ASK1 inhibits browning of white adipose tissue in obesity

Brown adipose tissue (BAT) and beige adipocytes dissipate energy via UCP1-mediated thermogenesis. In adult humans, limited BAT mass has shifted therapeutic interest toward inducing browning of white adipose tissue (WAT). Obesity is characterized by chronic low-grade adipose inflammation and elevated endotoxins/cytokines (e.g., TNFα, LPS), which have been reported to reduce UCP1 expression in adipocytes. Both TNFα and LPS activate apoptosis signal-regulating kinase 1 (ASK1), a MAP3K activated by inflammatory and oxidative stress and whose expression and activation are increased in adipose tissue in obesity and correlate with insulin resistance. The authors hypothesize that obesity-induced expression/activation of ASK1 in adipocytes negatively regulates UCP1 expression in WAT and thereby inhibits adipose browning, contributing to reduced energy expenditure and metabolic dysfunction.

Prior work established BAT-mediated thermogenesis and the capacity of WAT to acquire beige, UCP1+ characteristics upon β3-adrenergic stimulation, offering a potential anti-obesity strategy. Inflammatory mediators increased in obesity (TNFα, LPS) suppress UCP1 in adipocytes and attenuate adaptive thermogenesis. ASK1 is a stress-responsive MAP3K activated downstream of TLR4/LPS and other stressors, and is elevated in obese human adipose tissue where it predicts insulin resistance. These data led to the question whether ASK1 acts as a molecular brake on WAT browning in obesity. Additional studies identified neuronal PPARγ and other pathways (Notch, MKK6, PRDM16, LAMA4) as modulators of WAT browning, often without affecting BAT, underscoring depot-specific regulatory mechanisms. IRF3 has been implicated in promoting adipose inflammation, insulin resistance, and repressing browning, and can be activated downstream of LPS via ASK1-dependent signaling. Together, these findings frame ASK1–IRF3 signaling as a candidate pathway mediating inflammation- and obesity-linked suppression of WAT browning.

The study combined in vivo mouse genetics, metabolic phenotyping, tissue analyses, and in vitro adipocyte experiments. Mouse models: (1) Adipocyte-specific ASK1 knockout (ASK1Δadipo) generated by crossing ASK1 floxed mice (loxP-flanked exon 14) with Adipoq-Cre mice; deletion confirmed by Western blot in adipocytes/BAT; (2) Myeloid-specific ASK1 knockout (ASK1Δmye) using LysM-Cre; (3) Adipocyte-specific ASK1 overexpression (CAG-ASK1+adipo) with ASK1 cDNA inserted at Rosa26 under CAG promoter with loxP-stop cassette, activated by Adipoq-Cre. Diets and environmental challenges: mice fed chow or high-fat diet (HFD; 58% kcal fat) for up to 20 weeks; cold exposure (7 °C for 7 days) to induce WAT browning; thermoneutrality (30 °C for 20 days). LPS exposure: osmotic mini-pumps (300 µg/kg/day for 24 days) delivering LPS or saline; cold exposure followed to assess effects on browning. Metabolic phenotyping: body weight/weight gain; intraperitoneal glucose tolerance tests (ipGTT); hyperinsulinemic–euglycemic clamp studies with jugular catheterization to determine glucose infusion rate (GIR), endogenous glucose production (EGP), and tissue-specific glucose uptake (2-[14C]deoxyglucose); indirect calorimetry (VO2, heat production) at room temperature and thermoneutrality; rectal temperature. Tissue analyses: fat pad weights and total body fat mass; liver triglycerides (Bligh and Dyer extraction and enzymatic assay); immunohistochemistry for UCP1 in inguinal WAT; Western blotting for UCP1, PGC1α, ASK1, phospho-ASK1 (Thr845), phospho-IRF3; RT-qPCR for Ucp1, Cidea, Pgc1a, Prdm16, inflammatory markers (Tnfa, Il-6, Mcp1, Il-1β, Il-10, F4/80), Ask1; plasma measurements (insulin, cytokines, adiponectin, LPS). In vitro adipocyte studies: immortalized subcutaneous white preadipocytes differentiated to adipocytes; treatments included LPS (100 ng/ml, 24 h) and β-adrenergic agonists (isoproterenol 0.1 µM for 6 h) to induce Ucp1; lentiviral shRNA knockdown of Ask1 (shASK1) or Irf3 (shIRF3), and control shLuc; siRNA knockdown of ASK1 or IRF3; lentiviral overexpression of wild-type ASK1 or kinase-dead ASK1-K716R; assessment of Ucp1 mRNA and IRF3 phosphorylation. Biochemistry: in vitro kinase assay incubating recombinant IRF3 with active ASK1 to test direct phosphorylation. Statistics: two-sided t-tests, ANOVA with multiple comparisons, Mann–Whitney for non-parametric data; p<0.05 considered significant; data as mean±SEM.

• LPS suppresses WAT browning: Chronic LPS reduced cold-induced UCP1 protein in inguinal WAT of mice (n=3 per group; p=0.004) and blunted isoproterenol-induced Ucp1 mRNA in inguinal fat explants (n=3; p=0.036) and in cultured subcutaneous white adipocytes (~50% reduction; n=7–8; p=0.007). • ASK1 expression/activation increases with obesity and LPS: ASK1 protein levels were elevated in inguinal WAT of HFD vs chow mice (n=8 chow, n=7 HFD; p=0.037) with increased phospho-ASK1; LPS increased Ask1 mRNA in subcutaneous adipocytes (n=7; p=0.046) and ASK1 phosphorylation in vitro and in vivo. • ASK1 mediates LPS-induced suppression of Ucp1: ASK1 knockdown (shASK1) in adipocytes blunted LPS-mediated downregulation of isoproterenol-induced Ucp1 (n=11–12; p=0.027). • Adipocyte ASK1 deletion improves metabolic health on HFD: ASK1Δadipo mice gained less weight on HFD vs controls (p=0.02 to <0.001), with improved glucose tolerance (ipGTT; p=0.03 to <0.001). After 4 days HFD, glucose tolerance improved without body weight differences. Hyperinsulinemic–euglycemic clamps showed increased GIR (n=6–8; p=0.011), increased glucose uptake into inguinal WAT (p=0.034) and a trend in epididymal WAT (p=0.062), unchanged uptake in BAT and skeletal muscle, reduced EGP (p=0.040), and reduced hepatic triglycerides (p=0.012). Adipose inflammation markers and circulating cytokines were similar between genotypes. • Myeloid ASK1 deletion does not recapitulate benefits: ASK1Δmye mice showed no differences in body weight, fat mass, glucose tolerance, or insulin sensitivity on chow or HFD. • Energy expenditure increases with adipocyte ASK1 deletion: ASK1Δadipo mice exhibited higher VO2 at room temperature (light and dark; p=0.020, p=0.010) and at thermoneutrality (p=0.042 trend), increased heat production (p=0.009), and higher rectal temperature (p=0.015), without differences in food intake, activity, or reduced nutrient absorption. • WAT browning is enhanced by ASK1 deletion: HFD-fed ASK1Δadipo mice had reduced fat pad masses and ~30% lower total body fat; inguinal WAT appeared darker with stronger UCP1 immunostaining; inguinal WAT showed higher Ucp1 and Cidea mRNA (p=0.027, p=0.028) and higher UCP1 and PGC1α protein (p=0.031, p=0.015); BAT UCP1 was unchanged. LPS reduced UCP1 in cold-exposed controls but not in ASK1Δadipo mice (p=0.003). • Adipocyte ASK1 overexpression suppresses WAT browning: In chow-fed, cold-exposed CAG-ASK1+adipo mice, inguinal WAT had reduced Ucp1, Cidea, and Pgc1a mRNA (p=0.042, p=0.017; trend for Ucp1) and reduced UCP1 protein (p=0.037), with no change in BAT UCP1. • Mechanism via IRF3: Phospho-IRF3 was reduced in isolated adipocytes from ASK1Δadipo mice (n=6; p=0.048). Active ASK1 directly phosphorylated IRF3 in vitro (n=6; p=0.0006). Overexpressing wild-type vs kinase-dead ASK1 increased IRF3 phosphorylation (trend p=0.070). ASK1 siRNA blunted LPS-induced IRF3 phosphorylation (****p<0.0001). IRF3 knockdown (shIRF3 or siRNA) attenuated LPS-mediated suppression of Ucp1 in adipocytes (p=0.019), indicating ASK1 reduces Ucp1 via IRF3.

The data identify adipocyte ASK1 as a negative regulator of WAT browning in the context of obesity and inflammatory signaling. Elevated ASK1 expression and activation in adipocytes during HFD feeding or LPS exposure correlate with reduced UCP1 and browning markers in subcutaneous/inguinal WAT, reduced energy expenditure, and worse metabolic outcomes. Genetic ASK1 deletion in adipocytes enhances energy expenditure and browning without affecting BAT thermogenic protein levels, indicating a depot-selective effect on beige adipocytes. The improved systemic insulin sensitivity and reduced hepatic steatosis in ASK1Δadipo mice likely stem from increased fuel utilization due to browning, thereby lowering ectopic lipid deposition and hepatic insulin resistance. Mechanistically, ASK1 phosphorylates IRF3, and IRF3 is necessary for the LPS-induced suppression of Ucp1 in adipocytes; as p38 and JNK can modulate IRF3 activation, ASK1 may act both directly and via downstream kinases. The absence of metabolic benefits in myeloid-specific ASK1 deletion points to adipocytes as the critical cell type. Together, the findings suggest that in obesity, inflammatory and stress stimuli (TNFα, Fas ligand, oxidative stress, LPS) upregulate and activate ASK1 in adipocytes, acting as a brake on diet- or cold-induced thermogenesis by repressing UCP1 via IRF3, thus contributing to reduced diet-induced thermogenesis and metabolic dysfunction.

Adipocyte-expressed ASK1 suppresses the browning of white adipose tissue by phosphorylating IRF3 and reducing Ucp1 expression. In HFD-fed mice, adipocyte-specific ASK1 deletion increases WAT browning, elevates energy expenditure, limits weight gain, reduces hepatic steatosis, and improves glucose tolerance and insulin sensitivity, while ASK1 overexpression diminishes cold-induced WAT browning. The effects are selective for WAT, with BAT thermogenic protein levels largely unaffected under chronic conditions. These findings position adipocyte ASK1 as a potential pharmacological target to treat obesity and its metabolic complications by promoting WAT browning. Future research should evaluate selective pharmacologic inhibition of ASK1 in adipocytes, assess translational relevance in human adipose tissue, delineate the ASK1–p38/JNK–IRF3 signaling axis in vivo, and define depot- and context-specific effects across sexes and diets.

The study relies on mouse genetic models and controlled environmental challenges (HFD, LPS infusion, cold exposure), which may limit direct translation to humans. Browning outcomes were primarily assessed in inguinal (subcutaneous) WAT, and depot-specific responses may vary. BAT thermogenic responses were evaluated under chronic conditions and may differ under acute stimulation. Some experiments have modest sample sizes, and no a priori power calculation was performed. LPS infusion models endotoxemia but may not fully recapitulate the complexity of obesity-associated inflammation. Pharmacologic inhibition of ASK1 was not tested, and long-term safety/efficacy in vivo remains to be established.