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Protein phosphorylation dynamically integrates environmental and cellular information to control biological processes. Identifying functional phosphorylation amongst the thousands of phosphosites regulated by a perturbation at a global scale is a major challenge. Here we introduce ‘personalized phosphoproteomics’, a combination of experimental and computational analyses to link signaling with biological function by utilizing human phenotypic variance. We measure individual subject phosphoproteome responses to interventions with corresponding phenotypes measured in parallel. Applying this approach to investigate how exercise potentiates insulin signaling in human skeletal muscle, we identify both known and previously unidentified phosphosites on proteins involved in glucose metabolism. This includes a cooperative relationship between mTOR and AMPK whereby the former directly phosphorylates the latter on S377, for which we find a role in metabolic regulation. These results establish personalized phosphoproteomics as a general approach for investigating the signal transduction underlying complex biology. Functionally relevant phosphorylation sites are detected by integrating phosphoproteomic and phenotypic data.

Objective The role of selective β2-adrenergic stimulation in regulation of leg glucose uptake and free fatty acid (FFA) balance is inadequately explored in humans. The objective of this study was to investigate β2-adrenergic effects on net leg glucose uptake and clearance, as well as FFA balance at rest and during exercise. Design The study was a randomized, placebo-controlled crossover trial where 10 healthy men received either infusion of β2-agonist terbutaline (0.2 to 0.4 mg) or placebo. Net leg glucose uptake and clearance and FFA balance were determined at rest and during 8 minutes of knee extensor exercise using Fick’s principle. Vastus lateralis muscle biopsies were collected at rest and at cessation of exercise. The primary outcome measure was net leg glucose uptake. Results At rest, net leg glucose uptake and clearance were 0.35 (±0.16) mmol/min and 41 (±17) mL/min (mean ± 95% CI) higher (P < 0.001) for terbutaline than placebo, corresponding to increases of 84% and 70%. During exercise, no treatment differences were observed in net leg glucose uptake, whereas clearance was 101 (±86) mL/min lower (P < 0.05) for terbutaline than placebo. At rest, terbutaline induced a net leg FFA release of 21 (±14) µmol/min, being different from placebo (P = 0.04). During exercise, net leg FFA uptake was not different between the treatments. Conclusions These observations indicate that β2-agonist alters net leg glucose uptake and clearance, as well as FFA balance in humans, which is associated with myocellular β2-adrenergic and insulin-dependent signaling. Furthermore, the study shows that exercise confounds the β2-adrenergic effect on net leg glucose uptake and FFA balance.

Context: Given the promising effects of prolonged treatment with [beta.sub.2]-agonist on insulin sensitivity in animals and nondiabetic individuals, the [beta.sub.2]-adrenergic receptor has been proposed as a target to counter peripheral insulin resistance. On the other hand, rodent studies also reveal that [beta.sub.2]-agonists acutely impair insulin action, posing a potential caveat for their use in treating insulin resistance. Objective: To assess the impact of [beta.sub.2]-agonist on muscle insulin action and glucose metabolism and identify the underlying mechanism(s) in 10 insulin-resistant subjects. Methods and participants: In a crossover design, we assessed the effect of [beta.sub.2]-agonist on insulin-stimulated muscle glucose uptake during a 3-hour hyperinsulinemic isoglycemic clamp with and without intralipid infusion in 10 insulin-resistant, overweight subjects. Two hours into the clamp, we infused [beta.sub.2]-agonist. We collected muscle biopsies before, 2 hours into, and by the end of the clamp and analyzed them using metabolomic and lipidomic techniques. Results: We establish that [beta.sub.2]-agonist, independently from and additively to intralipid, impairs insulin-stimulated muscle glucose uptake via different mechanisms. In combination, [beta.sub.2]-agonist and intralipid nearly eliminates insulin-dependent muscle glucose uptake. Although both [beta.sub.2]-agonist and intralipid elevated muscle glucose-6-phosphate, only intralipid caused accumulation of downstream muscle glycolytic intermediates, whereas [beta.sub.2]-agonist attenuated incorporation of glucose into glycogen. Conclusion: Our findings suggest that [beta.sub.2]-agonist inhibits glycogenesis, whereas intralipid inhibits glycolysis in skeletal muscle of insulin-resistant individuals. These results should be addressed in future treatment of insulin resistance with [beta.sub.2]-agonist. Key Words: [beta.sub.2]-adrenergic agonists, muscle glucose uptake, intralipid, glycogenesis, glucose-6-phosphate, insulin resistance

Abstract Context Given the promising effects of prolonged treatment with beta2-agonist on insulin sensitivity in animals and nondiabetic individuals, the beta2-adrenergic receptor has been proposed as a target to counter peripheral insulin resistance. On the other hand, rodent studies also reveal that beta2-agonists acutely impair insulin action, posing a potential caveat for their use in treating insulin resistance. Objective To assess the impact of beta2-agonist on muscle insulin action and glucose metabolism and identify the underlying mechanism(s) in 10 insulin-resistant subjects. Methods and participants In a crossover design, we assessed the effect of beta2-agonist on insulin-stimulated muscle glucose uptake during a 3-hour hyperinsulinemic isoglycemic clamp with and without intralipid infusion in 10 insulin-resistant, overweight subjects. Two hours into the clamp, we infused beta2-agonist. We collected muscle biopsies before, 2 hours into, and by the end of the clamp and analyzed them using metabolomic and lipidomic techniques. Results We establish that beta2-agonist, independently from and additively to intralipid, impairs insulin-stimulated muscle glucose uptake via different mechanisms. In combination, beta2-agonist and intralipid nearly eliminates insulin-dependent muscle glucose uptake. Although both beta2-agonist and intralipid elevated muscle glucose-6-phosphate, only intralipid caused accumulation of downstream muscle glycolytic intermediates, whereas beta2-agonist attenuated incorporation of glucose into glycogen. Conclusion Our findings suggest that beta2-agonist inhibits glycogenesis, whereas intralipid inhibits glycolysis in skeletal muscle of insulin-resistant individuals. These results should be addressed in future treatment of insulin resistance with beta2-agonist.

Objective: The role of selective [[beta] sub.2]-adrenergic stimulation in regulation of leg glucose uptake and free fatty acid (FFA) balance is inadequately explored in humans. The objective of this study was to investigate [[beta] sub.2]-adrenergic effects on net leg glucose uptake and clearance, as well as FFA balance at rest and during exercise. Design: The study was a randomized, placebo-controlled crossover trial where 10 healthy men received either infusion of [[beta] sub.2]-agonist terbutaline (0.2 to 0.4 mg) or placebo. Net leg glucose uptake and clearance and FFA balance were determined at rest and during 8 minutes of knee extensor exercise using Fick's principle. Vastus lateralis muscle biopsies were collected at rest and at cessation of exercise. The primary outcome measure was net leg glucose uptake. Results: At rest, net leg glucose uptake and clearance were 0.35 ([+ or -]0.16) mmol/min and 41 ([+ or -]17) mL/min (mean [+ or -] 95% CI) higher (P < 0.001) for terbutaline than placebo, corresponding to increases of 84% and 70%. During exercise, no treatment differences were observed in net leg glucose uptake, whereas clearance was 101 ([+ or -] 86) mL/min lower (P < 0.05) for terbutaline than placebo. At rest, terbutaline induced a net leg FFA release of 21 ([+ or -]14) [micro]mol/min, being different from placebo (P = 0.04). During exercise, net leg FFA uptake was not different between the treatments. Conclusions: These observations indicate that [[beta].sub.2]-agonist alters net leg glucose uptake and clearance, as well as FFA balance in humans, which is associated with myocellular [[beta].sub.2]-adrenergic and insulin-dependent signaling. Furthermore, the study shows that exercise confounds the [[beta].sub.2]-adrenergic effect on net leg glucose uptake and FFA balance. (J Clin Endocrinol Metab 104: 647-657, 2019)

A single bout of exercise improves muscle insulin sensitivity for up to 48 hours via AMPK. Limb ischemia activates AMPK in muscle, and subsequent reperfusion enhances insulin-stimulated vasodilation, potentially eliciting a more pronounced exercise effect with reduced workload. We investigated the combined effect of upper leg intermittent ischemia/reperfusion (IIR) and continuous knee-extension exercise on muscle insulin sensitivity regulation. We found that IIR exercise potentiated AMPK activation and muscle insulin sensitivity. The potentiating effect of ПВ exercise on muscle insulin sensitivity was associated with increased insulin-stimulated blood flow in parallel with enhanced phosphorylation of endothelial nitric oxide synthase. Metabolomics analyses demonstrated a suppression of muscle medium-chain acylcarnitines during IIR exercise, which correlated with insulin sensitivity and was consistent with findings in isolated rat muscle treated with decanoyl-.carnitine. Collectively, combining IIR with low- to moderate-intensity exercise may represent a promising intervention to effectively enhance muscle insulin sensitivity. This approach could offer potential for mitigating muscle insulin resistance in clinical settings and among individuals with lower physical activity levels.

Purpose β 2 -Agonists have been proposed as weight-loss treatment, because they elevate energy expenditure. However, it is unknown what effect β 2 -agonists have on energy expenditure in overweight individuals. Furthermore, the influence of β 2 -agonist R- and S-enantiomer ratio for the increased energy expenditure is insufficiently explored. Methods Nineteen males were included in the study of which 14 completed. Subjects were 31.6 (±3.5) years [mean (±95% CI)] and had a fat percentage of 22.7 (±2.1)%. On separate days, subjects received either placebo or inhaled racemic ( rac -) formoterol (2 × 27 µg). After an overnight fast, energy expenditure and substrate oxidation were estimated by indirect calorimetry at rest and during submaximal exercise. Plasma ( R , R )- and ( S , S )-formoterol enantiomer levels were measured by ultra-performance liquid chromatograph–mass spectrometry. Results At rest, energy expenditure and fat oxidation were 12% ( P  ≤ 0.001) and 38% ( P  = 0.006) higher for rac -formoterol than placebo. Systemic ( R , R ):( S , S ) formoterol ratio was correlated with change in energy expenditure at rest in response to rac -formoterol ( r  = 0.63, P  = 0.028), whereas no association was observed between fat percentage and rac -formoterol-induced change in energy expenditure. During exercise, energy expenditure was not different between treatments, although carbohydrate oxidation was 15% higher ( P  = 0.021) for rac -formoterol than placebo. Rac -formoterol-induced shift in substrate choice from rest to exercise was related to plasma ln- rac -formoterol concentrations ( r  = 0.75, P  = 0.005). Conclusion Selective β 2 -adrenoceptor agonism effectively increases metabolic rate and fat oxidation in overweight individuals. The potential for weight loss induced by β 2 -agonists may be greater for R -enantiopure formulations.

A single bout of exercise improves muscle insulin sensitivity for up to 48 hours via AMPK. Limb ischemia activates AMPK in muscle, and subsequent reperfusion enhances insulin-stimulated vasodilation, potentially eliciting a more pronounced exercise effect with reduced workload. We investigated the combined effect of upper leg intermittent ischemia/reperfusion (IIR) and continuous knee-extension exercise on muscle insulin sensitivity regulation. We found that IIR exercise potentiated AMPK activation and muscle insulin sensitivity. The potentiating effect of IIR exercise on muscle insulin sensitivity was associated with increased insulin-stimulated blood flow in parallel with enhanced phosphorylation of endothelial nitric oxide synthase. Metabolomics analyses demonstrated a suppression of muscle medium-chain acylcarnitines during IIR exercise, which correlated with insulin sensitivity and was consistent with findings in isolated rat muscle treated with decanoyl-l-carnitine. Collectively, combining IIR with low- to moderate-intensity exercise may represent a promising intervention to effectively enhance muscle insulin sensitivity. This approach could offer potential for mitigating muscle insulin resistance in clinical settings and among individuals with lower physical activity levels.

Purpose [beta]2-Agonists have been proposed as weight-loss treatment, because they elevate energy expenditure. However, it is unknown what effect [beta]2-agonists have on energy expenditure in overweight individuals. Furthermore, the influence of [beta]2-agonist R- and S-enantiomer ratio for the increased energy expenditure is insufficiently explored. Methods Nineteen males were included in the study of which 14 completed. Subjects were 31.6 (±3.5) years [mean (±95% CI)] and had a fat percentage of 22.7 (±2.1)%. On separate days, subjects received either placebo or inhaled racemic (rac-) formoterol (2 × 27 µg). After an overnight fast, energy expenditure and substrate oxidation were estimated by indirect calorimetry at rest and during submaximal exercise. Plasma (R,R)- and (S,S)-formoterol enantiomer levels were measured by ultra-performance liquid chromatograph-mass spectrometry. Results At rest, energy expenditure and fat oxidation were 12% (P [less than or equal to] 0.001) and 38% (P = 0.006) higher for rac-formoterol than placebo. Systemic (R,R):(S,S) formoterol ratio was correlated with change in energy expenditure at rest in response to rac-formoterol (r = 0.63, P = 0.028), whereas no association was observed between fat percentage and rac-formoterol-induced change in energy expenditure. During exercise, energy expenditure was not different between treatments, although carbohydrate oxidation was 15% higher (P = 0.021) for rac-formoterol than placebo. Rac-formoterol-induced shift in substrate choice from rest to exercise was related to plasma ln-rac-formoterol concentrations (r = 0.75, P = 0.005). Conclusion Selective [beta]2-adrenoceptor agonism effectively increases metabolic rate and fat oxidation in overweight individuals. The potential for weight loss induced by [beta]2-agonists may be greater for R-enantiopure formulations.

In the Greenlandic Inuit population, 4% are homozygous carriers of a genetic nonsense TBC1D4 p.Arg684Ter variant leading to loss of the muscle-specific isoform of TBC1D4 and an approximately tenfold increased risk of type 2 diabetes 1 . Here we show the metabolic consequences of this variant in four female and four male homozygous carriers and matched controls. An extended glucose tolerance test reveals prolonged hyperglycaemia followed by reactive hypoglycaemia in the carriers. Whole-body glucose disposal is impaired during euglycaemic-hyperinsulinaemic clamp conditions and associates with severe insulin resistance in skeletal muscle only. Notably, a marked reduction in muscle glucose transporter GLUT4 and associated proteins is observed. While metabolic regulation during exercise remains normal, the insulin-sensitizing effect of a single exercise bout is compromised. Thus, loss of the muscle-specific isoform of TBC1D4 causes severe skeletal muscle insulin resistance without baseline hyperinsulinaemia. However, physical activity can ameliorate this condition. These observations offer avenues for personalized interventions and targeted preventive strategies. In Greenlandic Inuit, a TBC1D4 loss-of-function mutation increases type 2 diabetes risk by tenfold. Carriers show severe muscle insulin resistance, impaired glucose disposal and reduced muscle GLUT4, yet exercise mitigates these defects, offering potential for personalized lifestyle interventions.