 3T3-L1 adipocytes upon subsequent challenge with PDGF. Finally, the relative amounts of glutamine:fructose-6-phosphate amidotransferase and O-linked N-acetylglucosamine transferase, two important hexosamine pathway enzymes, were similar in both treatments when compared with controls. Thus, 3T3-L1 adipocytes can be used as a model system for studying insulin resistance induced by increased influx of glucose. Under appropriate experimental conditions, glucosamine treatment can mimic the effects of increased glucose flux without impairment of tyrosine phosphorylation-based signaling.

It has been hypothesized that increased production of tumor necrosis factor-alpha (TNF-alpha) plays a role in causing the insulin resistance associated with obesity. Obesity with insulin resistance is associated with increased production of TNF-alpha by fat cells. Exposure of 3T3-L1 adipocytes to TNF-alpha for 3-4 days makes them insulin resistant. TNF-alpha has also been reported to rapidly (15-60 min) cause insulin resistance, with a decrease in insulin-stimulated tyrosine phosphorylation, in a number of cultured cell lines. Because skeletal muscle is the major tissue responsible for insulin-stimulated glucose disposal, we performed the present study to determine if acute exposure to TNF-alpha causes insulin resistance in muscle. We found that exposure of soleus muscles to 6 nmol/l TNF-alpha for 45 min in vitro had no inhibitory effect on insulin-stimulated tyrosine phosphorylation of the insulin receptor or insulin receptor substrate 1 (IRS-1) or on phosphatidylinositol 3-kinase association with IRS-1. Incubation of epitrochlearis and soleus muscles with 6 nmol/l TNF-alpha for 45 min or 4 h had no effect on insulin-stimulated 2-deoxyglucose (2-DG) uptake. Treatment of epitrochlearis muscles with 2 nmol/l TNF-alpha for 8 h also had no effect on insulin-stimulated 2-DG uptake. We conclude that in contrast to Fao hepatoma cells and 3T3-L1 fibroblasts, skeletal muscle does not become insulin resistant in response to short-term exposure to TNF-alpha.

Using drift fences, the spatial movement patterns of the pool frog (Rana lessonae Camerano), the moor frog (R arvalis Nilsson) and the common frog (R, temporaria L.)were studied with respect to ori ...

The spatial movement pattern of pool frogs (Rana lessonae Camerano) was studied at two breeding ponds surrounded by drift fences at 60˚n in Sweden. Fence traps were classified according to (i) surrounding vegetation, or (ii) whether they were situated in a direction leading towards forest, adjacent marshes or neighboring pool frog localities. Observed captures per trap category were tested against numbers expected from random movements. Trap records of individually marked frogs were used in the analyses, which were performed separately for three time periods throughout the active season, each of two study-years. By separating captures during days with singly moving frogs (dispersers) from those during days with simultaneous group movements, the orientation of dispersal versus group (migration) movements were also compared. The movements of both adults and juveniles were significantly and consistently directed during all time periods, and generally the movements of singly-moving frogs were as targe...

beta-Adrenergic stimulation has been reported to inhibit insulin-stimulated glucose transport in adipocytes. This effect has been attributed to a decrease in the intrinsic activity of the GLUT-4 isoform of the glucose transporter that is mediated by phosphorylation of GLUT-4. Early studies showed no inhibition of insulin-stimulated glucose transport by epinephrine in skeletal muscle. The purpose of this study was to determine the effect of epinephrine on GLUT-4 phosphorylation, and reevaluate the effect of beta-adrenergic stimulation on insulin-activated glucose transport, in skeletal muscle. We found that 1 microM epinephrine, which raised adenosine 3',5'-cyclic monophosphate approximately ninefold, resulted in GLUT-4 phosphorylation in rat skeletal muscle but had no inhibitory effect on insulin-stimulated 3-O-methyl-D-glucose (3-MG) transport. In contrast to 3-MG transport, the uptakes of 2-deoxyglucose and glucose were markedly inhibited by epinephrine treatment. This inhibitory effect was presumably mediated by stimulation of glycogenolysis, which resulted in an increase in glucose 6-phosphate concentration to levels known to severely inhibit hexokinase. We conclude that 1) beta-adrenergic stimulation decreases glucose uptake by raising glucose 6-phosphate concentration, thus inhibiting hexokinase, but does not inhibit insulin-stimulated glucose transport and 2) phosphorylation of GLUT-4 has no effect on glucose transport in skeletal muscle.

Enhanced glucose flux via the hexosamine biosynthetic pathway has been implicated in insulin resistance. We measured products of this pathway, UDP-N-acetyl hexosamines (UDP-HexNAc), and activity of the rate-limiting enzyme L-glutamine:D-fructose-6-phosphate amidotransferase (GFAT) in tissues of ob/ob mice and lean controls. Ob/ob mice were obese, hyperglycemic, and hyperinsulinemic. Resistance to the effect of insulin on glucose transport was demonstrated in isolated soleus muscles, although total GLUT-4 concentration was mildly increased in muscles from ob/ob mice. UDP-HexNAc concentrations in hindlimb muscles decreased between 8 and 17 wk but were always higher in ob/ob vs. controls (P < 0.001, mean increase 67%). Concentrations of UDP-hexoses and GDP-mannose were similar in ob/ob and control muscles. Muscle GFAT activity declined with age but was increased in ob/ob vs. controls at each age examined (P < 0.001, mean increase 108%). UDP-HexNAc concentrations and GFAT activity were similar in livers of ob/ob and controls. These data suggest that glucose flux via the hexosamine pathway is selectively increased in muscle but not liver of ob/ob mice and may contribute to muscle insulin resistance in this model of non-insulin-dependent diabetes mellitus.

Policy makers and managers are increasingly called upon to assess the state of biodiversity, and make decisions regarding potential interventions. Genetic tools are well-recognised in the research community as a powerful approach to evaluate species and population status, reveal ecological and demographic processes, and inform nature conservation decisions. The wealth of genetic data and power of genetic methods are rapidly growing, but the consideration of genetic information and concerns in policy and management is limited by the currently low capacity of decision-makers to access and apply genetic resources. Here we describe a freely available, user-friendly online resource for decision-makers at local and national levels (http://congressgenetics.eu), which increases access to current knowledge, facilitates implementation of studies and interpretation of available data, and fosters collaboration between researchers and practitioners. This resource was created in partnership with conservation practitioners across the European Union, and includes a spectrum of taxa, ecosystems and conservation issues. Our goals here are to (1) introduce the rationale and context, (2) describe the specific tools (knowledge summaries, publications database, decision making tool, project planning tool, forum, community directory), and the challenges they help solve, and (3) summarise lessons learned. This article provides an outlook and model for similar efforts to build policy and management capacity

There is evidence that an increase in sarcoplasmic Ca2+ stimulates glucose transport in muscle. Recent studies have provided the apparently conflicting finding that a sustained increase in cytosolic Ca2+ has little effect on basal glucose transport but inhibits insulin-stimulated transport. This study was done to try to explain this discrepancy. Continuous exposure of rat epitrochlearis and soleus muscles to the Ca2+ ionophore ionomycin (2 microM) had no effect on basal 2-deoxyglucose (2-DG) transport but blunted, by approximately 40%, stimulation of 2-DG transport by insulin. Decreasing Ca2+ in the medium to a very low level prevented this inhibition. Ionomycin induced a small increase in adenosine 3',5'-cyclic monophosphate (cAMP); however, studies with the protein kinase A (PKA) inhibitor HA-1004 provided evidence that activation of PKA by cAMP does not mediate the inhibition of glucose transport. When muscles were allowed to recover in the absence of ionomycin for 15 min, basal 2-DG transport was significantly increased. Our results agree with previous studies showing that a sustained influx of Ca2+ into the cytoplasm can inhibit insulin-stimulated glucose transport. They further show that stimulation of glucose transport by Ca2+ is also inhibited. A recovery period that allows this inhibition to wear off unmasks the stimulation of glucose transport by an increase in sarcoplasmic Ca2+.

Both the anabolic hormone insulin and contractile activity stimulate the uptake of glucose into mammalian skeletal muscle. In this study, we examined the role of phosphatidylinositol 3-kinase (PI 3-kinase), a putative mediator of insulin actions, in the stimulation of hexose uptake in response to hormone and contraction. Phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-triphosphate accumulate in skeletal muscle exposed to insulin but not hypoxia, which mimics stimulation of the contractile-dependent pathway of hexose transport activation. The fungal metabolite wortmannin, an inhibitor of PI 3-kinase, completely blocks the appearance of 3′-phospholipids in response to insulin. Moreover, wortmannin entirely prevented the increase in hexose uptake in muscle exposed to insulin but was without effect on muscle stimulated by repetitive contraction or hypoxia. These results support the view that PI 3-kinase is involved in the signaling pathways mediating insulin-responsive glucose transport in skeletal muscle but is not required for stimulation by hypoxia or contraction. Furthermore, these data indicate that there exist at least two signaling pathways leading to activation of glucose transport in skeletal muscle with differential sensitivities to wortmannin.

Skeletal muscle glucose transport and metabolism were studied in a line of transgenic mice overexpressing the human Glut4 facilitative glucose transporter. Skeletal muscle Glut4 protein levels were increased 2-4-fold in transgenic animals relative to their nontransgenic litter mates. Glut4 overexpression increased total transport activity (measured with 1 mM 2-deoxy-D-glucose) in the isolated extensor digitorum brevis muscle in the presence of insulin; this increase was due to 1) an increase in basal glucose transport (0.8 +/- 0.1 versus 0.5 +/- 0.1 mumol.ml-1.20 min-1 in transgenic and control mice, respectively) and 2) an increase in insulin-stimulated transport (1.5 +/- 0.1 versus 0.8 +/- 0.1 mumol.ml-1.20 min-1 above basal transport in transgenic and control mice, respectively). Glut4 overexpression also increased glucose transport stimulated by muscle contractions. In addition, glycolysis and glucose incorporation into glycogen were enhanced in muscle isolated from transgenic mice compared to controls. These data demonstrate that Glut4 overexpression in skeletal muscle increases insulin- and contraction-stimulated glucose transport activity and glucose metabolism. These findings are consistent with the role of Glut4 as the primary mediator of transport stimulated by insulin or contractions.

Lithium is thought to have an insulin-like effect on glucose transport and metabolism in skeletal muscle and adipocytes. However, we found that lithium had only a minimal effect on basal glucose transport activity in rat epitrochlearis muscles. Instead, lithium markedly increased the sensitivity of glucose transport to insulin, so that the increase in glucose transport activity induced by 300 pM insulin was ∼2.5-fold greater in the presence of lithium than in its absence. Lithium also caused a modest increase in insulin responsiveness. This enhancement of the susceptibility of the glucose transport process to stimulation was not limited to insulin, because lithium induced increases in the susceptibility of glucose transport to stimulation by contractile activity, hypoxia, a phorbol ester, and phospholipase C. Lithium also blunted the activation of glycogen phosphorylase by epinephrine. These effects were not mediated by inhibition of adenylate cyclase, because neither basal- nor epinephrine-stimulated muscle cAMP concentration was affected by lithium treatment. The effects of lithium on glucose transport and metabolism in skeletal muscle are strikingly similar to the persistent effects of exercise. These results support the possibility that lithium might be useful in the treatment of insulin resistance in patients with non-insulin-dependent diabetes mellitus.

The distribution and extinction patterns within a northern metapopulation of the pool frog (Rana lessonae) were analyzed with reference to a metapopulation theory. Occupied ponds were permanent and differed from unoccupied ones in terms of higher water temperature during May—June and a closer proximity to neighboring pool—frog localities, but local climate was not spatially autocorrelated. Two types of population extinctions occurred (average rate = 2% per population and year): (1) deterministic extinctions due to succession or draining pool—frog pond, and (2) extinctions of populations whose isolation had increased to a critical degree because of Type 1 extinctions of neighboring populations, increasing their susceptibility to predation and combined demographic/environmental stochasticity. The Type 2 extinctions were spatially correlated to a moderate degree, which may reflect the great impact of environmental stochasticity in the system. The results confirm and emphasize the importance of interpopulation proximity and connectivity of metapopulation persistence.

Glucose transport activity was examined in transgenic mice overexpressing the human GLUT1 glucose transporter in skeletal muscles. Basal transport activity measured in vitro with the glucose analog 2-deoxy-D-glucose (1 mM) was increased 2-8-fold in four different muscle preparations. Incubation of muscles from control nontransgenic littermates with a maximally effective concentration of insulin or with insulin-like growth factor-1 resulted in glucose transport rates that were 2-3-fold higher than basal. In contrast, insulin did not stimulate glucose transport activity in three different muscle preparations from transgenic animals; insulin-like growth factor-1 was similarly ineffective. Activation of System A amino acid transport activity (measured with the nonmetabolizable analog alpha-methylaminoisobutyrate) by insulin was not impaired in muscles from transgenic mice, indicating that the defect does not involve the insulin receptor. In skeletal muscle, glucose transport can be activated by muscle contractions or hypoxia via a pathway separate from that activated by insulin. Incubation of muscles under hypoxic conditions or stimulation of muscles to contract in situ did not increase glucose transport activity in muscles from GLUT1-overexpressing mice, in contrast to the stimulatory effects measured in muscles from control animals. These data suggest that increased glucose flux per se into skeletal muscle results in resistance of GLUT4 to activation by insulin and various other stimuli that activate glucose transport by mechanisms distinct from that of insulin. GLUT1-overexpressing mice thus provide a new model system for studying the effects of glucose-induced resistance to activation of glucose transport.

GLUT4 glucose transporter content and glucose transport capacity are closely correlated in skeletal muscle. In this study, we tested the hypothesis that a rapid increase in GLUT4 expression occurs as part of the early adaptive response of muscle to exercise and serves to enhance glycogen storage. Rats exercised by swimming had a approximately 2-fold increase in GLUT4 mRNA and a 50% increase in GLUT4 protein expression in epitrochlearis muscle 16 h after one prolonged exercise session. After a 2nd day of exercise, muscle GLUT4 protein was increased further to approximately 2-fold while there was no additional increase in GLUT4 mRNA. Muscle hexokinase activity also doubled in response to 2 days of exercise. Glucose transport activity maximally stimulated with insulin, contractions, or hypoxia was increased roughly in proportion to the adaptive increase in GLUT4 protein in epitrochlearis muscles. Treatment with insulin prior to subcellular fractionation of muscle resulted in a approximately 2-fold greater increase in GLUT4 content of a plasma membrane fraction in the 2-day swimmers than in controls. When epitrochlearis muscles were incubated with glucose and insulin, glycogen accumulation over 3 h was twice as great in muscles from 2-day swimmers as in control muscles. Our results show that a rapid increase in GLUT4 expression is an early adaptive response of muscle to exercise. This adaptation appears to be mediated by pretranslational mechanisms. We hypothesize that the physiological role of this adaptation is to enhance replenishment of muscle glycogen stores.

It is widely thought that aging results in development of insulin resistance in skeletal muscle. In this study, we examined the effects of growth and aging on the concentration of the GLUT-4 glucose transporter and on glucose transport activity in skeletal muscles of female Long-Evans rats. Relative amounts of immunoreactive GLUT-4 protein were measured in muscle homogenates of 1-, 10-, and 25-mo-old rats by immunoblotting with a polyclonal antibody directed against GLUT-4. In the epitrochlearis, plantaris, and the red and white regions of the quadriceps muscles, GLUT-4 immunoreactivity decreased by 14-33% between 1 and 10 mo of age and thereafter remained constant. In flexor digitorum brevis (FDB) and soleus muscles, GLUT-4 concentration was similar at all three ages studied. Glucose transport activity was assessed in epitrochlearis and FDB muscles by incubation with 2-deoxyglucose under the following conditions: basal, submaximal insulin, and either maximal insulin or maximal insulin combined with contractile activity. Glucose transport in the epitrochlearis muscle decreased by approximately 60% between 1 and 4 mo of age and then did not decline further between 4 and 25 mo of age. Transport activity in the FDB assessed with a maximally effective insulin concentration decreased only slightly (< 20%) between 1 and 7 mo of age. Aging, i.e., the transition from young adulthood to old age, was not associated with a decrease in glucose transport activity in either the epitrochlearis or the FDB.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of skeletal muscle to contract activates phosphorylase b-to-a conversion and glycogenolysis. Despite reversal of the increase in percentage of phosphorylase a after a few minutes, continued glycogen breakdown can occur during strenuous exercise. Hypoxia causes sustained glycogenolysis in skeletal muscle without an increase in percentage of phosphorylase a. We used this model to obtain insights regarding how glycogenolysis is mediated in the absence of an increase in percentage of phosphorylase a. Hypoxia caused a 70% decrease in glycogen in epitrochlearis muscles during an 80-min incubation despite no increase in percentage of phosphorylase a above the basal level of approximately 10%. Muscle Pi concentration increased from 3.8 to 8.6 mumol/g muscle after 5 min and 15.7 mumol/g after 20 min. AMP concentration doubled, attaining a steady state of 0.23 mumol/g in 5 min. Incubation of oxygenated muscles with 0.1 microM epinephrine induced an approximately sixfold increase in percentage of phosphorylase a but resulted in minimal glycogenolysis. Muscle Pi concentration was not altered by epinephrine. Despite no increase in percentage of phosphorylase a, hypoxia resulted in a fivefold greater depletion of glycogen over 20 min than did epinephrine. To evaluate the role of phosphorylase b, muscles were loaded with 2-deoxyglucose 6-phosphate, which inhibits phosphorylase b. The rate of glycogenolysis during 60 min of hypoxia was reduced by only approximately 14% in 2-deoxyglucose 6-phosphate-loaded muscles.(ABSTRACT TRUNCATED AT 250 WORDS)

During experiments involving prolonged incubation of skeletal muscle, we observed large increases in glucose transport activity. The basal rate of 3-O-methylglucose (3-MG) transport increased two- to fourfold in rat epitrochlearis muscles incubated for 9 h without insulin in Krebs-Henseleit buffer supplemented with 8 mM glucose. The stimulatory effect of a low concentration of insulin (30 microU/ml, added during the final 30 or 60 min of incubation) on glucose transport activity was enhanced 2.5-fold after 6 h and approximately 5-fold after 9 h of incubation. Exposure of muscles to 100 microU/ml of insulin for the first 8 h inhibited slightly but significantly the increase in insulin-stimulated 3-MG transport over a 9-h incubation period. Incubation of muscles in minimal essential medium (MEM) for 9 h inhibited the time-dependent rise in basal and insulin-stimulated transport by approximately 45%. The effect of MEM was reproduced with MEM essential, but not nonessential, amino acids. Incubation of muscles with MEM plus 100 microU/ml of insulin for the first 8 h prevented the increases in 3-MG transport activity measured after a 9-h incubation period. Muscles incubated for 9 h maintained ATP and phosphocreatine concentrations, and changes in glycogen concentrations were small. Thus we have defined conditions for long-term incubation of skeletal muscle under which a progressive increase in glucose transport is prevented.

Exercise stimulates insulin-independent glucose transport in skeletal muscle and also increases the sensitivity of the glucose transport process in muscle to insulin. A previous study [D. A. Young, H. Wallberg-Henriksson, M. D. Sleeper, and J. O. Holloszy. Am. J. Physiol. 253 (Endocrinol. Metab. 16): E331–E335, 1987] showed that the exercise-induced increase in glucose transport activity disappears rapidly when rat epitrochlearis muscles are incubated for 3 h in vitro in the absence of insulin and that 7.5 microU/ml insulin in the incubation medium apparently slowed the loss of enhanced sugar transport. We examined whether addition of insulin several hours after exercise increases glucose transport to the same extent as continuous insulin exposure. Addition of 7.5 microU/ml insulin 2.5 h after exercise (when glucose transport has returned to basal levels) increased sugar transport to the same level as that which resulted from continuous insulin exposure. This finding provides evidence for an increase in insulin sensitivity rather than a slowing of reversal of the exercise-induced increase in insulin-independent glucose transport activity. Glucose transport was enhanced only at submaximal, not at maximal, insulin concentrations. Exposure to a high concentration of glucose and a low insulin concentration reduced the exercise-induced increase in insulin-sensitive glucose transport. Incubation with a high concentration of 2-deoxy-D-glucose (2-DG) did not alter the increase in insulin sensitivity, even though a large amount of 2-DG entered the muscle and was phosphorylated.(ABSTRACT TRUNCATED AT 250 WORDS)

The cause of reduced insulin-stimulated glucose transport in skeletal muscle of diabetic rats was investigated. Basal and insulin-stimulated glucose uptake into hindquarter muscles of 7-day diabetic rats were 70% and 50% lower, respectively, than in nondiabetic controls. Subcellular fractionation of hindquarter muscles yielded total crude membranes, plasma membranes and intracellular membranes. The number of GLUT-4 glucose transporters was lower in crude membranes, plasma membranes and intracellular membranes, relative to non-diabetic rat muscles. These results were paralleled by reductions in D-glucose-protectable binding of cytochalasin B. Insulin caused a redistribution of GLUT-4 transporters from intracellular membranes to plasma membranes, in both control and diabetic rat muscles. This redistribution was also recorded using binding of cytochalasin B. The insulin-dependent decrement in glucose transporters in intracellular membranes was similar for both animal groups, but the gain and final amount of transporters in the plasma membrane were 50% lower in the diabetic group. The results suggest that insulin signalling and recruitment of GLUT-4 glucose transporters occur in diabetic rat muscle, and that the diminished insulin response may be due to fewer glucose transporters operating in the muscle plasma membrane.

Adiponectin plays important roles in regulating insulin sensitivity and atherogenesis. Adiponectin has been shown to suppress hepatic glucose production in rodents. It has not been reported whether ectopically expressed adiponectin could regulate glucose metabolism in cultured hepatocyte-like cells. In the current study, the effect of adiponectin on glucose production was analyzed by ectopically expressing the protein in hepatoma H4IIE cells using an adenovirus delivery system to generate both human full-length and the globular domain of the protein. Expression of adiponectin in hepatoma H4IIE cells, in the absence of insulin, suppressed expression of the genes encoding glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, rate-limiting enzymes in the gluconeogenic pathway. Furthermore, expression of adiponectin in H4IIE cells suppressed glucose production from lactate and pyruvate. Purified recombinant human adiponectin also reduced glucose production in H4IIE cells and in rat primary hepatocytes in the absence of insulin. These data suggest that adiponectin protein could exert its function independent of the presence of insulin in these culture systems.

Non-receptor proline-rich tyrosine kinase-2 (PYK2), which is activated by phosphorylation of one or more of its tyrosine residues, has been implicated in the regulation of GLUT4 glucose transporter translocation and glucose transport. Some data favor a positive role of PYK2 in stimulating glucose transport, whereas other studies suggest that PYK2 may participate in the induction of insulin resistance. To ascertain the importance of PYK2 in the setting of obesity and insulin resistance, we (1) evaluated the regulation of PYK2 in mice fed a high-fat diet and (2) characterized body and glucose homeostasis in wild type (WT) and PYK2(−/−) mice on different diets. We found that both PYK2 expression and phosphorylation were significantly increased in liver and adipose tissues harvested from high-fat diet fed mice. Wild type and PYK2(−/−) mice were fed a high-fat diet for 8 weeks to induce insulin resistance/obesity. Surprisingly, in response to this diet PYK2(−/−) mice gained significantly more weight than WT mice (18.7 ± 1.2 g vs. 9.5 ± 0.6 g). Fasting serum leptin and insulin and blood glucose levels were significantly increased in high-fat diet fed mice irrespective of the presence of PYK2 protein. There was a close correlation between serum leptin and body weight. Intraperitoneal glucose tolerance tests revealed that as expected, the high-fat diet resulted in increased blood glucose levels following glucose administration in wild type mice compared to those fed normal chow. An even greater increase in blood glucose levels was observed in PYK2(−/−) mice compared to wild type mice. These results demonstrate that a lack of PYK2 exacerbates weight gain and development of glucose intolerance/insulin resistance induced by a high-fat diet, suggesting that PYK2 may play a role in slowing the development of obesity, insulin resistance, and/or frank diabetes.

The enzyme 5'AMP-activated protein kinase (AMPK) is activated by increases in intracellular AMP concentration through a complex interaction of phosphorylation and allosteric regulation. Actions of AMPK elucidated thus far suggest that AMPK may be a viable target for pharmacologic intervention in type II diabetes. Activation of AMPK is believed to mediate both the acute increase in skeletal muscle glucose uptake during exercise, as well as the adaptive responses to chronic exercise such as regulation of expression of components of the muscle glucose uptake system. In addition, AMPK is known to inhibit key enzymes involved in lipid and cholesterol synthesis, suggesting that activation of this kinase may also ameliorate dyslipidemia. To investigate the effects of AMPK activation in animal models of type II diabetes, db/db and ob/ob mice were administered 5-aminoimidazole-4-carboxamide 1-beta-ribofuranoside (AICAR) subcutaneously either acutely (single injection) or twice per day for 8 days (chronic treatment). Blood glucose was lowered transiently in both db/db and ob/ob mice by acute AICAR treatment, returning to basal levels approximately 3 h after AICAR administration. In response to chronic treatment, blood glucose (measured 18 h post-AICAR administration) was significantly decreased in both mouse models when compared to vehicle control groups, with morning blood glucose values on Day 8 being decreased approximately 30-35% in both mouse models. Chronic AICAR administration also resulted in an elevation of total Glut4 concentration in skeletal muscle from ob/ob mice, but not db/db mice. In contrast to the beneficial effects on glucose metabolism, AICAR treatment of db/db and ob/ob mice led to approximately a 2.5-3-fold increase in serum triglyceride levels compared to vehicle-treated controls. These data suggest that pharmacological activation of AMPK may enhance glucose uptake in individuals with type II diabetes, however, this benefit may be offset by the concomitant elevation in triglycerides.

A line of transgenic mice was constructed in which the human Glut1 glucose transporter is overexpressed in skeletal muscle. Overexpression of Glut1 protein was evident in epitrochlearis, extensor digitorum longus (EDL), and quadriceps muscles, and resulted in 6.6-7.4-fold elevations in basal glucose transport activity as measured in isolated muscles in vitro. The elevated glucose transporter activity in the skeletal muscles of transgenic mice was associated with a 10-fold increase in glycogen concentration in EDL and quadriceps muscles that was not due to an increase in muscle glycogen synthase activity or a decrease in glycogen phosphorylase activity. The increased glucose transport activity also resulted in a 2-fold increase in muscle lactate concentration, with no increase in muscle glucose 6-phosphate. Despite a slight (10%) increase in muscle hexokinase activity, there was a 4-fold increase in total muscle free glucose in transgenic mice, indicating that hexokinase becomes rate-limiting for glucose uptake when the rate of glucose transport is very high. These results demonstrate that the muscle glycogen content can be dramatically elevated by increasing the muscle Glut1 protein level and that glucose transport is a rate-limiting step for muscle glucose disposal in normal, resting mice.

The enzyme glutamine:fructose 6-phosphate amidotransferase ( l -glutamine: d -fructose-6-phosphate amidotransferase; EC 2.6.1.16, GFAT) catalyzes the formation of glucosamine 6-phosphate from fructose 6-phosphate and glutamine. In view of the important role of GFAT in the hexosamine biosynthetic pathway, we have purified the enzyme from rat liver and characterized its physicochemical properties in comparison to those from the published microbial enzymes. The purified enzyme has a molecular mass of about 75 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. On a Sephacryl S-200 gel filtration column, the purified enzyme eluted in a single peak corresponding to a molecular mass of about 280 kDa, indicating that the active enzyme may be composed of four subunits. The N-terminal amino acid sequence of the purified enzyme was determined as X-G-I-F-A-Y-L-N-Y-H-X-P-R, where X indicates an unidentified residue. The KM values of the purified enzyme for fructose 6-phosphate and glutamine were 0.4 and 0.8 mM, respectively. The purified enzyme was inactivated by 4,4′-dithiodipyridine, and the activity of the inactivated enzyme was restored by dithiothreitol. The inactivation followed pseudo first-order and saturation kinetics with the Kinact of 5.0 μM. Kinetic studies also indicated that 4,4′-dithiodipyridine is a competitive inhibitor of the enzyme with respect to glutamine. Isolation and analysis of the cysteine-modified peptide indicated that Cys-1 was the modified site. Cys-1 has been suggested to play an important role in enzymatic activity of the Escherichia coli enzyme (M. N. Isupov, G. Obmolova, S. Butterworth, M. Badet-Denisot, B. Badet, I. Polikarpov, J. A. Littlechild, and A. Teplyakov, 1996, Structure 4, 801–810).

Glutamine:fructose-6-phosphate amidotransferase (GFA) is the rate-limiting enzyme in hexosamine biosynthesis, an important pathway for cellular glucose sensing. Human GFA has two potential sites for phosphorylation by cAMP-dependent protein kinase A (PKA). To test whether GFA activity is regulated by cAMP-dependent phosphorylation, rat aortic smooth muscle cells were treated in vivo with cAMP-elevating agents, 10 micromol/l forskolin, 1 mmol/l 8-Br-cAMP, or 3-isobutyl-1-methylxanthine. All treatments resulted in rapid and significant increases (2- to 2.4-fold) in GFA activity assayed in cytosolic extracts. Maximal effects of forskolin were observed at 10 micromol/l and 60 min. Preincubation of cells with cycloheximide did not abolish the effect of forskolin. Incubation of cytosolic extracts at 37 degrees C for 10 min in a buffer without phosphatase inhibitors led to a 79% decrease of GFA activity. This loss of activity was inhibited by the addition of phosphatase inhibitors (5 mmol/l sodium orthovanadate, 50 mmol/l sodium fluoride, or 5 mmol/l EDTA, but not 100 nmol/l okadaic acid), suggesting that GFA undergoes rapid dephosphorylation by endogenous phosphatases. Purified GFA is phosphorylated in vitro by purified PKA, resulting in a 1.7-fold increase in GFA activity. Treatment of GFA with purified protein kinase C had no effect. We conclude that GFA activity may be modulated by cAMP-dependent phosphorylation.

The present study examined the effects of 6 wk of ovarian endocrine deficiency on skeletal muscle GLUT-4 glucose transporter protein and glucose transport activity in sedentary and endurance-trained rats. Female Wistar rats (10 wk old) underwent bilateral ovariectomy (OVX) or sham surgery followed by a 5-wk swim-training protocol. OVX resulted in no significant changes in glycogen or GLUT-4 glucose transporter concentration in the soleus, epitrochlearis, or flexor digitorum brevis (FDB) muscles or in basal and maximally insulin-stimulated 2-deoxy-D-[1,2-3H]glucose (2-[3H]DG) transport in the soleus or epitrochlearis, suggesting that moderate-duration ovarian hormone deficiency does not significantly impair insulin action in skeletal muscle. In contrast, OVX decreased the maximal activation of 2-[3H]DG transport in the FDB by in vitro electrical stimulation. OVX had no significant effect on the training-induced changes in oxidative enzyme activities, GLUT-4 protein expression, glycogen content, or insulin-stimulated 2-[3H]DG transport in the soleus or epitrochlearis. These findings provide the first evidence that ovarian hormone deficiency decreases contraction-stimulated glucose transport in skeletal muscle.

There is some controversy regarding whether insulin or contractile activity alters the affinity of skeletal muscle glucose transporters for glucose and its analogues. The effects of insulin and contractions on the kinetics of glucose transport were therefore reexamined in isolated rat skeletal muscles. Concentration-dependent rates of 2-deoxyglucose (2-DG) transport were measured in the absence or presence of insulin (2 mU/ml) in the epitrochlearis and split soleus muscles. The apparent half-maximal saturating substrate concentration (Km) for basal 2-DG transport (approximately 12 mM) was similar for the split soleus and epitrochlearis, and the apparent Km was not changed by insulin in either muscle type. The presence of 2 mU/ml insulin increased the maximal transport velocity (Vmax) approximately fourfold in the epitrochlearis and approximately eightfold in the split soleus. In the epitrochlearis, in vitro muscle contractions also resulted in an approximately fourfold increases in Vmax with no change in apparent Km. The combined effects of insulin and contractions on Vmax were completely additive, but the apparent Km was not different from insulin alone. The apparent Km values for basal and insulin-stimulated glucose transport were further characterized in the epitrochlearis isolated from transgenic mice overexpressing the GLUT-1 isoform in the sarcolemma and their nontransgenic littermates. The apparent Km for basal 2-DG transport in the transgenic muscle (9 mM) was not significantly different from the apparent Km for insulin-stimulated transport in the control muscle (10 mM). The present study provides evidence that insulin and contractions, either alone or in combination, increase glucose transport activity in skeletal muscle by increasing Vmax, with no significant change in Km. Our results also suggest that, in intact skeletal muscle, the Km for basal glucose transport (a process mediated primarily by GLUT-1) is similar to the Km values for stimulated transport, mediated predominantly by GLUT-4.

The maximal effects of insulin and muscle contractions on glucose transport are additive. GLUT-4 is the major glucose transporter isoform expressed in skeletal muscle. Muscle contraction and insulin each induce translocation of GLUT-4 from intracellular sites into the plasma membrane. The purpose of this study was to test the hypothesis that the incremental effect of contractions and insulin on glucose transport is mediated by additivity of the maximal effects of these stimuli on GLUT-4 translocation into the sarcolemma. Anesthetized rats were given insulin by intravenous infusion to raise plasma insulin to 2,635 +/- 638 microU/ml. The gastrocnemius-plantaris-soleus group was stimulated to contract via the sciatic nerve by using a protocol that maximally activates glucose transport. After treatment with insulin, contractions, or insulin plus contractions or no treatment, the gastrocnemius-plantaris-soleus muscle group was dissected out and was subjected to subcellular fractionation to separate the plasma membrane and intracellular membrane fractions. Insulin induced a 70% increase and contractions induced a 113% increase in the GLUT-4 content of the plasma membrane fraction. The effects of insulin and contractions were additive, as evidenced by a 185% increase in the GLUT-4 content of the sarcolemmal fraction. This finding provides evidence that the incremental effect of maximally effective insulin and contractile stimuli on glucose transport is mediated by additivity of their effects on GLUT-4 translocation into the sarcolemma.

The insulin sensitivity of glucose transport is enhanced in skeletal muscle after a bout of exercise. In a previous study, stimulation of washed muscles to contract in vitro, in contrast to exercise, did not result in an increase in insulin sensitivity. The purpose of the present study was to explain this apparent discrepancy. We found that, although rat epitrochlearis muscles stimulated to contract in vitro after 15 min of incubation in Krebs-Henseleit buffer did not develop increased insulin sensitivity, muscles stimulated to contract immediately after being dissected showed a small but significant enhancement of the stimulation of 3-O-methyl-D-glucose transport by 30 microU/ml insulin. Furthermore, muscles stimulated to contract in situ and then allowed to recover in vitro showed as large an increase in insulin sensitivity as that which occurs after a bout of swimming. To follow up these findings suggesting involvement of a humoral factor, we incubated epitrochlearis muscles in serum before and during contractile activity in vitro. Epitrochlearis muscle insulin sensitivity was enhanced to as great an extent after in vitro contractile activity in serum as after swimming. Experiments involving charcoal treatment, ultrafiltration, or trypsin digestion provided evidence that the serum factor that interacts with contractions to enhance insulin sensitivity is a protein.

We examined the effects of voluntary exercise on glucose transporter concentration in skeletal muscle from young adult and old female Long-Evans rats. Rats had free access to voluntary running wheels beginning at 4 months of age or remained sedentary. Exercising rats ran approximately 7.5, 6.2, 5.6 and 5.3 km/day during their 6th, 8th, 9th and 10th month of age, respectively. During the 23rd, 24th and 25th month of age running distance averaged 3.0, 2.8 and 2.4 km/day, respectively. At 10 and 25 months of age, glucose transporter protein concentration was assessed in epitrochlearis and flexor digitorum brevis muscles with a polyclonal antibody directed against the GLUT4 transporter isoform. GLUT4 protein concentration was not altered by the aging process (i.e., comparing 10- and 25-month-old rats) in either muscle type. Wheel running increased GLUT4 protein concentration by 45% in epitrochlearis muscles of 10-month-old rats relative to age-matched sedentary controls. The training-induced adaptation in GLUT4 protein was no longer present at age 25 months, probably because the running distance had declined by 50%. In the flexor digitorum brevis, exercise did not alter GLUT4 concentration at either 10 or 25 months, presumably due to insufficient recruitment of this muscle during wheel running as assessed by measurement of citrate synthase and hexokinase enzyme activities. Wheel running induced cardiac and soleus muscle hypertrophy in 10- and 25-month-old rats. In summary, voluntary wheel running can induce an increase in skeletal muscle GLUT4 protein concentration in adult rats. Older rats that run less exhibit cardiac and soleus muscle hypertrophy, but do not maintain an elevated GLUT4 protein concentration in the epitrochlearis muscle. Aging does not alter GLUT4 protein concentration in the epitrochlearis or FDB muscles.

Voluntary wheel running induces an increase in the concentration of the regulatable glucose transporter (GLUT4) in rat plantaris muscle but not in soleus muscle (K. J. Rodnick, J. O. Holloszy, C. E. Mondon, and D. E. James. Diabetes 39: 1425–1429, 1990). Wheel running also causes hypertrophy of the soleus in rats. This study was undertaken to ascertain whether endurance training that induces enzymatic adaptations but no hypertrophy results in an increase in the concentration of GLUT4 protein in rat soleus (slow-twitch red) muscle and, if it does, to determine whether there is a concomitant increase in maximal glucose transport activity. Female rats were trained by treadmill running at 25 m/min up a 15% grade, 90 min/day, 6 days/wk for 3 wk. This training program induced increases of 52% in citrate synthase activity, 66% in hexokinase activity, and 47% in immunoreactive GLUT4 protein concentration in soleus muscles without causing hypertrophy. Glucose transport activity stimulated maximally with insulin plus contractile activity was increased to roughly the same extent (44%) as GLUT4 protein content in soleus muscle by the treadmill exercise training. In a second set of experiments, we examined whether a swim-training program increases glucose transport activity in the soleus in the presence of a maximally effective concentration of insulin. The swimming program induced a 44% increase in immunoreactive GLUT4 protein concentration. Glucose transport activity maximally stimulated with insulin was 62% greater in soleus muscle of the swimmers than in untrained controls. Training did not alter the basal rate of 2-deoxyglucose uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

We compared the breakdown of total cellular protein with that of the contractile protein, myosin, in cultured C2 mouse skeletal myotubes. The degradation of long-lived cellular proteins (which comprise the vast majority of myotube proteins) was inhibited by serum, insulin, and rat insulin-like growth factor-2. A physiological concentration of insulin was effective, but most of the effect of insulin occurred at concentrations well above the physiological range. IGF-2 inhibited protein breakdown at concentrations well within the range of total IGF-2 known to be present in the serum of fetal and neonatal rats. The breakdown of short-lived proteins was not altered by insulin or serum. We measured myosin degradation using a monoclonal antibody directed against myosin heavy chain. The half-life of myosin was 27 hours, and myosin breakdown was not altered by withdrawal of serum. Therefore, the enhanced protein degradation in response to serum withdrawal applies to certain proteins, but not to others.