AMPK activated protein kinase (AMPK), a master regulator of energy homeostasis, is activated in response to an energy shortage imposed by physical activity and caloric restriction. We here report on the identification of PAN-AMPK activator O304, which — in diet-induced obese mice — increased glucose uptake in skeletal muscle, reduced β cell stress, and promoted β cell rest. Accordingly, O304 reduced fasting plasma glucose levels and homeostasis model assessment of insulin resistance (HOMA-IR) in a proof-of-concept phase IIa clinical trial in type 2 diabetes (T2D) patients on Metformin. T2D is associated with devastating micro- and macrovascular complications, and O304 improved peripheral microvascular perfusion and reduced blood pressure both in animals and T2D patients. Moreover, like exercise, O304 activated AMPK in the heart, increased cardiac glucose uptake, reduced cardiac glycogen levels, and improved left ventricular stroke volume in mice, but it did not increase heart weight in mice or rats. Thus, O304 exhibits a great potential as a novel drug to treat T2D and associated cardiovascular complications.

Altered ceramide accumulation contributes to skeletal muscle insulin resistance, but mechanisms underlying fibre-type-specific susceptibility remain unclear. We hypothesized that fibre-type-specific ceramide metabolism governs vulnerability to lipid-induced insulin resistance. Lipidomics and quantification of ceramide-pathway enzymes were performed in mouse skeletal muscles with distinct fibre-type composition (oxidative, mixed and glycolytic) from control-diet (n = 12) and high-fat-diet (HFD; n = 12) mice. In humans, lipidomics and enzyme profiling were done in vastus lateralis biopsies from 36 adults stratified into oxidative or glycolytic phenotypes; insulin sensitivity was determined by glucose tolerance testing. siRNA-mediated silencing of SGMS1 and SGMS2 followed by lipidomics probed sphingomyelin-ceramide cycling in human myoblasts. In mouse muscle, ceramide composition rather than total content, differed by fibre type: oxidative muscle was enriched in very-long-chain ceramides, whereas glycolytic and mixed muscles contained higher C18-ceramides, paralleled by fibre-type-specific expression of enzymes involved in de novo synthesis and sphingomyelin-ceramide cycling. HFD induced ceramide remodelling, with C18-ceramides accumulating in oxidative and mixed muscles and very-long-chain species decreasing in glycolytic muscle; among all assessed enzymes, only SGMS2 was significantly downregulated in oxidative muscle. In humans, an oxidative phenotype associated with higher very-long-chain ceramides and insulin sensitivity, whereas a glycolytic phenotype displayed higher C16-18 ceramides, higher SGMS1 and SMPD2 expression, and lower insulin sensitivity. Elastic net regression identified C16-18 ceramides and galactosylceramides as negative predictors of insulin sensitivity. SGMS2 silencing caused broader ceramide accumulation than SGMS1 silencing, supporting a central role for SGMS2-mediated sphingomyelin-ceramide cycling in limiting ceramide burden.

Background: Circulating S-Klotho is increasingly recognized as an anti-aging protein linked to cardiovascular protection and metabolic regulation. Although exercise elevates S-Klotho, the dose–response threshold and metabolic determinants of this response in young adults with obesity, a population at heightened risk for cardiometabolic dysfunction, remain poorly characterized. To examine the acute effects of submaximal and maximal cycling on circulating S-Klotho in young adults with obesity and to identify key metabolic predictors of exercise-induced changes. Methods: Eighteen young (26 ± 3 years, BMI 30 ± 2 kg/m²) males and females completed incremental cycling to exhaustion. Serum S-Klotho was assessed at rest, after submaximal (SubMax), and post-maximal (Max) exercise. Covariates included cardiorespiratory fitness (VO_2max), iDXA body composition, HOMA-IR, and hsCRP. Predictor Screening and Partial Least Squares (PLS) regression with fivefold cross-validation were used to identify determinants of exercise-induced S-Klotho responses. Results: A significant acute increase in S-Klotho was observed (Main Effect of Time: F_(2,32) = 9.4, p = 0.0006). Post hoc analysis revealed an intensity-dependent threshold, with a significant surge occurring only between submaximal and maximal exertion (p = 0.021; Rest to Max: p = 0.0005). No significant sex differences or interactions were observed (p > 0.05). The PLS model explained 74% of the variance in S-Klotho changes, identifying systemic inflammation (Log₁₀[hsCRP]) and visceral adiposity as the primary predictive factors. Conclusions: Exhaustive exercise acutely increases circulating S-Klotho in young adults with obesity, independent of sex. This response is intensity-dependent and primarily driven by baseline metabolic risk factors. S-Klotho may represent an acute exercise-responsive biomarker linked to metabolic health in at-risk populations. These exploratory findings warrant confirmation in larger and adequately powered studies.