The Impact of Sex and 25(OH)D Deficiency on Metabolic Function in Mice.

Both dietary fat and vitamin D deficiency have been linked with increased incidence of non-alcoholic fatty liver disease and insulin resistance. While sex differences in disease prevalence and severity are well known, the impact on disease pathogenesis remains unclear. To further explore the effect of these exposures on metabolic function, C57BL/6 male and female mice were weaned onto one of four diets; low fat vitamin D replete, low fat vitamin D deficient, or two high fat diets, one vitamin D replete and one deficient. Visceral fat, hepatic adiposity, and insulin resistance were measured after five and a half weeks. Vitamin D deficiency, independent of dietary fat, increased hepatic fat accumulation in both sexes (p = 0.003), although did not increase hepatic expression of interleukin-6 (p = 0.92) or tumor necrosis factor-α (p = 0.78). Males were observed to have greater insulin resistance (glucose area under the curve: p < 0.001, homeostatic model assessment for insulin resistance: p = 0.046), and have greater visceral adiposity (p < 0.001), while female mice had greater hepatic fat accumulation (p < 0.001). This study is the first to demonstrate vitamin D deficiency alone can cause hepatic accumulation while also being the first to observe higher liver fat percentages in female mice.

Skeletal Muscle Microvascular-Linked Improvements in Glycemic Control From Resistance Training in Individuals With Type 2 Diabetes.

OBJECTIVE: Insulin increases glucose disposal in part by enhancing microvascular blood flow (MBF) and substrate delivery to myocytes. Insulin's microvascular action is impaired with insulin resistance and type 2 diabetes. Resistance training (RT) improves glycemic control and insulin sensitivity, but whether this improvement is linked to augmented skeletal muscle microvascular responses in type 2 diabetes is unknown. RESEARCH DESIGN AND METHODS: Seventeen (11 male and 6 female; 52 ± 2 years old) sedentary patients with type 2 diabetes underwent 6 weeks of whole-body RT. Before and after RT, participants who fasted overnight had clinical chemistries measured (lipids, glucose, HbA1c, insulin, and advanced glycation end products) and underwent an oral glucose challenge (OGC) (50 g × 2 h). Forearm muscle MBF was assessed by contrast-enhanced ultrasound, skin MBF by laser Doppler flowmetry, and brachial artery flow by Doppler ultrasound at baseline and 60 min post-OGC. A whole-body DEXA scan before and after RT assessed body composition. RESULTS: After RT, muscle MBF response to the OGC increased, while skin microvascular responses were unchanged. These microvascular adaptations were accompanied by improved glycemic control (fasting blood glucose, HbA1c, and glucose area under the curve [AUC] during OGC) and increased lean body mass and reductions in fasting plasma triglyceride, total cholesterol, advanced glycation end products, and total body fat. Changes in muscle MBF response after RT significantly correlated with reductions in fasting blood glucose, HbA1c, and OGC AUC with adjustment for age, sex, % body fat, and % lean mass. CONCLUSIONS: RT improves OGC-stimulated muscle MBF and glycemic control concomitantly, suggesting that MBF plays a role in improved glycemic control from RT.

Determination of Skeletal Muscle Microvascular Flowmotion with Contrast-Enhanced Ultrasound.

Most methods of assessing flowmotion (rhythmic oscillation of blood flow through tissue) are limited to small sections of tissue and are invasive in tissues other than skin. To overcome these limitations, we adapted the contrast-enhanced ultrasound (CEUS) technique to assess microvascular flowmotion throughout a large region of tissue, in a non-invasive manner and in real time. Skeletal muscle flowmotion was assessed in anaesthetised Sprague Dawley rats, using CEUS and laser Doppler flowmetry (LDF) for comparison. Wavelet transformation of CEUS and LDF data was used to quantify flowmotion. The α-adrenoceptor antagonist phentolamine was infused to predictably blunt the neurogenic component of flowmotion. Both techniques identified similar flowmotion patterns, validating the use of CEUS to assess flowmotion. This study demonstrates for the first time that the novel technique of CEUS can be adapted for determination of skeletal muscle flowmotion in large regions of skeletal muscle.

Regulation of microvascular flow and metabolism: An overview.

Skeletal muscle is an important site for insulin to regulate blood glucose levels. It is estimated that skeletal muscle is responsible for ~80% of insulin-mediated glucose disposal in the post-prandial period. The classical action of insulin to increase muscle glucose uptake involves insulin binding to insulin receptors on myocytes to stimulate glucose transporter 4 (GLUT 4) translocation to the cell surface membrane, enhancing glucose uptake. However, an additional role of insulin that is often under-appreciated is its action to increase muscle perfusion thereby improving insulin and glucose delivery to myocytes. Either of these responses (myocyte and/or vascular) may be impaired in insulin resistance, and both impairments are apparent in type 2 diabetes, resulting in diminished glucose disposal by muscle. The aim of this review is to report on the growing body of literature suggesting that insulin-mediated control of skeletal muscle perfusion is an important regulator of muscle glucose uptake and that impairment of microvascular insulin action has important physiological consequences early in the pathogenesis of insulin resistance. This work was discussed at the 2015 Australian Physiological Society Symposium "Physiological mechanisms controlling microvascular flow and muscle metabolism".

Muscle microvascular blood flow responses in insulin resistance and ageing.

Insulin resistance plays a key role in the development of type 2 diabetes. Skeletal muscle is the major storage site for glucose following a meal and as such has a key role in maintenance of blood glucose concentrations. Insulin resistance is characterised by impaired insulin-mediated glucose disposal in skeletal muscle. Multiple mechanisms can contribute to development of muscle insulin resistance and our research has demonstrated an important role for loss of microvascular function within skeletal muscle. We have shown that insulin can enhance blood flow to the microvasculature in muscle thus improving the access of glucose and insulin to the myocytes to augment glucose disposal. Obesity, insulin resistance and ageing are all associated with impaired microvascular responses to insulin in skeletal muscle. Impairments in insulin-mediated microvascular perfusion in muscle can directly cause insulin resistance, and this event can occur early in the aetiology of this condition. Understanding the mechanisms involved in the loss of microvascular function in muscle has the potential to identify novel treatment strategies to prevent or delay progression of insulin resistance and type 2 diabetes.

No effect of NOS inhibition on skeletal muscle glucose uptake during in situ hindlimb contraction in healthy and diabetic Sprague-Dawley rats.

Nitric oxide (NO) has been shown to be involved in skeletal muscle glucose uptake during contraction/exercise, especially in individuals with Type 2 diabetes (T2D). To examine the potential mechanisms, we examined the effect of local NO synthase (NOS) inhibition on muscle glucose uptake and muscle capillary blood flow during contraction in healthy and T2D rats. T2D was induced in Sprague-Dawley rats using a combined high-fat diet (23% fat wt/wt for 4 wk) and low-dose streptozotocin injections (35 mg/kg). Anesthetized animals had one hindlimb stimulated to contract in situ for 30 min (2 Hz, 0.1 ms, 35 V) with the contralateral hindlimb rested. After 10 min, the NOS inhibitor, N(G)-nitro-l-arginine methyl ester (l-NAME; 5 µM) or saline was continuously infused into the femoral artery of the contracting hindlimb until the end of contraction. Surprisingly, there was no increase in skeletal muscle NOS activity during contraction in either group. Local NOS inhibition had no effect on systemic blood pressure or muscle contraction force, but it did cause a significant attenuation of the increase in femoral artery blood flow in control and T2D rats. However, NOS inhibition did not attenuate the increase in muscle capillary recruitment during contraction in these rats. Muscle glucose uptake during contraction was significantly higher in T2D rats compared with controls but, unlike our previous findings in hooded Wistar rats, NOS inhibition had no effect on glucose uptake during contraction. In conclusion, NOS inhibition did not affect muscle glucose uptake during contraction in control or T2D Sprague-Dawley rats, and this may have been because there was no increase in NOS activity during contraction.

Integrating muscle cell biochemistry and whole-body physiology in humans:(31)P-MRS data from the InSight trial.

We acquired (31)P-MRS data from skeletal muscle of subjects of mixed gender and ethnicity, combined with a panel of physiological characteristics, and tested several long-standing hypotheses regarding relationships between muscle cell biochemistry and whole-body physiology with unusually high statistical power. We hypothesized that i) whole-body VO(2)max would correlate with muscle respiratory capacity, ii) resting muscle phosphocreatine concentration ([PCr]) would negatively correlate with delta efficiency and iii) muscle mitochondrial function would positively correlate with both resting VO(2) and total daily energy expenditure (TDEE). Muscle respiratory capacity explained a quarter of the variation in VO(2)max (r(2) = 26, p < .001, n = 87). There was an inverse correlation between muscle [PCr] and delta efficiency (r = -23, p = 046, n = 87). There was also a correlation between [PCr] recovery halftime and TDEE (r = -23, p = 035, n = 87). Our data not only provide insights into muscle cell chemistry and whole-body physiology but our mixed cohort means that our findings are broadly generalizable.

The reproducibility of 31-phosphorus MRS measures of muscle energetics at 3 Tesla in trained men.

OBJECTIVE: Magnetic resonance spectroscopy (MRS) provides an exceptional opportunity for the study of in vivo metabolism. MRS is widely used to measure phosphorus metabolites in trained muscle, although there are no published data regarding its reproducibility in this specialized cohort. Thus, the aim of this study was to assess the reproducibility of (31)P-MRS in trained skeletal muscle. METHODS: We recruited fifteen trained men (VO(2)peak = 4.7±0.8 L min(-1)/58±8 mL kg(-1) min(-1)) and performed duplicate MR experiments during plantar flexion exercise, three weeks apart. RESULTS: Measures of resting phosphorus metabolites were reproducible, with 1.7 mM the smallest detectable difference in phosphocreatine (PCr). Measures of metabolites during exercise were less reliable: exercising PCr had a coefficient of variation (CV) of 27% during exercise, compared with 8% at rest. Estimates of mitochondrial function were variable, but experimentally useful. The CV of PCr(1/2t) was 40%, yet much of this variance was inter-subject such that differences of <20% were detectable with n = 15, given a significance threshold of p<0.05. CONCLUSIONS: 31-phosphorus MRS provides reproducible and experimentally useful measures of phosphorus metabolites and mitochondrial function in trained human skeletal muscle.

Decreased microvascular vasomotion and myogenic response in rat skeletal muscle in association with acute insulin resistance.

In addition to increased glucose uptake, insulin action is associated with increased total and microvascular blood flow, and vasomotion in skeletal muscle. The aim of this study was to determine the effect of acute insulin resistance caused by the peripheral vasoconstrictor alpha-methylserotonin (alphaMT) on microvascular vasomotion in muscle. Heart rate (HR), mean arterial pressure (MAP), femoral blood flow (FBF), whole body glucose infusion (GIR) and hindleg glucose uptake (HGU) were determined during control and hyperinsulinaemic euglycaemic clamp conditions in anaesthetized rats receiving alphaMT infusion. Changes in muscle microvascular perfusion were measured by laser Doppler flowmetry (LDF) and vasomotion was assessed by applying wavelet analysis to the LDF signal. Insulin increased GIR and HGU. Five frequency bands corresponding to cardiac, respiratory, myogenic, neurogenic and endothelial activities were detected in the LDF signal. Insulin infusion alone increased FBF (1.18 +/- 0.10 to 1.78 +/- 0.12 ml min(-1), P < 0.05), LDF signal strength (by 16% compared to baseline) and the relative amplitude of the myogenic component of vasomotion (0.89 +/- 0.09 to 1.18 +/- 0.06, P < 0.05). When infused alone alphaMT decreased LDF signal strength and the myogenic component of vasomotion by 23% and 27% respectively compared to baseline, but did not affect HGU or FBF. Infusion of alphaMT during the insulin clamp decreased the stimulatory effects of insulin on GIR, HGU, FBF and LDF signal and blocked the myogenic component of vasomotion. These data suggest that insulin action to recruit microvascular flow may in part involve action on the vascular smooth muscle to increase vasomotion in skeletal muscle to thereby enhance perfusion and glucose uptake. These processes are impaired with this model of alphaMT-induced acute insulin resistance.

Interleukin-6 attenuates insulin-mediated increases in endothelial cell signaling but augments skeletal muscle insulin action via differential effects on tumor necrosis factor-alpha expression.

OBJECTIVE: The cytokine interleukin-6 (IL-6) stimulates AMP-activated protein kinase (AMPK) and insulin signaling in skeletal muscle, both of which result in the activation of endothelial nitric oxide synthase (eNOS). We hypothesized that IL-6 promotes endothelial cell signaling and capillary recruitment in vivo, contributing to increased glucose uptake. RESEARCH DESIGN AND METHODS: The effect of IL-6 with and without insulin on AMPK, insulin, and eNOS signaling in and nitric oxide (NO) release from human aortic endothelial cells (HAECs) was examined. The physiological significance of these in vitro signaling events was assessed by measuring capillary recruitment in rats during control and euglycemic-hyperinsulinemic clamps with or without IL-6 infusion. RESULTS: IL-6 blunted increases in insulin signaling, eNOS phosphorylation (Ser1177), and NO production and reduced phosphorylation of AMPK in HAEC in vitro and capillary recruitment in vivo. In contrast, IL-6 increased Akt phosphorylation (Ser473) in hindlimb skeletal muscle and enhanced whole-body glucose disappearance and glucose uptake during the clamp. The differences in endothelial cell and skeletal muscle signaling were mediated by the cell-specific, additive effects of IL-6 and insulin because this treatment markedly increased tumor necrosis factor (TNF)-alpha protein expression in HAECs without any effect on TNF-alpha in skeletal muscle. When HAECs were incubated with a TNF-alpha-neutralizing antibody, the negative effects of IL-6 on eNOS signaling were abolished. CONCLUSIONS: In the presence of insulin, IL-6 contributes to aberrant endothelial cell signaling because of increased TNF-alpha expression.