Ketogenic diet leads to O-GlcNAc modification in the BTBRT+tf/j mouse model of autism.

BACKGROUND: Protein O-linked-ß-N-acetyl glucosamine (O-GlcNAc) is a post-translational modification to Ser/Thr residues that integrates energy supply with demand. Abnormal O-GlcNAc patterning is evident in several neurological disease states including epilepsy, Alzheimer's disease and autism spectrum disorder (ASD). A potential treatment option for these disorders includes the high-fat, low-carbohydrate, ketogenic diet (KD). The goal of this study was to determine whether the KD induces changes in O-GlcNAc in the BTBRT+tf/j (BTBR) mouse model of ASD. METHODS: Juvenile male (5weeks), age-matched C57 or BTBR mice consumed a chow diet (13% kcal fat) or KD (75% kcal fat) for 10-14days. Following these diets, brain (prefrontal cortex) and liver were examined for gene expression levels of key O-GlcNAc mediators, global and protein specific O-GlcNAc as well as indicators of energy status. RESULTS: The KD reduced global O-GlcNAc in the livers of all animals (p<0.05). Reductions were likely mediated by lower protein levels of O-GlcNAc transferase (OGT) and increased O-GlcNAcase (OGA) (p<0.05). In contrast, no differences in global O-GlcNAc were noted in the brain (p>0.05), yet OGT and OGA expression (mRNA) were elevated in both C57 and BTBR animals (p<0.05). CONCLUSIONS: The KD has tissue specific impacts on O-GlcNAc. Although levels of O-GlcNAc play an important role in neurodevelopment, levels of this modification in the juvenile mouse brain were stable with the KD despite large fluctuations in energy status. This suggests that it is unlikely that the KD exerts it therapeutic benefit in the BTBR model of ASD by O-GlcNAc related pathways.

The ACTN3 R577X Polymorphism Is Associated with Cardiometabolic Fitness in Healthy Young Adults.

Homozygosity for a premature stop codon (X) in the ACTN3 "sprinter" gene is common in humans despite the fact that it reduces muscle size, strength and power. Because of the close relationship between skeletal muscle function and cardiometabolic health we examined the influence of ACTN3 R577X polymorphism over cardiovascular and metabolic characteristics of young adults (n = 98 males, n = 102 females; 23 ± 4.2 years) from our Assessing Inherent Markers for Metabolic syndrome in the Young (AIMMY) study. Both males and females with the RR vs XX genotype achieved higher mean VO2 peak scores (47.8 ± 1.5 vs 43.2 ±1.8 ml/O2/min, p = 0.002) and exhibited higher resting systolic (115 ± 2 vs 105 ± mmHg, p = 0.027) and diastolic (69 ± 3 vs 59 ± 3 mmHg, p = 0.005) blood pressure suggesting a role for ACTN3 in the maintenance of vascular tone. We subsequently identified the expression of alpha-actinin 3 protein in pulmonary artery smooth muscle, which may explain the genotype-specific differences in cardiovascular adaptation to acute exercise. In addition, we utilized targeted serum metabolomics to distinguish between RR and XX genotypes, suggesting an additional role for the ACTN3 R577X polymorphism in human metabolism. Taken together, these results identify significant cardiometabolic effects associated with possessing one or more functional copies of the ACTN3 gene.

Myostatin inhibits proliferation and insulin-stimulated glucose uptake in mouse liver cells.

Although myostatin functions primarily as a negative regulator of skeletal muscle growth and development, accumulating biological and epidemiological evidence indicates an important contributing role in liver disease. In this study, we demonstrate that myostatin suppresses the proliferation of mouse Hepa-1c1c7 murine-derived liver cells (50%; p < 0.001) in part by reducing the expression of the cyclins and cyclin-dependent kinases that elicit G1-S phase transition of the cell cycle (p < 0.001). Furthermore, real-time PCR-based quantification of the long noncoding RNA metastasis associated lung adenocarcinoma transcript 1 (Malat1), recently identified as a myostatin-responsive transcript in skeletal muscle, revealed a significant downregulation (25% and 50%, respectively; p < 0.05) in the livers of myostatin-treated mice and liver cells. The importance of Malat1 in liver cell proliferation was confirmed via arrested liver cell proliferation (p < 0.05) in response to partial Malat1 siRNA-mediated knockdown. Myostatin also significantly blunted insulin-stimulated glucose uptake and Akt phosphorylation in liver cells while increasing the phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS), a protein that is essential for cancer cell proliferation and insulin-stimulated glucose transport. Together, these findings reveal a plausible mechanism by which circulating myostatin contributes to the diminished regenerative capacity of the liver and diseases characterized by liver insulin resistance.

Enhanced stem cell engraftment and modulation of hepatic reactive oxygen species production in diet-induced obesity.

OBJECTIVE: The impact of dietary-induced obesity (DIO) on stem cell engraftment and the respective therapeutic potential of stem cell engraftment in DIO have not been reported. The objectives of this study were to examine the impact of DIO on the survival and efficacy of intravenous bone marrow-derived mesenchymal stem cell (MSC) administration in the conscious C57BL/6 mouse. METHODS: Male mice consumed either a chow (CH) or high fat (HF, 60% kcal) diet for 18 weeks and were subsequently treated with MSC over a 6-day period. Key measurements included tissue-specific cell engraftment, glucose and insulin sensitivity, inflammation, and oxidative stress. RESULTS: MSC administration had no effect on inflammatory markers, glucose, or insulin sensitivity. DIO mice showed increases in MSC engraftment in multiple tissues compared with their CH counterparts. Engraftment was increased in the HF liver where MSC administration attenuated DIO-induced oxidative stress. These liver-specific alterations in HF-MSC were associated with increases in stanniocalcin-1 (STC1) and uncoupling protein 2 (UCP2), which contribute to cell survival and modulate mitochondrial bioenergetics. CONCLUSION: Results suggest that MSC administration in DIO promotes engraftment and mitigates hepatic oxidative stress. These data invite further exploration into the therapeutic potential of stem cells for the treatment of DIO oxidative stress in the liver.

Myostatin-induced inhibition of the long noncoding RNA Malat1 is associated with decreased myogenesis.

Myostatin, a member of the transforming growth factor-ß (TGF-ß) superfamily of secreted proteins, is a potent negative regulator of myogenesis. Free myostatin induces the phosphorylation of the Smad family of transcription factors, which, in turn, regulates gene expression, via the canonical TGF-ß signaling pathway. There is, however, emerging evidence that myostatin can regulate gene expression independent of Smad signaling. As such, we acquired global gene expression data from the gastrocnemius muscle of C57BL/6 mice following a 6-day treatment with recombinant myostatin compared with vehicle-treated animals. Of the many differentially expressed genes, the myostatin-associated decrease (-11.20-fold; P < 0.05) in the noncoding metastasis-associated lung adenocarcinoma transcript 1 (Malat1) was the most significant and the most intriguing because of numerous reports describing its novel role in regulating cell growth. We therefore sought to further characterize the role of Malat1 expression in skeletal muscle myogenesis. RT-PCR-based quantification of C2C12 and primary human skeletal muscle cells revealed a significant and persistent upregulation (4- to 7-fold; P < 0.05) of Malat1 mRNA during the differentiation of myoblasts into myotubes. Conversely, targeted knockdown of Malat1 using siRNA suppressed myoblast proliferation by arresting cell growth in the G(0)/G(1) phase. These results reveal Malat1 as novel downstream target of myostatin with a considerable ability to regulate myogenesis. The identification of new targets of myostatin will have important repercussions for regenerative biology through inhibition and/or reversal of muscle atrophy and wasting diseases.

Exercise training mitigates aberrant cardiac protein O-GlcNAcylation in streptozotocin-induced diabetic mice.

AIMS: Increased protein O-GlcNAcylation occurs in response to increased availability of glucose and fatty acids and is a hallmark of diabetes. Previous studies have demonstrated an improvement in heart function associated with decreased protein O-GlcNAcylation. Our group has recently demonstrated a capacity for exercise to decrease protein O-GlcNAcylation in the heart of normal mice; however, the impact of such training under diabetic conditions has not been examined. MAIN METHODS: Diabetes was induced in mice through injection of streptozotocin. Animals either remained sedentary or were subjected to 6 weeks of swim training protocol. At the end of 6 weeks in vivo cardiac function was assessed and the hearts were harvested for gene expression and Western blotting in relation to O-GlcNAcylation KEY FINDINGS: Diabetes resulted in elevated blood glucose relative to non-diabetic mice. Relative to the sedentary diabetic group, the rate of relaxation (Tau) was significantly improved in the exercised group. Western blot analysis revealed an increase in protein O-GlcNAcylation in the diabetic group which was reversed through exercise despite persistent hyperglycemia. No change in the expression of O-GlcNAc transferase (OGT) was noted between sedentary and exercised diabetic mice; however an increase in the expression and activity of O-GlcNAcase (OGA) was apparent in the exercised group. SIGNIFICANCE: This study demonstrates the potential for exercise training to decrease intracellular protein O-GlcNAcylation in the heart even under conditions of persistent hyperglycemia associated with diabetes. Our results suggest the beneficial effects of regular aerobic exercise extend beyond simple regulation of blood glucose levels.

Enhanced cardiac protein glycosylation (O-GlcNAc) of selected mitochondrial proteins in rats artificially selected for low running capacity.

O-linked ß-N-acetyl glucosamine (O-GlcNAc) is a posttranslational modification consisting of a single N-acetylglucosamine moiety attached by an O-ß-glycosidic linkage to serine and threonine residues of both nuclear and cytosolic proteins. Analogous to phosphorylation, the modification is reversible and dynamic, changing in response to stress, nutrients, hormones, and exercise. Aims of this study were to examine differences in O-GlcNAc protein modification in the cardiac tissue of rats artificially selected for low (LCR) or high (HCR) running capacity. Hyperinsulinemic-euglycemic clamps in conscious animals assessed insulin sensitivity while 2-[(14)C] deoxyglucose tracked both whole body and tissue-specific glucose disposal. Immunoblots of cardiac muscle examined global O-GlcNAc modification, enzymes that control its regulation (OGT, OGA), and specific proteins involved in mitochondrial oxidative phosphorylation. LCR rats were insulin resistant disposing of 65% less glucose than HCR. Global tissue O-GlcNAc, OGT, OGA, and citrate synthase were similar between groups. Analysis of cardiac proteins revealed enhanced O-GlcNAcylation of mitochondrial Complex I, Complex IV, VDAC, and SERCA in LCR compared with HCR. These results are the first to establish an increase in specific protein O-GlcNAcylation in LCR animals that may contribute to progressive mitochondrial dysfunction and the pathogenesis of insulin resistance observed in the LCR phenotype.

Mesenchymal stem cell transplantation for the infarcted heart: a role in minimizing abnormalities in cardiac-specific energy metabolism.

Intense interest has been focused on cell-based therapy for the infarcted heart given that stem cells have exhibited the ability to reduce infarct size and mitigate cardiac dysfunction. Despite this, it is unknown whether mesenchymal stem cell (MSC) therapy can prevent metabolic remodeling following a myocardial infarction (MI). This study examines the ability of MSCs to rescue the infarcted heart from perturbed substrate uptake in vivo. C57BL/6 mice underwent chronic ligation of the left anterior descending coronary artery to induce a MI. Echocardiography was performed on conscious mice at baseline as well as 7 and 23 days post-MI. Twenty-eight days following the ligation procedure, hyperinsulinemic euglycemic clamps assessed in vivo insulin sensitivity. Isotopic tracer administration evaluated whole body, peripheral tissue, and cardiac-specific glucose and fatty acid utilization. To gain insight into the mechanisms by which MSCs modulate metabolism, mitochondrial function was assessed by high-resolution respirometry using permeabilized cardiac fibers. Data show that MSC transplantation preserves insulin-stimulated fatty acid uptake in the peri-infarct region (4.25 ± 0.64 vs. 2.57 ± 0.34 vs. 3.89 ± 0.54 µmol·100 g(-1)·min(-1), SHAM vs. MI + PBS vs. MI + MSC; P < 0.05) and prevents increases in glucose uptake in the remote left ventricle (3.11 ± 0.43 vs. 3.81 ± 0.79 vs. 6.36 ± 1.08 µmol·100 g(-1)·min(-1), SHAM vs. MI + PBS vs. MI + MSC; P < 0.05). This was associated with an enhanced efficiency of mitochondrial oxidative phosphorylation with a respiratory control ratio of 3.36 ± 0.18 in MSC-treated cardiac fibers vs. 2.57 ± 0.14 in the infarct-only fibers (P < 0.05). In conclusion, MSC therapy exhibits the potential to rescue the heart from metabolic aberrations following a MI. Restoration of metabolic flexibility is important given the metabolic demands of the heart and the role of energetics in the progression to heart failure.

Respirometric oxidative phosphorylation assessment in saponin-permeabilized cardiac fibers.

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, oxidative stress, apoptosis, aging, mitochondrial encephalomyopathies and drug toxicity. Given this, technologies to measure cardiac mitochondrial function are in demand. One technique that employs an integrative approach to measure mitochondrial function is respirometric oxidative phosphorylation (OXPHOS) analysis. The principle of respirometric OXPHOS assessment is centered around measuring oxygen concentration utilizing a Clark electrode. As the permeabilized fiber bundle consumes oxygen, oxygen concentration in the closed chamber declines. Using selected substrate-inhibitor-uncoupler titration protocols, electrons are provided to specific sites of the electron transport chain, allowing evaluation of mitochondrial function. Prior to respirometric analysis of mitochondrial function, mechanical and chemical preparatory techniques are utilized to permeabilize the sarcolemma of muscle fibers. Chemical permeabilization employs saponin to selectively perforate the cell membrane while maintaining cellular architecture. This paper thoroughly describes the steps involved in preparing saponin-skinned cardiac fibers for oxygen consumption measurements to evaluate mitochondrial OXPHOS. Additionally, troubleshooting advice as well as specific substrates, inhibitors and uncouplers that may be used to determine mitochondria function at specific sites of the electron transport chain are provided. Importantly, the described protocol may be easily applied to cardiac and skeletal tissue of various animal models and human samples.

Exercise training does not correct abnormal cardiac glycogen accumulation in the db/db mouse model of type 2 diabetes.

Substrate imbalance is a well-recognized feature of diabetic cardiomyopathy. Insulin resistance effectively limits carbohydrate oxidation, resulting in abnormal cardiac glycogen accumulation. Aims of the present study were to 1) characterize the role of glycogen-associated proteins involved in excessive glycogen accumulation in type 2 diabetic hearts and 2) determine if exercise training can attenuate abnormal cardiac glycogen accumulation. Control (db(+)) and genetically diabetic (db/db) C57BL/KsJ-lepr(db)/lepr(db) mice were subjected to sedentary or treadmill exercise regimens. Exercise training consisted of high-intensity/short-duration (10 days) and low-intensity/long-duration (6 wk) protocols. Glycogen levels were elevated by 35-50% in db/db hearts. Exercise training further increased (2- to 3-fold) glycogen levels in db/db hearts. Analysis of soluble and insoluble glycogen pools revealed no differential accumulation of one glycogen subspecies. Phosphorylation (Ser(640)) of glycogen synthase, an indicator of enzymatic fractional activity, was greater in db/db mice subjected to sedentary and exercise regimens. Elevated glycogen levels were accompanied by decreased phosphorylation (Thr(172)) of 5'-AMP-activated kinase and phosphorylation (Ser(79)) of its downstream substrate acetyl-CoA carboxylase. Glycogen concentration was not associated with increases in other glycogen-associated proteins, including malin and laforin. Novel observations show that exercise training does not correct diabetes-induced elevations in cardiac glycogen but, rather, precipitates further accumulation.

Hyperinsulinemic-euglycemic clamp in the conscious rat.

Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, T2D results in elevated blood glucose levels. The "gold-standard" in assessing insulin sensitivity is a hyperinsulinemic-euglycemic clamp or insulin clamp. In this procedure, insulin is infused at a constant rate resulting in a drop in blood glucose. To maintain blood glucose at a constant level, exogenous glucose (D50) is infused into the venous circulation. The amount of glucose infused to maintain homeostasis is indicative of insulin sensitivity. Here, we show the basic clamp procedure in the chronically catheterized, unrestrained, conscious rat. This model allows blood to be collected with minimal stress to the animal. Following the induction of anesthesia, a midline incision is made and the left common carotid artery and right jugular vein are catheterized. Inserted catheters are flushed with heparinized saline, then exteriorized and secured. Animals are allowed to recover for 4-5 days prior to experiments, with weight gain monitored daily. Only those animals who regain weight to pre-surgery levels are used for experiments. On the day of the experiment, rats are fasted and connected to pumps containing insulin and D50. Baseline glucose is assessed from the arterial line and used a benchmark throughout the experiment (euglycemia). Following this, insulin is infused at a constant rate into the venous circulation. To match the drop in blood glucose, D50 is infused. If the rate of D50 infusion is greater than the rate of uptake, a rise in glucose will occur. Similarly, if the rate is insufficient to match whole body glucose uptake, a drop will occur. Titration of glucose continues until stable glucose readings are achieved. Glucose levels and glucose infusion rates during this stable period are recorded and reported. Results provide an index of whole body insulin sensitivity. The technique can be refined to meet specific experimental requirements. It is further enhanced by the use of radioactive tracers that can determine tissue specific insulin-stimulated glucose uptake as well as whole body glucose turnover.