Regulation of mitochondrial calcium uniporter expression and calcium-dependent cell signaling by lncRNA Tug1 in cardiomyocytes.

Cardiomyocyte calcium homeostasis is a tightly regulated process. The mitochondrial calcium uniporter (MCU) complex can buffer elevated cytosolic Ca2+ levels and consists of pore-forming proteins including MCU, and various regulatory proteins such as mitochondrial calcium uptake proteins 1 and 2 (MICU1/2). The stoichiometry of these proteins influences the sensitivity to Ca2+ and the activity of the complex. However, the factors that regulate their gene expression remain incompletely understood. Long noncoding RNAs (lncRNAs) regulate gene expression through various mechanisms, and we recently found that the lncRNA Tug1 increased the expression of Mcu and associated genes. To further explore this, we performed antisense LNA knockdown of Tug1 (Tug1 KD) in H9c2 rat cardiomyocytes. Tug1 KD increased MCU protein expression, yet pyruvate dehydrogenase dephosphorylation, which is indicative of mitochondrial Ca2+ uptake, was not enhanced. However, RNA-seq revealed that Tug1 KD increased Mcu along with differential expression of >1,000 genes including many related to Ca2+ regulation pathways in the heart. To understand the effect of this on Ca2+ signaling, we measured phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and its downstream target cAMP Response Element-Binding protein (CREB), a transcription factor known to drive Mcu gene expression. In response to a Ca2+ stimulus, the increase in CaMKII and CREB phosphorylation was attenuated by Tug1 KD. Inhibition of CaMKII, but not CREB, partially prevented the Tug1 KD-mediated increase in Mcu. Together, these data suggest that Tug1 modulates MCU expression via a mechanism involving CaMKII and regulates cardiomyocyte Ca2+ signaling, which could have important implications for cardiac function.NEW & NOTEWORTHY Calcium is essential for signaling, excitation contraction, and energy homeostasis in the heart. Despite this, molecular regulators of these processes are not completely understood. We report that knockdown of lncRNA Tug1 alters the calcium handling transcriptome and increases mitochondrial calcium uniporter expression via a mechanism involving CaMKII. As overexpression of MCU is known to be protective against pathological cardiac remodeling, targeting Tug1 may be a potential strategy for treating cardiovascular disease.

Skeletal muscle cell-specific differences in type 2 diabetes.

Major stores of glucose are found as glycogen in skeletal muscle and liver. Skeletal muscle is a heterogenous tissue, with cellular metabolic and contractile distinctions dependent on whether the cell (fibre) is slow-twitch (Type I) or fast-twitch (Type II). We hypothesised that proteins important for glycogen metabolism would be differentially abundant between these diverse fibres. We further hypothesised that the cellular location of these proteins would be different in muscle samples between control (CON) and individuals with type 2 diabetes (T2D). We dissected individual muscle fibre segments from vastus lateralis skeletal muscle biopsy samples from CON and T2D and used cell-type-specific approaches to address muscle heterogeneity. We measured glycogen and glycogen-related proteins by immunoblotting techniques. A lower proportion of Type I fibres was found in muscle in T2D compared with CON. AMPK-ß2, glycogen branching enzyme (GBE), glycogen debranching enzyme (GDE), and glycogen phosphorylase (GP) were differentially localized between fibre types and in fibres from CON and T2D individuals. A key novel finding was that the majority of glycogen is loosely bound or cytosolic in location in human skeletal muscle. The proportion of this diffusible pool of glycogen was significantly lower in Type I fibres in T2D compared to CON. A hyperinsulinaemic, euglycaemic clamp in people with type 2 diabetes had no effect on the proportion of diffusible glycogen. We identify cell-type as an important consideration when assessing glycogen metabolism in muscle. Our findings demonstrate varying glucose handling abilities in specific muscle fibre types in type 2 diabetes. A model is presented to provide an overview of the cell-specific differences in glycogen metabolism in type 2 diabetes.

Long non-coding RNA Tug1 modulates mitochondrial and myogenic responses to exercise in skeletal muscle.

BACKGROUND: Mitochondria have an essential role in regulating metabolism and integrate environmental and physiological signals to affect processes such as cellular bioenergetics and response to stress. In the metabolically active skeletal muscle, mitochondrial biogenesis is one important component contributing to a broad set of mitochondrial adaptations occurring in response to signals, which converge on the biogenesis transcriptional regulator peroxisome proliferator-activated receptor coactivator 1-alpha (PGC-1α), and is central to the beneficial effects of exercise in skeletal muscle. We investigated the role of long non-coding RNA (lncRNA) taurine-upregulated gene 1 (TUG1), which interacts with PGC-1α in regulating transcriptional responses to exercise in skeletal muscle. RESULTS: In human skeletal muscle, TUG1 gene expression was upregulated post-exercise and was also positively correlated with the increase in PGC-1α gene expression (PPARGC1A). Tug1 knockdown (KD) in differentiating mouse myotubes led to decreased Ppargc1a gene expression, impaired mitochondrial respiration and morphology, and enhanced myosin heavy chain slow isoform protein expression. In response to a Ca2+-mediated stimulus, Tug1 KD prevented an increase in Ppargc1a expression. RNA sequencing revealed that Tug1 KD impacted mitochondrial Ca2+ transport genes and several downstream PGC-1α targets. Finally, Tug1 KD modulated the expression of ~300 genes that were upregulated in response to an in vitro model of exercise in myotubes, including genes involved in regulating myogenesis. CONCLUSIONS: We found that TUG1 is upregulated in human skeletal muscle after a single session of exercise, and mechanistically, Tug1 regulates transcriptional networks associated with mitochondrial calcium handling, muscle differentiation and myogenesis. These data demonstrate that lncRNA Tug1 exerts regulation over fundamental aspects of skeletal muscle biology and response to exercise stimuli.

Unpacking and validating the "physiological adaptation" core concept of physiology.

A national Task Force of 25 Australian physiology educators used the Delphi protocol to develop seven physiology core concepts that were agreed to nationally. The aim of the current study was to unpack the "physiological adaptation" core concept with the descriptor "organisms adjust and adapt to acute and chronic changes in the internal and external environments across the lifespan." This core concept was unpacked by three Task Force members and a facilitator into four themes and nine subthemes that encompass the role of stressors and disturbed homeostasis in adaptation and the capacity for, and the nature of, the physiological adaptation. Twenty-two Task Force members then provided feedback and rated the themes and subthemes for level of importance and difficulty for students to learn via an online survey using a five-point Likert scale. Seventeen respondents completed all survey questions. For all themes/subthemes, importance was typically rated 1 (Essential) or 2 (Important) (n = 17, means ±SD ranged from 1.1 ± 0.3 to 2.2 ± 0.9), and difficulty was typically rated 3 (Moderately Difficult) (n = 17, means ranged from 2.9 ± 0.7 to 3.4 ± 0.9). Subtle differences in the proportion of importance scores (n = 17, Fisher's exact: P = 0.004, ANOVA: F12,220 = 2.630, P = 0.003; n = 22, Fisher's exact: P = 0.002, ANOVA: F12,281 = 2.743, P < 0.001), but not difficulty scores, were observed between themes/subthemes, and free-text feedback was minor. The results suggest successful unpacking of the physiological adaptation core concept. The themes and subthemes can inform the design of learning outcomes, assessment, and teaching and learning activities that have commonality and consistency across curricula.NEW & NOTEWORTHY An Australian Task Force of physiology educators identified physiological adaptation as a core concept of physiology. It was subsequently unpacked into four themes and nine subthemes. These were rated, by the Task Force, Essential or Important and Moderately Difficult for students to learn. The themes and subthemes can inform the design of learning outcomes, assessments, and teaching and learning activities that have commonality and consistency across curricula.

A Comparison of Sodium Citrate and Sodium Bicarbonate Ingestion: Blood Alkalosis and Gastrointestinal Symptoms.

This study compared the recommended dose of sodium citrate (SC, 500 mg/kg body mass) and sodium bicarbonate (SB, 300 mg/kg body mass) for blood alkalosis (blood [HCO3-]) and gastrointestinal symptoms (GIS; number and severity). Sixteen healthy individuals ingested the supplements in a randomized, crossover design. Gelatin capsules were ingested over 15 min alongside a carbohydrate-rich meal, after which participants remained seated for forearm venous blood sample collection and completion of GIS questionnaires every 30 min for 300 min. Time-course and session value (i.e., peak and time to peak) comparisons of SC and SB supplementation were performed using linear mixed models. Peak blood [HCO3-] was similar for SC (mean 34.2, 95% confidence intervals [33.4, 35.0] mmol/L) and SB (mean 33.6, 95% confidence intervals [32.8, 34.5] mmol/L, p = .308), as was delta blood [HCO3-] (SC = 7.9 mmol/L; SB = 7.3 mmol/L, p = .478). Blood [HCO3-] was ≥6 mmol/L above baseline from 180 to 240 min postingestion for SC, significantly later than for SB (120-180 min; p < .001). GIS were mostly minor, and peaked 80-90 min postingestion for SC, and 35-50 min postingestion for SB. There were no significant differences for the number or severity of GIS reported (p > .05 for all parameters). In summary, the recommended doses of SC and SB induce similar blood alkalosis and GIS, but with a different time course.

Impaired postprandial skeletal muscle vascular responses to a mixed meal challenge in normoglycaemic people with a parent with type 2 diabetes.

AIMS/HYPOTHESIS: Microvascular blood flow (MBF) increases in skeletal muscle postprandially to aid in glucose delivery and uptake in muscle. This vascular action is impaired in individuals who are obese or have type 2 diabetes. Whether MBF is impaired in normoglycaemic people at risk of type 2 diabetes is unknown. We aimed to determine whether apparently healthy people at risk of type 2 diabetes display impaired skeletal muscle microvascular responses to a mixed-nutrient meal. METHODS: In this cross-sectional study, participants with no family history of type 2 diabetes (FH-) for two generations (n = 18), participants with a positive family history of type 2 diabetes (FH+; i.e. a parent with type 2 diabetes; n = 16) and those with type 2 diabetes (n = 12) underwent a mixed meal challenge (MMC). Metabolic responses (blood glucose, plasma insulin and indirect calorimetry) were measured before and during the MMC. Skeletal muscle large artery haemodynamics (2D and Doppler ultrasound, and Mobil-O-graph) and microvascular responses (contrast-enhanced ultrasound) were measured at baseline and 1 h post MMC. RESULTS: Despite normal blood glucose concentrations, FH+ individuals displayed impaired metabolic flexibility (reduced ability to switch from fat to carbohydrate oxidation vs FH-; p < 0.05) during the MMC. The MMC increased forearm muscle microvascular blood volume in both the FH- (1.3-fold, p < 0.01) and FH+ (1.3-fold, p < 0.05) groups but not in participants with type 2 diabetes. However, the MMC increased MBF (1.9-fold, p < 0.01), brachial artery diameter (1.1-fold, p < 0.01) and brachial artery blood flow (1.7-fold, p < 0.001) and reduced vascular resistance (0.7-fold, p < 0.001) only in FH- participants, with these changes being absent in FH+ and type 2 diabetes. Participants with type 2 diabetes displayed significantly higher vascular stiffness (p < 0.001) compared with those in the FH- and FH+ groups; however, vascular stiffness did not change during the MMC in any participant group. CONCLUSIONS/INTERPRETATION: Normoglycaemic FH+ participants display impaired postprandial skeletal muscle macro- and microvascular responses, suggesting that poor vascular responses to a meal may contribute to their increased risk of type 2 diabetes. We conclude that vascular insulin resistance may be an early precursor to type 2 diabetes in humans, which can be revealed using an MMC.

Oral and intravenous glucose administration elicit opposing microvascular blood flow responses in skeletal muscle of healthy people: role of incretins.

Insulin infusion increases skeletal muscle microvascular blood flow (MBF) in healthy people but is impaired during insulin resistance. However, we have shown that eliciting insulin secretion via oral glucose loading in healthy people impairs muscle MBF, whilst others have demonstrated intravenous glucose infusion stimulates MBF. We aimed to show that the route of glucose administration (oral versus intravenous) influences muscle MBF, and explore potential gut-derived hormones that may explain these divergent responses. Ten healthy individuals underwent a 120 min oral glucose tolerance test (OGTT; 75 g glucose) and on a subsequent occasion an intravenous glucose tolerance test (IVGTT, bypassing the gut) matched for similar blood glucose excursions. Femoral artery and thigh muscle microvascular (contrast-enhanced ultrasound) haemodynamics were measured at baseline and during the OGTT/IVGTT. Plasma insulin, C-peptide, glucagon, non-esterified fatty acids and a range of gut-derived hormones and incretins (gastric inhibitory polypeptide (GIP) and glucagon-like peptide-1(GLP-1)) were measured at baseline and throughout the OGTT/IVGTT. The IVGTT increased whereas the OGTT impaired MBF (1.3-fold versus 0.5-fold from baseline, respectively, P = 0.0006). The impairment in MBF during the OGTT occurred despite producing 2.8-fold higher plasma insulin concentrations (P = 0.0001). The change in MBF from baseline (ΔMBF) negatively correlated with ΔGIP concentrations (r = -0.665, P < 0.0001). The natural log ratio of incretins GLP-1:GIP was positively associated with ΔMBF (r = 0.658, P < 0.0001), suggesting they have opposing actions on the microvasculature. Postprandial hyperglycaemia per se does not acutely determine opposing microvascular responses between OGTT and IVGTT. Incretins may play a role in modulating skeletal muscle MBF in humans. KEY POINTS: Insulin or mixed nutrient meals stimulate skeletal muscle microvascular blood flow (MBF) to aid in the delivery of nutrients; however, this vascular effect is lost during insulin resistance. Food/drinks containing large glucose loads impair MBF in healthy people; however, this impairment is not observed when glucose is infused intravenously (bypassing the gut). We investigated skeletal muscle MBF responses to a 75 g oral glucose tolerance test and intravenous glucose infusion and aimed to identify potential gut hormones responsible for glucose-mediated changes in MBF. Despite similar blood glucose concentrations, orally ingested glucose impaired, whereas intravenously infused glucose augmented, skeletal muscle MBF. The incretin gastric inhibitory polypeptide was negatively associated with MBF, suggestive of an incretin-mediated MBF response to oral glucose ingestion. This work provides new insight into why diets high in glucose may be detrimental to vascular health and provides new avenues for novel treatment strategies targeting microvascular dysfunction.

Impaired postprandial adipose tissue microvascular blood flow responses to a mixed-nutrient meal in first-degree relatives of adults with type 2 diabetes.

Adipose tissue microvascular blood flow (MBF) is stimulated postprandially to augment delivery of nutrients and hormones to adipocytes. Adipose tissue MBF is impaired in type 2 diabetes (T2D). Whether healthy individuals at-risk of T2D show similar impairments is unknown. We aimed to determine whether adipose tissue MBF is impaired in apparently healthy individuals with a family history of T2D. Overnight-fasted individuals with no family history of T2D for two generations (FH-, n = 13), with at least one parent with T2D (FH+, n = 14) and clinically diagnosed T2D (n = 11) underwent a mixed meal challenge (MMC). Metabolic responses [blood glucose, plasma insulin, plasma nonesterified fatty acids (NEFAs), and fat oxidation] were measured before and during the MMC. MBF in truncal subcutaneous adipose tissue was assessed by contrast ultrasound while fasting and 60 min post-MMC. FH+ had normal blood glucoses, increased adiposity, and impaired post-MMC adipose tissue MBF (Δ0.70 ± 0.22 vs. 2.45 ± 0.60 acoustic intensity/s, P = 0.007) and post-MMC adipose tissue insulin resistance (Adipo-IR index; Δ45.5 ± 13.9 vs. 7.8 ± 5.1 mmol/L × pmol/L, P = 0.007) compared with FH-. FH+ and T2D had an impaired ability to suppress fat oxidation post-MMC. Fat oxidation incremental area under the curve (iAUC) (35-55 min post-MMC, iAUC) was higher in FH+ and T2D than in FH- (P = 0.005 and 0.009, respectively). Postprandial MBF was negatively associated with postprandial fat oxidation iAUC (P = 0.01). We conclude that apparently healthy FH+ individuals display blunted postprandial adipose tissue MBF that occurs in parallel with adipose tissue insulin resistance and impaired suppression of fat oxidation, which may help explain their heightened risk for developing T2D.NEW & NOTEWORTHY Adipose tissue blood flow plays a key role in postprandial nutrient storage. People at-risk of type 2 diabetes have impaired postmeal adipose tissue blood flow. Impaired adipose tissue blood flow is associated with altered fat oxidation. Risk of type 2 diabetes may be elevated by poor adipose tissue blood flow.

Prior exercise enhances skeletal muscle microvascular blood flow and mitigates microvascular flow impairments induced by a high-glucose mixed meal in healthy young men.

KEY POINTS: Exercise, insulin-infusion and low-glucose mixed-nutrient meal ingestion increases muscle microvascular blood flow which in part facilitates glucose delivery and disposal. In contrast, high-glucose ingestion impairs muscle microvascular blood flow which may contribute to impaired postprandial metabolism. We investigated the effects of prior cycling exercise on postprandial muscle microvascular blood flow responses to a high-glucose mixed-nutrient meal ingested 3 and 24 h post-exercise. Prior exercise enhanced muscle microvascular blood flow and mitigated microvascular impairments induced by a high-glucose mixed meal ingested 3 h post-exercise, and to a lesser extent 24 h post-exercise. High-glucose ingestion 3 h post-exercise leads to greater postprandial blood glucose, non-esterified fatty acids, and fat oxidation, and a delay in the insulin response to the meal compared to control. Effects of acute exercise on muscle microvascular blood flow persist well after the cessation of exercise which may be beneficial for conditions characterized by microvascular and glycaemic dysfunction. ABSTRACT: Exercise, insulin-infusion and low-glucose mixed-nutrient meal ingestion lead to increased muscle microvascular blood flow (MBF), whereas high-glucose ingestion impairs MBF. We investigated whether prior cycling exercise could enhance postprandial muscle MBF and prevent MBF impairments induced by high-glucose mixed-nutrient meal ingestion. In a randomized cross-over design, eight healthy young men ingested a high-glucose mixed-nutrient meal (1.1 g glucose/kg body weight; 45% carbohydrate, 20% protein and 35% fat) after an overnight fast (no-exercise control) and 3 h and 24 h after moderate-intensity cycling exercise (1 h at 70-75% V̇O2peak ). Skeletal muscle MBF, measured directly by contrast-enhanced ultrasound, was lower at 60 min and 120 min postprandially compared to baseline in all conditions (P < 0.05), with a greater decrease occurring from 60 min to 120 min in the control (no-exercise) condition only (P < 0.001). Despite this meal-induced decrease, MBF was still markedly higher compared to control in the 3 h post-exercise condition at 0 min (pre-meal; 74%, P = 0.004), 60 min (112%, P = 0.002) and 120 min (223%, P < 0.001), and in the 24 h post-exercise condition at 120 min postprandially (132%, P < 0.001). We also report that in the 3 h post-exercise condition postprandial blood glucose, non-esterified fatty acids (NEFAs), and fat oxidation were substantially elevated, and the insulin response to the meal delayed compared to control. This probably reflects a combination of increased post-exercise exogenous glucose appearance, substrate competition, and NEFA-induced insulin resistance. We conclude that prior cycling exercise elicits long-lasting effects on muscle MBF and partially mitigates MBF impairments induced by high-glucose mixed-nutrient meal ingestion.

Exercise alters cardiovascular and renal pregnancy adaptations in female rats born small on a high-fat diet.

Intrauterine growth restriction programs adult cardiorenal disease, which may be exacerbated by pregnancy and obesity. Importantly, exercise has positive cardiovascular effects. This study determined if high-fat feeding exacerbates the known adverse cardiorenal adaptations to pregnancy in rats born small and whether endurance exercise can prevent these complications. Uteroplacental insufficiency was induced by bilateral uterine vessel ligation (Restricted) or sham (Control) surgery on embryonic day 18 (E18) in Wistar-Kyoto rats. Female offspring consumed a Chow or high-fat diet (HFD) from weaning and were randomly allocated to either a sedentary (Sedentary) or an exercise protocol at 16 wk; exercised before and during pregnancy (Exercise), or exercised during pregnancy only (PregEx). Systolic blood pressure was measured prepregnancy and rats were mated at 20 wk. During pregnancy, systolic blood pressure (E18) and renal function (E19) were assessed. Sedentary HFD Control females had increased estimated glomerular filtration rate (eGFR) compared with Chow. Compared with Control, Sedentary-Restricted females had increased eGFR, which was not influenced by HFD. Renal function was not affected by exercise and prepregnancy blood pressure was not altered. Restricted Chow-fed dams and dams fed a high-fat diet had a greater reduction in systolic blood pressure during late gestation, which was only prevented by Exercise. In summary, high-fat fed females born small are at a greater risk of altered cardiorenal adaptations to pregnancy. Although cardiovascular dysfunction was prevented by Exercise, renal dysfunction was not affected by exercise interventions. This study highlights that modifiable risk factors can have beneficial effects in the mother during pregnancy, which may impact fetal growth and development.

Exercise improves metabolic function and alters the microbiome in rats with gestational diabetes.

Gestational diabetes mellitus (GDM) is a common pregnancy complication, particularly prevalent in obese women. Importantly, exercise has beneficial impacts on maternal glucose control and may prevent GDM in "at-risk" women. We aimed to determine whether a high-fat diet (HFD) exacerbates metabolic dysfunction and alters gut microbiome in GDM and whether endurance exercise prevents these changes. Uteroplacental insufficiency was induced by bilateral uterine vessel ligation (Restricted) or sham (Control) surgery on E18 in Wistar-Kyoto rats. Female offspring were fed a Chow or HFD (23% fat) from weaning (5 weeks) and at 16 weeks randomly allocated to remain Sedentary or to an exercise protocol of either Exercise prior to and during pregnancy (Exercise); or Exercise during pregnancy only (PregEx). Females were mated (20 weeks) and underwent indirect calorimetry (embryonic day 16; E16), glucose tolerance testing (E18), followed by 24-hr feces collection at E19 (n = 8-10/group). HFD consumption in female rats with GDM exacerbated the adverse metabolic adaptations to pregnancy and altered gut microbial populations. Specifically, the Firmicutes-to-Bacteroidetes ratio was increased, due to an underlying change in abundance of the orders Clostridiales and Bacteroidales. Maternal Exercise, but not PregEx, prevented the development of metabolic dysfunction, increased pancreatic ß-cell mass, and prevented the alteration of the gut microbiome in GDM females. Our findings suggest that maternal exercise and diet influence metabolic and microbiome dysfunction in females with GDM, which may impact long-term maternal and offspring health.

Factors Influencing Blood Alkalosis and Other Physiological Responses, Gastrointestinal Symptoms, and Exercise Performance Following Sodium Citrate Supplementation: A Review.

This review aimed to identify factors associated with (a) physiological responses, (b) gastrointestinal (GI) symptoms, and (c) exercise performance following sodium citrate supplementation. A literature search identified 33 articles. Observations of physiological responses and GI symptoms were categorized by dose (< 500, 500, and > 500 mg/kg body mass [BM]) and by timing of postingestion measurements (in minutes). Exercise performance following sodium citrate supplementation was compared with placebo using statistical significance, percentage change, and effect size. Performance observations were categorized by exercise duration (very short < 60 s, short ≥ 60 and ≤ 420 s, and longer > 420 s) and intensity (very high > 100% VO2max and high 90-100% VO2max). Ingestion of 500 mg/kg BM sodium citrate induced blood alkalosis more frequently than < 500 mg/kg BM, and with similar frequency to >500 mg/kg BM. The GI symptoms were minimized when a 500 mg/kg BM dose was ingested in capsules rather than in solution. Significant improvements in performance following sodium citrate supplementation were reported in all observations of short-duration and very high-intensity exercise with a 500 mg/kg BM dose. However, the efficacy of supplementation for short-duration, high-intensity exercise is less clear, given that only 25% of observations reported significant improvements in performance following sodium citrate supplementation. Based on the current literature, the authors recommend ingestion of 500 mg/kg BM sodium citrate in capsules to induce alkalosis and minimize GI symptoms. Supplementation was of most benefit to performance of short-duration exercise of very high intensity; further investigation is required to determine the importance of ingestion duration and timing.

Skeletal muscle AMPK is not activated during 2 h of moderate intensity exercise at ∼65% V̇O2peak in endurance trained men.

KEY POINTS: AMP-activated protein kinase (AMPK) is considered a major regulator of skeletal muscle metabolism during exercise. However, we previously showed that, although AMPK activity increases by 8-10-fold during ∼120 min of exercise at ∼65% V̇O2peak in untrained individuals, there is no increase in these individuals after only 10 days of exercise training (longitudinal study). In a cross-sectional study, we show that there is also a lack of activation of skeletal muscle AMPK during 120 min of cycling exercise at 65% V̇O2peak in endurance-trained individuals. These findings indicate that AMPK is not an important regulator of exercise metabolism during 120 min of exercise at 65% V̇O2peak in endurance trained men. It is important that more energy is directed towards examining other potential regulators of exercise metabolism. ABSTRACT: AMP-activated protein kinase (AMPK) is considered a major regulator of skeletal muscle metabolism during exercise. Indeed, AMPK is activated during exercise and activation of AMPK by 5-aminoimidazole-4-carboxyamide-ribonucleoside (AICAR) increases skeletal muscle glucose uptake and fat oxidation. However, we have previously shown that, although AMPK activity increases by 8-10-fold during ∼120 min of exercise at ∼65% V̇O2peak in untrained individuals, there is no increase in these individuals after only 10 days of exercise training (longitudinal study). In a cross-sectional study, we examined whether there is also a lack of activation of skeletal muscle AMPK during 120 min of cycling exercise at 65% V̇O2peak in endurance-trained individuals. Eleven untrained (UT; V̇O2peak = 37.9 ± 5.6 ml.kg-1  min-1 ) and seven endurance trained (ET; V̇O2peak = 61.8 ± 2.2 ml.kg-1  min-1 ) males completed 120 min of cycling exercise at 66 ± 4% V̇O2peak (UT: 100 ± 21 W; ET: 190 ± 15 W). Muscle biopsies were obtained at rest and following 30 and 120 min of exercise. Muscle glycogen was significantly (P < 0.05) higher before exercise in ET and decreased similarly during exercise in the ET and UT individuals. Exercise significantly increased calculated skeletal muscle free AMP content and more so in the UT individuals. Exercise significantly (P < 0.05) increased skeletal muscle AMPK α2 activity (4-fold), AMPK αThr172 phosphorylation (2-fold) and ACCß Ser222 phosphorylation (2-fold) in the UT individuals but not in the ET individuals. These findings indicate that AMPK is not an important regulator of exercise metabolism during 120 min of exercise at 65% V̇O2peak in endurance trained men.

High-glucose mixed-nutrient meal ingestion impairs skeletal muscle microvascular blood flow in healthy young men.

Oral glucose ingestion leads to impaired muscle microvascular blood flow (MBF), which may contribute to acute hyperglycemia-induced insulin resistance. We investigated whether incorporating lipids and protein into a high-glucose load would prevent postprandial MBF dysfunction. Ten healthy young men (age, 27 yr [24, 30], mean with lower and upper bounds of the 95% confidence interval; height, 180 cm [174, 185]; weight, 77 kg [70, 84]) ingested a high-glucose (1.1 g/kg glucose) mixed-nutrient meal (10 kcal/kg; 45% carbohydrate, 20% protein, and 35% fat) in the morning after an overnight fast. Femoral arterial blood flow was measured via Doppler ultrasound, and thigh MBF was measured via contrast-enhanced ultrasound, before meal ingestion and 1 h and 2 h postprandially. Blood glucose and plasma insulin were measured at baseline and every 15 min throughout the 2-h postprandial period. Compared with baseline, thigh muscle microvascular blood volume, velocity, and flow were significantly impaired at 60 min postprandial (-25%, -27%, and -46%, respectively; all P < 0.05) and to a greater extent at 120 min postprandial (-37%, -46%, and -64%; all P < 0.01). Heart rate and femoral arterial diameter, blood velocity, and blood flow were significantly increased at 60 min and 120 min postprandial (all P < 0.05). Higher blood glucose area under the curve was correlated with greater MBF dysfunction (R2 = 0.742; P < 0.001). Ingestion of a high-glucose mixed-nutrient meal impairs MBF in healthy individuals for up to 2 h postprandial.

Modest changes to glycemic regulation are sufficient to maintain glucose fluxes in healthy young men following overfeeding with a habitual macronutrient composition.

Currently, it is unclear whether short-term overfeeding in healthy people significantly affects postprandial glucose regulation, as most human overfeeding studies have utilized induced experimental conditions such as the euglycemic-hyperinsulinemic clamp technique to assess glucoregulation. The aim of this study was to quantify glucose fluxes [rates of meal glucose appearance (Ra), disposal (Rd), and endogenous glucose production (EGP)] in response to 5 and 28 days of overfeeding (+45% energy) while maintaining habitual macronutrient composition (31.0 ± 1.9% fat, 48.6 ± 2.2% carbohydrate, 16.7 ± 1.4% protein) in healthy, lean young men. Meal tolerance testing was combined with the triple-stable isotope glucose tracer approach. Visceral adipose volume increased by ~15% with 5 days of overfeeding, while there was no further change at 28 days. In contrast, body mass (+1.6 kg) and fat mass (+1.3 kg) were significantly increased only after 28 days of overfeeding. Fasting EGP, Rd, and insulin were increased at 5 but unchanged after 28 days. Postprandial glucose and insulin responses were unaltered by 5 days of overfeeding but were modestly increased after 28 days (P < 0.05). However, meal Ra and glucose Rd were significantly increased after both 5 and 28 days of overfeeding (P < 0.05). Despite this, overfeeding did not lead to alterations to postprandial EGP suppression. Thus, in contrast to findings from euglycemic-hyperinsulinemic clamp studies, chronic overfeeding did not affect the ability to suppress EGP or stimulate Rd under postprandial conditions. Rather, glucose flux was appropriately maintained following 28 days of overfeeding through modest increases in postprandial glycemia and insulinemia.

Ascorbic acid supplementation improves postprandial glycaemic control and blood pressure in individuals with type 2 diabetes: Findings of a randomized cross-over trial.

AIM: The primary aim of this study was to investigate whether ascorbic acid (AA) supplementation improves postprandial glucose responses under free-living conditions in individuals with type 2 diabetes. A secondary aim was to investigate the effect of AA supplementation on blood pressure. MATERIALS AND METHODS: A total of 31 individuals with type 2 diabetes (26 males and 5 females; aged 61.8 ± 6.8 years; duration of diabetes, 5.6 ± 4.6 years; HbA1c, 7.6% ± 0.7% [mean ± SD]) were enrolled in a randomized cross-over study involving 4 months of supplementation with oral AA (2 × 500 mg/d) or placebo. Participants wore continuous glucose monitors for 48 hours and consumed standardized meals pre- and post-supplementation. Measurements included postprandial glucose incremental areas under the curve (iAUC), duration of day in hyper- and hypo-glycaemia status, average 24-hour and daily postprandial glucose concentrations, HbA1c, insulin, blood pressure (BP) and oxidative stress (F2 -isoprostanes). RESULTS: Following AA supplementation, significant decreases were observed in daily postprandial glucose iAUC (-36%), in duration of day with hyperglycaemia (-2.8 h/d) and postprandial hyperglycaemia (-1.7 h/d), in average 24-hour glucose (-0.8 mmol/L) and daily postprandial glucose (-1.1 mmol/L) concentrations, in systolic (-7 mm Hg) and diastolic (-5 mm Hg) blood pressures and in a specific fraction of free plasma F2 -isoprostanes (-47 pg/mL) as compared to placebo. CONCLUSIONS: Individuals with type 2 diabetes experienced improved postprandial and 24-hour glycaemia and decreased BP after 4 months of AA supplementation as compared to placebo. These findings offer evidence for the proposed use of AA as an adjunct therapy to improve glycaemic and BP control in individuals with type 2 diabetes.

Measurement of postprandial glucose fluxes in response to acute and chronic endurance exercise in healthy humans.

The effect of endurance exercise on enhancing insulin sensitivity and glucose flux has been well established with techniques such as the hyperinsulinemic clamp. Although informative, such techniques do not emulate the physiological postprandial state, and it remains unclear how exercise improves postprandial glycaemia. Accordingly, combining mixed-meal tolerance testing and the triple-stable isotope glucose tracer approach, glucose fluxes [rates of meal glucose appearance (Ra), disposal (Rd), and endogenous glucose production (EGP)] were determined following acute endurance exercise (1 h cycling; ~70% V̇o2max) and 4 wk of endurance training (cycling 5 days/wk). Training was associated with a modest increase in V̇o2max (~7%, P < 0.001). Postprandial glucose and insulin responses were reduced to the same extent following acute and chronic training. Interestingly, this was not accompanied by changes to rates of meal Ra, Rd, or degree of EGP suppression. Glucose clearance (Rd relative to prevailing glucose) was, however, enhanced with acute and chronic exercise. Furthermore, the duration of EGP suppression was shorter with acute and chronic exercise, with EGP returning toward fasting levels more rapidly than pretraining conditions. These findings suggest that endurance exercise influences the efficiency of the glucoregulatory system, where pretraining rates of glucose disposal and production were achieved at lower glucose and insulin levels. Notably, there was no influence of chronic training over and above that of a single exercise bout, providing further evidence that glucoregulatory benefits of endurance exercise are largely attributed to the residual effects of the last exercise bout.

Skeletal muscle mitochondria: a major player in exercise, health and disease.

BACKGROUND: Maintaining skeletal muscle mitochondrial content and function is important for sustained health throughout the lifespan. Exercise stimulates important key stress signals that control skeletal mitochondrial biogenesis and function. Perturbations in mitochondrial content and function can directly or indirectly impact skeletal muscle function and consequently whole-body health and wellbeing. SCOPE OF REVIEW: This review will describe the exercise-stimulated stress signals and molecular mechanisms positively regulating mitochondrial biogenesis and function. It will then discuss the major myopathies, neuromuscular diseases and conditions such as diabetes and ageing that have dysregulated mitochondrial function. Finally, the impact of exercise and potential pharmacological approaches to improve mitochondrial function in diseased populations will be discussed. MAJOR CONCLUSIONS: Exercise activates key stress signals that positively impact major transcriptional pathways that transcribe genes involved in skeletal muscle mitochondrial biogenesis, fusion and metabolism. The positive impact of exercise is not limited to younger healthy adults but also benefits skeletal muscle from diseased populations and the elderly. Impaired mitochondrial function can directly influence skeletal muscle atrophy and contribute to the risk or severity of disease conditions. Pharmacological manipulation of exercise-induced pathways that increase skeletal muscle mitochondrial biogenesis and function in critically ill patients, where exercise may not be possible, may assist in the treatment of chronic disease. GENERAL SIGNIFICANCE: This review highlights our understanding of how exercise positively impacts skeletal muscle mitochondrial biogenesis and function. Exercise not only improves skeletal muscle mitochondrial health but also enables us to identify molecular mechanisms that may be attractive targets for therapeutic manipulation. This article is part of a Special Issue entitled Frontiers of mitochondrial research.

Exercise as an intervention to improve metabolic outcomes after intrauterine growth restriction.

Individuals born after intrauterine growth restriction (IUGR) are at an increased risk of developing diabetes in their adult life. IUGR impairs ß-cell function and reduces ß-cell mass, thereby diminishing insulin secretion. IUGR also induces insulin resistance, with impaired insulin signaling in muscle in adult humans who were small for gestational age (SGA) and in rodent models of IUGR. There is epidemiological evidence in humans that exercise in adults can reduce the risk of metabolic disease following IUGR. However, it is not clear whether adult IUGR individuals benefit to the same extent from exercise as do normal-birth-weight individuals, as our rat studies suggest less of a benefit in those born IUGR. Importantly, however, there is some evidence from studies in rats that exercise in early life might be able to reverse or reprogram the long-term metabolic effects of IUGR. Studies are needed to address gaps in current knowledge, including determining the mechanisms involved in the reprogramming effects of early exercise in rats, whether exercise early in life or in adulthood has similar beneficial metabolic effects in larger animal models in which insulin resistance develops after IUGR. Human studies are also needed to determine whether exercise training improves insulin secretion and insulin sensitivity to the same extent in IUGR adults as in control populations. Such investigations will have implications for customizing the recommended level and timing of exercise to improve metabolic health after IUGR.

A gene therapy targeting medium-chain acyl-CoA dehydrogenase (MCAD) did not protect against diabetes-induced cardiac pathology.

Diabetic cardiomyopathy describes heart disease in patients with diabetes who have no other cardiac conditions but have a higher risk of developing heart failure. Specific therapies to treat the diabetic heart are limited. A key mechanism involved in the progression of diabetic cardiomyopathy is dysregulation of cardiac energy metabolism. The aim of this study was to determine if increasing the expression of medium-chain acyl-coenzyme A dehydrogenase (MCAD; encoded by Acadm), a key regulator of fatty acid oxidation, could improve the function of the diabetic heart. Male mice were administered streptozotocin to induce diabetes, which led to diastolic dysfunction 8 weeks post-injection. Mice then received cardiac-selective adeno-associated viral vectors encoding MCAD (rAAV6:MCAD) or control AAV and were followed for 8 weeks. In the non-diabetic heart, rAAV6:MCAD increased MCAD expression (mRNA and protein) and increased Acadl and Acadvl, but an increase in MCAD enzyme activity was not detectable. rAAV6:MCAD delivery in the diabetic heart increased MCAD mRNA expression but did not significantly increase protein, activity, or improve diabetes-induced cardiac pathology or molecular metabolic and lipid markers. The uptake of AAV viral vectors was reduced in the diabetic versus non-diabetic heart, which may have implications for the translation of AAV therapies into the clinic. KEY MESSAGES: The effects of increasing MCAD in the diabetic heart are unknown. Delivery of rAAV6:MCAD increased MCAD mRNA and protein, but not enzyme activity, in the non-diabetic heart. Independent of MCAD enzyme activity, rAAV6:MCAD increased Acadl and Acadvl in the non-diabetic heart. Increasing MCAD cardiac gene expression alone was not sufficient to protect against diabetes-induced cardiac pathology. AAV transduction efficiency was reduced in the diabetic heart, which has clinical implications.