Mitochondrial Utilization of Competing Fuels is Altered in Insulin Resistant Skeletal Muscle of Non-Obese Rats (Goto-Kakizaki)

Aim: Insulin-resistant skeletal muscle is characterized by metabolic inflexibility with associated alterations in substrate selection, mediated by peroxisome-proliferator activated receptor (PPAR). Although it is established that PPAR contributes to the alteration of energy metabolism, it is not clear whether it plays a role in mitochondrial fuel competition. While nutrient overload may impair metabolic flexibility by fuel congestion within mitochondria, in absence of obesity defects at a mitochondrial level have not yet been excluded. We sought to determine whether reduced PPAR content in insulin-resistant rat skeletal muscle of a non-obese rat model of T2DM (Goto-Kakizaki, GK) ameliorate the inhibitory effect of fatty acid (i.e., palmitoylcarnitine) on mitochondrial carbohydrate oxidization (i.e., pyruvate) in muscle fibers. Methods: Bioenergetic function was characterized in oxidative soleus (S) and glycolytic white gastrocnemius (WG) muscles with measurement of respiration rates in permeabilized fibers in the presence of complex I, II, IV, and fatty acid substrates. Mitochondrial content was measured by citrate synthase (CS) and succinate dehydrogenase activity (SDH). Western blot was used to determine protein expression of PPAR, PDK isoform 2 and 4. Results: CS and SDH activity, key markers of mitochondrial content, were reduced by similar to 10-30% in diabetic vs. control, and the effect was evident in both oxidative and glycolytic muscles. PPAR (p\u3c 0.01), PDK2 (p\u3c 0.01), and PDK4 (p= 0.06) protein content was reduced in GK animals compared to Wistar rats (N= 6 per group). Ex vivorespiration rates in permeabilized muscle fibers determined in the presence of complex I, II, IV, and fatty acid substrates, suggested unaltered mitochondrial bioenergetic function in T2DM muscle. Respiration in the presence of pyruvate was higher compared to palmitoylcarnitine in both animal groups and fiber types. Moreover, respiration rates in the presence of both palmitoylcarnitine and pyruvate were reduced by 25 ± 6% (S), 37 ± 6% (WG) and 63 ± 6% (S), 57 ± 8% (WG) compared to pyruvate for both controls and GK, respectively. The inhibitory effect of palmitoylcarnitine on respiration was significantly greater in GK than controls (p \u3c 10-3). Conclusion: With competing fuels, the presence of fatty acids diminishes mitochondria ability to utilize carbohydrate derived substrates in insulin-resistant muscle despite reduced PPAR delta content

Mitochondrial Utilization of Competing Fuels Is Altered in Insulin Resistant Skeletal Muscle of Non-obese Rats (Goto-Kakizaki)

Aim: Insulin-resistant skeletal muscle is characterized by metabolic inflexibility with associated alterations in substrate selection, mediated by peroxisome-proliferator activated receptor δ (PPARδ). Although it is established that PPARδ contributes to the alteration of energy metabolism, it is not clear whether it plays a role in mitochondrial fuel competition. While nutrient overload may impair metabolic flexibility by fuel congestion within mitochondria, in absence of obesity defects at a mitochondrial level have not yet been excluded. We sought to determine whether reduced PPARδ content in insulin-resistant rat skeletal muscle of a non-obese rat model of T2DM (Goto-Kakizaki, GK) ameliorate the inhibitory effect of fatty acid (i.e., palmitoylcarnitine) on mitochondrial carbohydrate oxidization (i.e., pyruvate) in muscle fibers. Methods: Bioenergetic function was characterized in oxidative soleus (S) and glycolytic white gastrocnemius (WG) muscles with measurement of respiration rates in permeabilized fibers in the presence of complex I, II, IV, and fatty acid substrates. Mitochondrial content was measured by citrate synthase (CS) and succinate dehydrogenase activity (SDH). Western blot was used to determine protein expression of PPARδ, PDK isoform 2 and 4. Results: CS and SDH activity, key markers of mitochondrial content, were reduced by ∼10–30% in diabetic vs. control, and the effect was evident in both oxidative and glycolytic muscles. PPARδ (p < 0.01), PDK2 (p < 0.01), and PDK4 (p = 0.06) protein content was reduced in GK animals compared to Wistar rats (N = 6 per group). Ex vivo respiration rates in permeabilized muscle fibers determined in the presence of complex I, II, IV, and fatty acid substrates, suggested unaltered mitochondrial bioenergetic function in T2DM muscle. Respiration in the presence of pyruvate was higher compared to palmitoylcarnitine in both animal groups and fiber types. Moreover, respiration rates in the presence of both palmitoylcarnitine and pyruvate were reduced by 25 ± 6% (S), 37 ± 6% (WG) and 63 ± 6% (S), 57 ± 8% (WG) compared to pyruvate for both controls and GK, respectively. The inhibitory effect of palmitoylcarnitine on respiration was significantly greater in GK than controls (p < 10–3). Conclusion: With competing fuels, the presence of fatty acids diminishes mitochondria ability to utilize carbohydrate derived substrates in insulin-resistant muscle despite reduced PPARδ content

Multi-Scale Peripheral Vasculopathy with Metabolic Syndrome

The combination of cardiovascular and metabolic risk factors including obesity, dyslipidemia, hypertension, and insulin resistance, in combination with a prothrombotic and proinflammatory state, is a condition termed Metabolic Syndrome (METS). Twenty percent of the adult population is afflicted with METS which increases the risk of type-2 diabetes mellitus and cardiovascular disease. Further, the presence of peripheral vascular disease (PVD) is tightly coupled with METS which is a perfusion-demand mismatch of blood supply to active skeletal muscle resulting in painful claudication and a late-stage potential for amputation. The underlying contributors of METS associated micro-vasculopathies in the skeletal muscle, their impact on impaired perfusion, and the potential for reversibility remain unclear. Owing its hyperphagia to leptin signaling resistance, the obese Zucker rat (OZR) is a translationally relevant model for human METS and the associated micro-vasculopathies. The overall purpose of this thesis is to utilize a multi-scale approach, particularly intravital microscopy and isolate vessels, to garner a greater understanding of the observed OZR vasculopathies and to investigate the potential of therapeutic interventions for their reversibility. Project 1: The purpose was to identify any alterations in postcapillary and collecting venule function in the OZR compared to healthy controls. The OZR presented with impaired dilator reactivity and elevation in thromboxane A2 constrictor responses for both postcapillary and collecting venules. Project 2: The purpose was to identify the possible contributors of a disconnect for in-situ and ex-vivo vascular studies utilizing the OZR model. Using a multi-scale approach, Project 2 provides insight to this disconnect and reveals a heterogenous adrenergic response in the OZR, giving rise to new potential avenues of study. Project 3: The purpose was to determine the potential for reversibility or restoration of established PVD using the chronic ingestion of an HMG-CoA inhibitor, atorvastatin, and/or the implementation of regular exercise. Following a seven-week intervention, the intervention groups revealed vascular improvements with the combination group having the greatest capacity for reversibility (in specific indices). Significance: Therefore, this thesis further advances the understanding of METS associated PVD as well as potential modes for improvement following its establishment

Impaired overload-induced hypertrophy in Obese Zucker rat slow-twitch skeletal muscle

How insulin resistant muscle responds to mechanical overload (MOV) is not well understood. Using immunohistochemical analysis and immunoblotting, we investigated whether Lean Zucker (LZ) and insulin resistant Obese Zucker rats (OZ) respond in a similar fashion to MOV. Five young adult (2 months old) male LZ and OZ rats were subjected to a surgical ablation of the gastrocnemius muscle and overloaded for 8 weeks. MOV-induced increases in soleus muscle mass and average fiber cross-sectional area were attenuated in OZ compared to LZ animals. This reduction in OZ muscle adaptation was associated with decreased activity / phosphorylation levels of STAT3, β-catenin and calcineurin. These data suggest that insulin resistance may decrease the ability of skeletal muscle to hypertrophy and that this impairment may be due to alterations in the ability of insulin-resistant muscle to activate STAT3, β-catenin and calcineurin signaling

Treadmill training effect on the myokines content in skeletal muscles of mice with a metabolic disorder model

The effect of treadmill training loads on the content of cytokines in mice skeletal muscles with metabolic disorders induced by a 16 week high fat diet (HFD) was studied. The study included accounting the age and biorhythmological aspects. In the experiment, mice were used at the age of 4 and 32 weeks, by the end of the experiment—respectively 20 and 48 weeks. HFD feeding lasted 16 weeks. Treadmill training were carried out for last 4 weeks six times a week, the duration 60 min and the speed from 15 to 18 m/min. Three modes of loading were applied. The first subgroup was subjected to stress in the morning hours (light phase); the second subgroup was subjected to stress in the evening hours (dark phase); the third subgroup was subjected to loads in the shift mode (the first- and third-weeks treadmill training was used in the morning hours, the second and fourth treadmill training was used in the evening hours). In 20-weekold animals, the exercise effect does not depend on the training regime, however, in 48-week-old animals, the decrease in body weight in mice with the shift training regime was more profound. HFD affected muscle myokine levels. The content of all myokines, except for LIF, decreased, while the concentration of CLCX1 decreased only in young animals in response to HFD. The treadmill training caused multidirectional changes in the concentration of myokines in muscle tissue. The IL-6 content changed most profoundly. These changes were observed in all groups of animals. The changes depended to the greatest extent on the training time scheme. The effect of physical activity on the content of IL-15 in the skeletal muscle tissue was observed mostly in 48-week-old mice. In 20-week-old animals, physical activity led to an increase in the concentration of LIF in muscle tissue when applied under the training during the dark phase or shift training scheme. In the HFD group, this effect was significantly more pronounced. The content of CXCL1 did not change with the use of treadmill training in almost all groups of animals. Physical activity, introduced considering circadian rhythms, is a promising way of influencing metabolic processes both at the cellular and systemic levels, which is important for the search for new ways of correcting metabolic disorders

Impact of estradiol, estrogen receptor subtype-selective agonists and genistein on energy homeostasis: Impact of estradiol, estrogen receptor subtype-selective agonists and genistein on energy homeostasis

The prevalence of obesity is dramatically increasing and thus constitutes a major risk factor for developing chronic diseases such as type 2 diabetes, dyslipidemia, cardiovascular diseases, and certain forms of cancer. High-caloric nutrition and a lack of physical activity are the main contributing factors for this global epidemic. Estrogen receptors (ERs) are recognized to be involved in many processes related to the control of energy homeostasis. In my studies, I investigated the impact of estrogens (17beta-estradiol (E2)) on energy homeostasis. Special emphasis was given to the effects of two synthetic ER subtype-selective agonists, 16alpha-LE2 (Alpha) and 8beta-VE2 (Beta), to determine to what extend the two distinct ER subtypes are involved in the underlying molecular mechanisms. Because of its estrogenic activity and also its widespread use as a nutritional supplement the influence of the isoflavone genistein (Gen) was examined. For this purpose two different female rat models were used: Wistar rats with nutrition-induced obesity and leptin resistant Zucker diabetic fatty (ZDF) rats. In both experiments, the animals were ovariectomized (OVX) and treated with vehicle (untreated controls) or the estrogenic compounds. The most important finding was that treatment of OVX animals with Beta enlarges soleus muscle fiber sizes in both animal models compared to untreated OVX animals. This anabolic effect may in turn improve the muscle/fat ratio of the body that enhances muscular uptake and utilization of fuels. By contrast, in the gastrocnemius muscle of OVX ZDF rats substitution with Alpha increased expression and distribution of the insulin-dependent glucose transporter 4 (GLUT4). Consequently, systemic insulin sensitivity in both animal models was improved by treatment with estrogenic compounds compared to untreated OVX animals. The strongest effect was observed in E2-treated rats that indicate an additive effect through activation of both pathways. In all OVX rats, treatment with either ER subtype-selective agonist showed an anti-lipogenic effect in adipose tissue, liver, and skeletal muscle of nutrition-induced obese Wistar rats in comparison to OVX animals without treatment. Decreased visceral fat mass, adipocyte sizes, serum leptin levels, triglyceride accumulation in liver and muscle as well as mRNA expression of genes that are involved in lipo-/adipogenesis reflected this. Therefore, the lower visceral fat mass as well as decreased accumulation of triglycerides in non-adipose tissues such as liver and skeletal muscle most likely contributes to the improved insulin sensitivity in such treated animals. Gen exerted effects similar to those of the ER beta-selective agonist (except on adipose tissue in Wistar rats). Especially, the similar ability to induce anabolic activity in the soleus muscle might be highly relevant. Gen-treated animals might have a more effective utilization of fuels compared to untreated OVX animals because they showed a lower TG content in muscle and liver as well as improved glucose metabolism. In conclusion, because of my studies and the fact that ER beta signaling is not involved in proliferation of uterus and mammary gland, an effective way to treat obesity and co-morbidities in postmenopausal women might be substances that only activate ER beta. A combination with physical activity may support the therapy of obesity and co-morbidities. The isoflavone Gen is able to activate both ER-subtypes. This compound is already placed on the market for treatment of postmenopausal complaints, although adverse effects of Gen cannot be excluded so far (e.g., increased risk of breast cancer). However, Gen might be a natural alternative – not only to the conventional hormone replacement therapy, but also as a strategy for treatment of obesity and co-morbidities – that deserves further research with respect to these new data.Die dramatisch zunehmende Prävalenz der Adipositas und das damit verbundene Risiko für Folgeerkrankungen wie Diabetes mellitus, Hypertonie, Dyslipidämie und koronare Herzkrankheiten stellt eine große Herausforderung für das Gesundheitswesen dar. Als Hauptursache wird ein chronisches Missverhältnis der Energiehomöostase aufgrund permanenter Überernährung und Bewegungsmangel postuliert. Estrogene beeinflussen den Glukose- und Lipidstoffwechsel und sind somit in die Regulation des Energiehaushaltes involviert. Estrogene vermitteln ihre Effekte über zwei Estrogenrezeptor (ER)-Subtypen, den ER alpha und den ER beta. Ziel der vorliegenden Arbeit war es mittels tierexperimentellen Studien den Einfluss von Estrogenen, speziell 17beta-Estradiol, auf den Energiehaushalt zu untersuchen. Um einen tieferen Einblick in die zugrundeliegenden molekularen Mechanismen zu erhalten, wurden zwei Subtyp-selektive ER-Agonisten, 16alpha-LE2 (Alpha) and 8beta-VE2 (Beta), synthetischer Herkunft eingesetzt. Aufgrund der estrogenen Aktivität und der Verfügbarkeit als Nahrungsergänzungsmittel wurde des Weiteren der Einfluss des Isoflavons Genistein untersucht. Für die Studien wurden zwei Tiermodelle genutzt: zum einen weibliche Wistar-Ratten mit ernährungsinduzierter Adipositas und zum anderen weibliche leptinresistente „Zucker diabetic fatty“ (ZDF)-Ratten. Die Tiere wurden ovarektomiert (OVX) und entweder mit einem Vehikel (unbehandelte Kontrolltiere) oder mit der entsprechenden estrogenen Substanz behandelt. Die interessanteste Erkenntnis war, dass im Vergleich zu unbehandelten OVX-Tieren beider Tiermodelle die Behandlung mit Beta zur Vergrößerung der Faserquerschnitte im Soleusmuskel führte. Dieser anabole Effekt könnte die muskuläre Aufnahme und Verwertung von Brennstoffmolekülen verbessern und sich insgesamt positiv auf die Körperzusammensetzung auswirken. Den stärksten Effekt hinsichtlich einer erhöhten Expression und Translokation des insulinabhängigen Glukosetransporters 4 (GLUT4) in die Zellmembran des Gastrocnemiusmuskels zeigte sich dagegen durch die Behandlung von OVX ZDF-Ratten mit Alpha. Im Endergebnis zeigten die Tiere beider Modelle durch die Behandlung mit estrogenen Substanzen eine verbesserte systemische Insulinsensitivität im Vergleich zu unbehandelten Kontrolltieren. E2-behandelte Tiere tolerierten die Glukose am besten und lassen einen additiven Effekt aufgrund der Aktivierung beider Signalwege vermuten. Im Vergleich zu unbehandelten OVX Wistar-Ratten führte die Behandlung mit E2 oder mit jeweils einem der beiden ER-Subtyp-selektiven Agonisten zu einer geringeren viszeralen Fettmasse, kleineren Fettzellen, niedrigeren Leptinspiegeln im Serum und geringeren Triglyzeridwerten in Leber und Muskel. Auf der Ebene der Genexpression waren zudem geringere mRNA-Spiegel von lipo- und adipogenen Genen messbar. Somit scheinen beide ER-Subtypen in die antilipogene Wirkung von E2 involviert zu sein. Sowohl die reduzierte viszerale Fettmasse als auch die geringere Anreicherung von Triglyzeriden in Leber und Muskel tragen sehr wahrscheinlich ebenfalls zur verbesserten Insulinsensitivität bei. Die Behandlung von OVX Tieren mit Gen führte zu ähnlichen Ergebnissen wie die Behandlung mit Beta. Eine alleinige Ausnahme stellte das Fettgewebe dar, da hier eine Gen-Behandlung keine antilipogenen/-adipogenen Effekte zeigte. Speziell die Fähigkeit von Gen ebenfalls anabol zu wirken, könnte die molekulare Grundlage sein, weshalb Gen-behandelte Tiere im Vergleich zu unbehandelten Tiere eine verbesserte Toleranz gegenüber Glukose und eine geringere Anreicherung von Triglyzeriden in Muskel und Leber zeigten. Der ER beta ist nicht in die estrogenvermittelte Proliferation von Uterus und Brustdrüse involviert. Vor diesem Hintergrund lassen meine Ergebnisse vermuten, dass eine Behandlung mit ER beta-selektiven Substanzen eine effektive Möglichkeit darstellt, um Adipositas und deren Folgeerkrankungen in postmenopausalen Frauen zu behandeln, ohne deren Risiko für estrogenabhängige Krebsformen zu erhöhen. Eine Kombination mit regelmäßiger körperlicher Aktivität könnte die Erfolge bei der Behandlung von Adipositas und deren Folgeerkrankungen noch maximieren bzw. eine geringere Dosierung der verwendeten Substanz bei gleichbleibendem Behandlungserfolg ermöglichen. Das Isoflavon Gen mit seiner Fähigkeit beide ERs zu aktivieren ist eine bereits auf dem Markt befindliche Substanz und wird zur Behandlung von postmenopausalen Beschwerden eingesetzt, obwohl mögliche negative Effekte (z.B. ein erhöhtes Brustkrebsrisiko) noch nicht abschließend geklärt sind. Falls diese Risiken von Gen ausgeräumt werden können, könnte diese Substanz eventuell eine kostengünstige Alternative darstellen, um sowohl postmenopausale Beschwerden als auch Adipositas und deren Folgekrankheiten zu behandeln

Sprint interval training: the influence of exercise modality

2013 Fall.Includes bibliographical references.Sprint interval training (SIT), whether performed on a cycle ergometer or non-motorized treadmill, enhances exercise capacity and evokes favorable metabolic and cardiopulmonary adaptations. However, despite known differences between cycling and running, the influence of exercise modality on the adaptive response to SIT has not been directly addressed. Additionally, the effect of SIT on the angiogenic factors, pigment epithelial-derived factor (PEDF) and vascular endothelial growth factor (VEGF), has not been well characterized. PURPOSE: To examine the influence of exercise modality on the adaptive response to SIT, we compared the effects of SIT performed on one of three different exercise machines: non-motorized treadmill, cycle ergometer, or plyometrics platform. Additionally, we sought to characterize the changes in circulating and skeletal muscle PEDF and VEGF following three weeks of SIT. METHODS: Twenty-seven healthy, sedentary or recreationally active adults (age: 23 ± 5 years; body mass index: 25.7 ± 4.7 kg m-2; VO2peak: 36.7 ± 6.1 ml kg-1 min-1 (mean ± SE)) completed nine sessions of repeated (four to eight) 30-s bouts of maximal exercise on a non-motorized treadmill (RUN), cycle ergometer (CYC), or plyometrics platform (JMP) over 21 days. Prior to and following completion of SIT, peak oxygen uptake (VO2peak) and time to exhaustion at 80% VO2peak were measured. Additionally, blood and skeletal muscle was sampled prior to and following completion of SIT to measure PEDF and VEGF. RESULTS: Three weeks of SIT increased time to exhaustion (40.0 min ± 3.2 vs.51.3 ± 5.5 min, P = 0.006). The interaction with exercise modality did not achieve statistical significance (P = 0.08), however, it appears that time to exhaustion increased in the RUN (43.2 ± 5.2 vs. 57.4 ± 9.2 min) and CYC (41.7 ± 6.1 vs. 62.3 ± 11.6 min) groups, but not the JMP group (35.5 ± 5.6 vs. 35.0 ± 4.9 min). Circulating and skeletal muscle VEGF and PEDF were not altered by three weeks of SIT (P > 0.05). DISCUSSION: Independent of exercise modality, three weeks of SIT improves endurance exercise capacity and does not alter circulating or skeletal muscle PEDF or VEGF

THE EFFECT OF AEROBIC AND RESISTANCE EXERCISE ON INSULIN SENSITIVITY AND GLYCEMIC CONTROL IN TYPE 1 DIABETIC RATS

Type 1 Diabetic Mellitus (T1DM) patients can develop insulin insensitivity partially due to the daily requirement of exogenous insulin. Regular exercise has been shown to improve insulin sensitivity (IS) partially though the increased expression of skeletal muscle GLUT4 and insulin receptor (IR) content. Although exercise is beneficial in increasing IS, exercise presents a problem in the T1DM population, as it leads to uncontrolled fluctuations in blood glucose (BG) concentrations. This study examined the effect of aerobic and resistance training exercises on IS and fluctuations in BG concentrations in T1DM rats. Fifty animals were randomly divided into 5 groups; control (C), control diabetic (CD), diabetic resistance exercised (DR), diabetic high intensity aerobic exercised (DH) and diabetic low intensity aerobic exercised (DL). CD, DR, DH and DL were injected with streptozotocin (STZ) and insulin pellets were subcutaneously implanted to maintain blood glucose concentrations between 9 and 15 mmol/L. DR climbed a ladder with weights secured to the tail; they performed 6-10 climbs per exercise bout and weights were increased every 3 days to ensure that rats were lifting maximal weight. They performed exercise bouts 5 days/week for 6 weeks. DH ran on a treadmill at 27 m/min and 6% grade, 1 hour/day, 5 days/week for 6 weeks. DL ran on a treadmill at 15 m/min and 6% grade, 1hour/day, 5 days/week for 6 weeks. At 3 and 6 weeks, BG clearance rates in all groups were lower than C (p\u3c0.05). At 6 weeks, DR, DH and DL also had greater BG clearance rates than CD (p\u3c0.05). IR protein content in the white gastrocnemius muscle (WG) was elevated in DH, compared to C and CD (p\u3c0.05), and in DL, compared to C, CD and DR (p\u3c0.05). GLUT4 protein content in the white portion of the vastus lateralis muscle was increased in DH, compared to CD and DL (p\u3c0.05), and in DR, compared to DL (p\u3c0.05), while CD and DL demonstrated a decrease in GLUT4, compared to C (p\u3c0.05). GLUT4 protein content in the red portion of the vastus lateralis was elevated in DH, compared to C, CD, DR and DL (p\u3c0.05). GLUT4 content in DL was lower than in C (p\u3c0.05). GLUT4 protein content in the WG was increased in DR, compared to CD and DL (p\u3c0.05) and in DH, compared to CD (p\u3c0.05). In the DR group, exercise lead to a significant drop in BG concentrations 1 hour post exercise, when compared to pre-exercise BG concentrations, at weeks 3 and 6 (p\u3c0.05). At weeks 3 and 6, DH experienced a significant drop in BG concentrations post-exercise, which restored to pre-exercise values at 45 and 75 minutes (p\u3c0.05). In week 3, DL experienced a decrease in BG concentrations for 2 hours post exercise (p\u3c0.05). In week 6 however, DL experienced an increase in BG concentrations at 30, 105 and 120 minutes post exercise (p\u3c0.05). To conclude, T1DM rodents exhibit insulin insensitivity, which was alleviated through regular exercise. In addition, different training modalities lead to different alterations in protein content and exercise induced fluctuations in BG concentrations. Specifically, aerobic exercise lead to immediate falls in BG, where as resistance exercise lead to a more gradual fall in BG concentrations

Muscle Plasticity and Intramuscular signaling in the Insulin-resistant Obese Zucker Rat

The ability to increase skeletal muscle mass may have important implications for the treatment of insulin resistance (IR) and diabetes [1-3]. Recent data suggest that IR muscle may adapt differently than normal muscle; however, molecular mechanism(s) responsible for this finding are not well understood [4]. Herein, we investigate the molecular mechanisms underlying the skeletal muscle remodeling in the IR Obese Zucker (OZ) rat. The OZ rat is characterized by skeletal muscle insulin resistance, hyperglycemia, and hyperlipidemia. Compared to LZ rats, our data demonstrate that soleus muscle hypertrophy was significantly attenuated in the OZ rats after 3-weeks of muscle overload and that these findings appear to be accompanied by significant impairments in the ability of the soleus to undergo phosphorylation of mammalian target of rapamycin (mTOR), 70 kDa ribosomal protein S6 kinase (p70S6k), ribosomal protein S6 (rpS6) and protein kinase B (Akt). Recent in vitro and in vivo studies have suggested a role for AMP-activated protein kinase (AMPK) and dsRNA-dependent protein kinase (PKR) in skeletal muscle adaptation and their interactions with mTOR related signaling [5, 6]. Our data suggest that IR attenuates overload-induced skeletal muscle hypertrophy through the activation of AMPK and PKR, which appears to be associated with an inhibition of mTOR and eIF2α phosphorylation. This finding is consistent with the possible depression of protein synthesis. Other data demonstrate that IR resistance is associated with the PKR-mediated activation of p38 MAP kinase, which would be predicted to lead to increased protein degradation. Further, we demonstrated that the regulation of heat shock proteins (HSPs) and the mitogen-activated protein kinases (MAPKs) are altered during hypertrophy in OZ rat, which suggest that these molecules may play a role in explaining why IR may be associated with alterations in muscle plasticity. In addition to traditional biochemical signaling cascades, recent data have strongly suggested that muscle-specific miRNAs may participate in the regulation of load-induced skeletal muscle remodeling [7]. To this end, we demonstrate for the first time that miR-1 and miR133 expression levels are lower in IR muscle. Further, we also observed that overload decreased mir-1 expression in the LZ muscle to a greater extent to that measured in the OZ muscle. Combined, these results are the first to report evidence that overload-induced skeletal muscle remodeling in IR OZ rat is associated with multiple level decrements including changes in mTOR signaling, hyperphosphorylation of AMPK and PKR and altered regulation of muscle-specific miRNAs

Neurolysin knockout mice generation and initial phenotype characterization

The oligopeptidase neurolysin (EC 3.4.24.16; Nln) was first identified in rat brain synaptic membranes and shown to ubiquitously participate in the catabolism of bioactive peptides such as neurotensin and bradykinin. Recently, it was suggested that Nln reduction could improve insulin sensitivity. Here, we have shown that Nln knockout mice (KO) have increased glucose tolerance, insulin sensitivity and gluconeogenesis. KO mice have increased liver mRNA for several genes related to gluconeogenesis. Isotopic label semi-quantitative peptidomic analysis suggests increase in specific intracellular peptides in gastrocnemius and epididymal adipose tissue, which likely is involved with the increased glucose tolerance and insulin sensitivity in the KO mice. These results suggest the exciting new possibility that Nln is a key enzyme for energy metabolism and could be a novel therapeutic target to improve glucose uptake and insulin sensitivity