Reduced adipocyte glutaminase activity promotes energy expenditure and metabolic health

Obesity-associated insulin resistance and type 2 diabetes feature broad alterations in circulating amino acids. Prior work indicates that specific amino acids can actively influence metabolic health; for example, brown adipose tissue activation increases branched-chain amino acid consumption to mitigate insulin resistance. In humans with obesity and insulin resistance, several amino acids are altered in white adipose tissue (WAT), yet how white adipocyte amino acid metabolism contributes to disease remains unclear. Glutamine and glutamate are pivotal in cellular biosynthesis, anaplerosis and epigenetic regulation, and their interconversion is controlled by multiple enzymes. A high plasma glutamine-to-glutamate ratio associates with lower type 2 diabetes risk, and WAT contributes to this ratio by releasing glutamine and taking up glutamate. The authors hypothesized that obesity perturbs WAT glutamine/glutamate turnover—via altered glutaminolysis in adipocytes—thereby lowering the glutamine-to-glutamate ratio and contributing to insulin resistance. They aimed to define how white adipocyte glutamine metabolism impacts energy expenditure and metabolic health by integrating human clinical cohorts, in vitro adipocyte models and in vivo mouse studies.

The study builds on evidence that circulating branched-chain and aromatic amino acids correlate with insulin resistance, while serine, asparagine and glycine correlate positively with insulin sensitivity. Prior findings show that WAT is a key contributor to the plasma glutamine-to-glutamate ratio and that a higher ratio is protective in humans and mice. BAT activation enhances BCAA utilization, influencing systemic metabolism. Glutamine/glutamate participate in nucleotide and glutathione biosynthesis, TCA anaplerosis, and epigenetic regulation, and their interconversion is catalyzed by enzymes including GLS (glutaminase) and GLUL (glutamine synthase). Inflammatory signaling (e.g., TNF) has been implicated in metabolic reprogramming and was explored here as a potential regulator of GLS/GLUL in adipocytes. Prior reports also link p38 MAPK signaling to thermogenic gene expression in beige/brown adipocytes, and lactate has been implicated in adipocyte browning and metabolic signaling. These themes contextualize a potential role of adipocyte glutaminolysis in metabolic disease.

Human clinical cohorts: Plasma and subcutaneous WAT were collected from women with and without obesity across a range of insulin sensitivity assessed by hyperinsulinaemic euglycaemic clamps (M/I). Targeted metabolomics quantified plasma and WAT amino acids and computed glutamine-to-glutamate ratios. WAT gene expression and protein abundance of glutaminolysis-related enzymes were measured by qPCR and western blot; enzyme activities were assayed. Associations with anthropometrics (BMI, waist-to-hip ratio), fat cell volume, energy expenditure (indirect calorimetry) and insulin sensitivity were evaluated using correlation analyses. In vitro human adipocytes: Primary human adipocyte progenitors were differentiated in vitro. GLS was depleted by siRNA electroporation; pharmacologic GLS inhibition used BPTES or CB-839. GLS activity and intracellular glutamine/glutamate levels were measured. RNA-seq profiled transcriptomic changes, with pathway analysis (GSEA) and thermogenic scoring (ProFAT). Mitochondrial ETC and UCP1 protein levels were assessed by western blot and immunofluorescence. Seahorse assays measured oxygen consumption rates (OCR) and extracellular acidification rates (ECAR). Glycolytic and substrate dependency were tested via glucose depletion, 2-deoxyglucose, UK5099, etomoxir, and pyruvate supplementation. Stable isotope tracing: [U-13C6]glucose incorporation into pyruvate, lactate and TCA intermediates; [U-13C]glutamine conversion with BPTES. HIF1α involvement was probed via protein measurement, target gene expression, α-ketoglutarate (αKG) quantification, αKG supplementation, and HIF1α inhibitor VI. Lactate levels were measured in media; redox (NAD+/NADH) assessed. p38 MAPK pathway activity (phospho-p38, CREB, ATF2) was evaluated with/without lactate and inhibited using SB203580. TNF regulation of GLS/GLUL was tested in isolated adipocytes; signaling inhibitors for JNK, STAT3, and NF-κB dissected pathways; c-Jun binding to GLS promoter was tested by ChIP–qPCR. Mouse studies: Wild-type male and female C57BL/6J mice were fed chow or high-fat diet (HFD). WAT and plasma glutamine/glutamate ratios and depot-specific Gls/Glul mRNA were measured. Pharmacologic inhibition: HFD-fed mice received CB-839 (200 mg kg−1 daily, 19 days) or vehicle; body weight and depot weights recorded; iWAT Ucp1 mRNA measured; insulin and HOMA-IR assessed. Glutamine supplementation (i.p.) tested systemic glutamine effects on iWAT gene expression. Genetic model: Adipocyte-specific Gls knockout (GlsAdipoqCre) mice were generated by crossing Gls-floxed with adiponectin-Cre mice. Gls depletion was validated in depots and mature adipocytes; GLS protein/activity and iWAT glutamine/glutamate ratio quantified. Body composition by EchoMRI; depot weights and adipocyte size by histology/image analysis; iWAT/UCP1, COX4, TOM20 immunofluorescence. Single-nucleus RNA-seq of iWAT and BAT identified adipocyte subtypes and Gls expression. Ex vivo high-resolution respirometry measured O2 consumption in iWAT and BAT. Whole-body indirect calorimetry assessed energy expenditure, with food intake and locomotor activity monitoring. Glucose tolerance tests (i.p.) were performed under chow and HFD conditions. Statistical analyses included t-tests, Mann–Whitney, ANOVA with multiple comparisons, and correlations as appropriate.

• In human cohorts, plasma and WAT glutamine-to-glutamate ratios were reduced in women with obesity and insulin resistance, strongly correlating with clamp-derived insulin sensitivity (M/I), and associated with waist-to-hip ratio and adipocyte size. Plasma and WAT ratios were tightly correlated.
• In isolated human adipocytes from people with obesity, GLUL mRNA/protein/activity were decreased while GLS was increased compared to non-obese controls, indicating enhanced glutaminolysis. In bulk WAT transcriptomics, GLS and GLUL levels inversely associated with BMI, energy expenditure, and insulin sensitivity.
• TNF negatively correlated with the GLUL/GLS ratio in WAT. TNF treatment increased GLS and decreased GLUL mRNA in adipocytes and lowered the glutamine-to-glutamate ratio; inhibition of JNK (but not STAT3 or NF-κB) blocked TNF-induced GLS protein elevation. ChIP–qPCR showed TNF increased c-Jun binding at the GLS promoter.
• GLS knockdown in human adipocytes (siGLS) decreased GLS activity, increased glutamine-to-glutamate ratio, and upregulated pathways for oxidative phosphorylation, TCA cycle, and fatty acid oxidation. ProFAT analysis indicated a more BAT-like/thermogenic transcriptome. ETC proteins and UCP1 were increased; rescue by α-ketoglutarate supplementation or GLS re-expression reversed the phenotype; inducible GLS overexpression decreased UCP1 and ETC proteins.
• Functional metabolism: siGLS increased basal and maximal OCR (mitochondrial respiration), dependent on UCP1 and glucose oxidation (reversed by glucose depletion, 2-DG, or UK5099; amplified by pyruvate). siGLS increased glucose uptake, glycolytic intermediates, ECAR, and [U-13C6]glucose labeling into pyruvate, lactate, and TCA metabolites; glutamine deprivation phenocopied OCR increases. Pharmacologic GLS inhibitors (BPTES, CB-839) replicated increased glycolysis, ETC proteins, OCR/ECAR.
• Mechanism: siGLS lowered α-ketoglutarate, stabilized HIF1α, and increased HIF1α target genes. αKG supplementation or HIF1α inhibition abrogated the increases in ECAR and OCR. siGLS and CB-839 elevated lactate production; p38 MAPK, CREB, and ATF2 phosphorylation increased with siGLS or lactate treatment. p38 inhibition (SB203580) blocked lactate- and siGLS-induced ETC protein increases, placing HIF1α–glycolysis–lactate upstream of p38-mediated mitochondrial activation.
• Mice: HFD increased WAT Gls and decreased Glul, lowering plasma and WAT glutamine-to-glutamate ratios in both sexes. CB-839 treatment (19 days) in HFD-fed mice (male and female) reduced total and fat mass, increased iWAT Ucp1 expression, and lowered insulin and HOMA-IR; i.p. glutamine did not induce iWAT browning genes, arguing against systemic glutamine buildup as sole driver.
• Adipocyte-specific Gls KO (male) reduced GLS and activity in WAT, increased iWAT glutamine-to-glutamate ratio, reduced fat mass and adipocyte size (iWAT and eWAT), and increased multilocular adipocytes in iWAT. Single-nucleus RNA-seq revealed an expanded beige adipocyte subpopulation in iWAT of KO mice. iWAT showed higher lactate and p38 phosphorylation; HIF1α target genes were upregulated.
• Ex vivo iWAT respiration was higher in KO mice; BAT respiration was unchanged. Whole-body energy expenditure increased without changes in food intake or activity; glucose tolerance improved on chow. Under HFD, KO mice retained higher iWAT/plasma glutamine-to-glutamate ratio, higher iWAT lactate, elevated energy expenditure, iWAT browning (UCP1/ETC proteins), reduced fat mass gain, and improved glucose tolerance compared to controls.
• Overall, white adipocyte GLS activity controls glutamine-to-glutamate stoichiometry, drives HIF1α–glycolysis–lactate–p38 signaling to enhance mitochondrial capacity and thermogenic programming, increasing energy expenditure and improving glucose homeostasis.

The study addresses whether altered white adipocyte glutamine metabolism contributes causally to metabolic dysfunction in obesity. The authors demonstrate that obesity-associated insulin resistance features a lower glutamine-to-glutamate ratio in plasma and WAT, paralleling increased GLS and decreased GLUL in adipocytes, consistent with heightened glutaminolysis. Mechanistically, reducing GLS activity in human adipocytes rewires metabolism toward glucose-driven glycolysis and oxidative phosphorylation, upregulating ETC components and UCP1. This reprogramming requires HIF1α stabilization due to reduced α-ketoglutarate, leading to elevated glycolysis and lactate production. Lactate then activates p38 MAPK signaling to induce thermogenic gene programs and mitochondrial capacity. In vivo, pharmacologic GLS inhibition or adipocyte-specific Gls deletion promotes iWAT browning, elevates tissue and whole-body energy expenditure, reduces adiposity, and improves glucose tolerance, including protection against HFD-induced metabolic derangements. The data substantiate a model in which adipocyte glutaminolysis is a modifiable node regulating systemic energy balance and insulin sensitivity, highlighting WAT’s contribution to circulating glutamine/glutamate stoichiometry as both a biomarker and mediator of metabolic health.

This work identifies adipocyte glutaminase (GLS) activity as a key determinant of white adipose tissue metabolism and whole-body energy expenditure. Obesity increases adipocyte glutaminolysis (higher GLS, lower GLUL), lowering the glutamine-to-glutamate ratio and associating with insulin resistance. Genetic or pharmacological reduction of GLS stabilizes HIF1α (via reduced α-ketoglutarate), elevates glycolysis and lactate, and activates p38 MAPK to induce a thermogenic, mitochondria-rich program in white adipocytes, particularly iWAT. In mice, this enhances energy expenditure, limits fat mass gain, and improves glucose tolerance even during HFD. These findings suggest that targeting adipocyte glutamine metabolism—e.g., via GLS inhibitors such as CB-839—may be a therapeutic strategy for obesity, insulin resistance and related conditions. Future studies should delineate sex-specific factors influencing gene targeting efficacy, resolve potential compartmentalized redox dynamics, and evaluate GLS inhibition in controlled clinical trials for metabolic disease.

• Sex-specific gene editing: Adipocyte-specific Gls deletion could not be achieved in mature adipocytes from female mice despite Cre expression, limiting genetic in vivo conclusions to males and pointing to sex-, age- or genotype-dependent Cre-loxP differences.
• Mechanistic complexity: Although evidence supports the αKG–HIF1α–glycolysis–lactate–p38 axis, interconnected pathways and temporal dynamics (e.g., organelle-specific redox/NAD+/NADH) were not fully resolved; measurements were largely endpoint in whole-cell lysates.
• Depot and environmental specificity: Browning was robust in iWAT but not eWAT or BAT; environmental conditions (cold vs thermoneutrality) could modulate phenotypes and were not systematically tested.
• Systemic factors: While glutamine supplementation did not induce iWAT browning, broader systemic adaptations to GLS inhibition (beyond adipocytes) may contribute and were not exhaustively dissected.
• Translational scope: Short-term CB-839 treatment in mice showed benefits; long-term efficacy, safety, and metabolic outcomes in humans require randomized clinical trials.