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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.

Galectin-3 (Gal-3) is a galactose-binding lectin involved in pathologies such as inflammation, fibrosis, heart disease, and tumor progression. Here, we report N-aryl-N-(thio)lactosylamides as a novel class of Gal-3 inhibitors. A structure-activity study identified 6-carboxyindol-4-yl amide as a key pharmacophoric motif within this series. The most potent inhibitor based on this motif, compound 11, binds to Gal-3 with excellent affinity (K_d = 5.7 nM) and selectivity (390-fold over Gal-1). Further in vitro characterization of this compound demonstrated high metabolic stability and no cytotoxicity (CC₅₀ > 300 μM). Compound 11 effectively engages Gal-3 with greater activity in macrophage-like than monocyte-like THP1 cells, without affecting inflammation via LPS-induced release of TNFα. In TGFβ-stimulated LX2 hepatic stellate cells, it downregulates profibrotic signaling as assessed by the reduced expression of ACTA2, COL1A2, and FN1. These findings implicate compound 11 as a promising candidate for further preclinical development in the context of fibrotic disease.

Objectives: Here we use skeletal muscle fiber composition to investigate whether defects in amino acid metabolism are involved in the early development of IR in healthy young individuals before the onset of clinical manifestations.

Design: Two groups consisting of healthy young men and women, insulin-sensitive and insulin-resistant, were studied using a cross-sectional design.

Methods: Biopsies were obtained from the vastus lateralis muscle, and an intravenous glucose tolerance test was performed. Plasma and muscle tissue were analyzed by metabolomics. Results Subjects in group 1 (n = 20; age 28 ± 5 years; body mass index 22.3 ± 2.7 kg/m2) had an expression of type I muscle fibers and whole-body insulin sensitivity of 58.8% ± 5.7% and 1.8 ± 0.7 units, respectively. Subjects in group 2 (n = 16; age 25 ± 6 years; body mass index 22.6 ± 3.0 kg/m2) had an expression of type I muscle fibers and whole-body insulin sensitivity, respectively, of 29.8% ± 6.6% and 0.8 ± 0.3 units (P < .001 vs group 1 for both). Anserine and β-alanine contents in muscle were significantly higher and taurine lower in group 2 vs 1, consistent with the differences in muscle fiber composition between groups. Taurine correlated well with insulin sensitivity and expression of type I muscle fibers (r = 0.63; P < .001 for both). In contrast, there were no significant differences in plasma or tissue contents of glutamine, arginine, or branched-chain amino acids between groups.

Conclusions: These data demonstrate that the early development of IR is not a consequence of defects in amino acid metabolism. Rather, defects in amino acid metabolism in diseased states are more likely a consequence of IR.