The Rho guanine dissociation inhibitor α inhibits skeletal muscle Rac1 activity and insulin action.

The molecular events governing skeletal muscle glucose uptake have pharmacological potential for managing insulin resistance in conditions such as obesity, diabetes, and cancer. With no current pharmacological treatments to target skeletal muscle insulin sensitivity, there is an unmet need to identify the molecular mechanisms that control insulin sensitivity in skeletal muscle. Here, the Rho guanine dissociation inhibitor α (RhoGDIα) is identified as a point of control in the regulation of insulin sensitivity. In skeletal muscle cells, RhoGDIα interacted with, and thereby inhibited, the Rho GTPase Rac1. In response to insulin, RhoGDIα was phosphorylated at S101 and Rac1 dissociated from RhoGDIα to facilitate skeletal muscle GLUT4 translocation. Accordingly, siRNA-mediated RhoGDIα depletion increased Rac1 activity and elevated GLUT4 translocation. Consistent with RhoGDIα's inhibitory effect, rAAV-mediated RhoGDIα overexpression in mouse muscle decreased insulin-stimulated glucose uptake and was detrimental to whole-body glucose tolerance. Aligning with RhoGDIα's negative role in insulin sensitivity, RhoGDIα protein content was elevated in skeletal muscle from insulin-resistant patients with type 2 diabetes. These data identify RhoGDIα as a clinically relevant controller of skeletal muscle insulin sensitivity and whole-body glucose homeostasis, mechanistically by modulating Rac1 activity.

Combining mass spectrometry and machine learning to discover bioactive peptides.

Peptides play important roles in regulating biological processes and form the basis of a multiplicity of therapeutic drugs. To date, only about 300 peptides in human have confirmed bioactivity, although tens of thousands have been reported in the literature. The majority of these are inactive degradation products of endogenous proteins and peptides, presenting a needle-in-a-haystack problem of identifying the most promising candidate peptides from large-scale peptidomics experiments to test for bioactivity. To address this challenge, we conducted a comprehensive analysis of the mammalian peptidome across seven tissues in four different mouse strains and used the data to train a machine learning model that predicts hundreds of peptide candidates based on patterns in the mass spectrometry data. We provide in silico validation examples and experimental confirmation of bioactivity for two peptides, demonstrating the utility of this resource for discovering lead peptides for further characterization and therapeutic development.

Human T Cells Expressing a CD19 CAR-T Receptor Provide Insights into Mechanisms of Human CD19-Positive ß Cell Destruction.

Autoimmune destruction of pancreatic ß cells underlies type 1 diabetes (T1D). To understand T cell-mediated immune effects on human pancreatic ß cells, we combine ß cell-specific expression of a model antigen, CD19, and anti-CD19 chimeric antigen receptor T (CAR-T) cells. Coculturing CD19-expressing ß-like cells and CD19 CAR-T cells results in T cell-mediated ß-like cell death with release of activated T cell cytokines. Transcriptome analysis of ß-like cells and human islets treated with conditioned medium of the immune reaction identifies upregulation of immune reaction genes and the pyroptosis mediator GSDMD as well as its activator CASP4. Caspase-4-mediated cleaved GSDMD is detected in ß-like cells under inflammation and endoplasmic reticulum (ER) stress conditions. Among immune-regulatory genes, PDL1 is one of the most upregulated, and PDL1 overexpression partially protects human ß-like cells transplanted into mice. This experimental platform identifies potential mechanisms of ß cell destruction and may allow testing of therapeutic strategies.

Semaglutide lowers body weight in rodents via distributed neural pathways.

Semaglutide, a glucagon-like peptide 1 (GLP-1) analog, induces weight loss, lowers glucose levels, and reduces cardiovascular risk in patients with diabetes. Mechanistic preclinical studies suggest weight loss is mediated through GLP-1 receptors (GLP-1Rs) in the brain. The findings presented here show that semaglutide modulated food preference, reduced food intake, and caused weight loss without decreasing energy expenditure. Semaglutide directly accessed the brainstem, septal nucleus, and hypothalamus but did not cross the blood-brain barrier; it interacted with the brain through the circumventricular organs and several select sites adjacent to the ventricles. Semaglutide induced central c-Fos activation in 10 brain areas, including hindbrain areas directly targeted by semaglutide, and secondary areas without direct GLP-1R interaction, such as the lateral parabrachial nucleus. Automated analysis of semaglutide access, c-Fos activity, GLP-1R distribution, and brain connectivity revealed that activation may involve meal termination controlled by neurons in the lateral parabrachial nucleus. Transcriptomic analysis of microdissected brain areas from semaglutide-treated rats showed upregulation of prolactin-releasing hormone and tyrosine hydroxylase in the area postrema. We suggest semaglutide lowers body weight by direct interaction with diverse GLP-1R populations and by directly and indirectly affecting the activity of neural pathways involved in food intake, reward, and energy expenditure.

Molecular Mechanisms in Skeletal Muscle Underlying Insulin Resistance in Women Who Are Lean With Polycystic Ovary Syndrome.

CONTEXT: Skeletal muscle molecular mechanisms underlying insulin resistance in women with polycystic ovary syndrome (PCOS) are poorly understood. OBJECTIVE: To provide insight into mechanisms regulating skeletal muscle insulin resistance in women who are lean with PCOS. PARTICIPANTS AND METHODS: A hyperinsulinemic-euglycemic clamp with skeletal muscle biopsies was performed. Thirteen women who are lean who have hyperandrogenism and PCOS and seven age- and body mass index-matched healthy control subjects were enrolled. Skeletal muscle protein expression and phosphorylation were analyzed by Western blotting and intramuscular lipid content was measured by thin-layer chromatography. RESULTS: Women with PCOS had 25% lower whole-body insulin sensitivity and 40% lower plasma adiponectin concentration than in control subjects. Intramuscular triacylglycerol, sn-1.3 diacylglycerol, and ceramide contents in skeletal muscle were higher (40%, 50%, and 300%, respectively) in women with PCOS than in control subjects. Activation of insulin signaling did not differ between groups. In women with PCOS, the insulin-stimulated glucose oxidation was reduced and insulin-stimulated dephosphorylation of pyruvate dehydrogenase (PDH) Ser293 was absent. AMP-activated protein kinase (AMPK) α2 protein expression and basal Thr172 phosphorylation were 45% and 50% lower in women with PCOS than in control subjects, respectively. CONCLUSIONS: Whole-body insulin resistance in women who are lean who have hyperandrogenism and PCOS was not related to changes in the proximal part of the insulin signaling cascade in skeletal muscle despite lipid accumulation. Rather, reduced insulin sensitivity was potentially related to plasma adiponectin levels playing a modulating role in human skeletal muscle via AMPK. Furthermore, abnormal PDH regulation may contribute to reduced whole-body metabolic flexibility and thereby insulin resistance.