**Background:** Obesity results from impaired energy balance and is a global epidemic. Non-shivering thermogenesis via brown adipose tissue (BAT) and browning of white adipose tissue (WAT) represents a promising anti-obesity strategy by dissipating energy as heat through uncoupling protein 1 (UCP1). Gut microbiota dysbiosis, particularly an increased Firmicutes-to-Bacteroidetes (F/B) ratio, is also implicated in obesity pathophysiology. Calebin A, a non-curcuminoid compound from Curcuma longa rhizomes (purity >99% by HPLC), has known anti-inflammatory and anti-tumor properties, and prior work showed it inhibits adipogenesis and hepatic steatosis via AMPK signaling. Its effects on thermogenesis and gut microbiota had not been characterized.
**Methods:** Four-week-old male C57BL/6J mice (n=9/group) were assigned to: (1) normal diet (ND; 15% energy from fat), (2) high-fat diet (HFD; 50% energy from fat, primarily lard), (3) HFD + 0.1% calebin A (LCA), and (4) HFD + 0.5% calebin A (HCA). Diets were based on Purina 5001, and mice had free access to food and water for 12 weeks. Body weight and food intake were recorded daily. Fasting blood glucose was measured after 8 h of fasting at week 12 using a glucose analyzer. Acute cold tolerance was assessed by exposing individually housed mice to 4 °C for 7 h; rectal temperature was measured at 0, 2, 4, 5, and 7 h. Gut microbiota was profiled by 16S rRNA gene sequencing (V4 region, Illumina platform) from fecal DNA (n=3/group). OTUs were clustered at ≥97% similarity. Alpha diversity (ACE and Shannon indices) and beta diversity (PCA) were calculated. Statistical analyses used one-way ANOVA with Duncan's multiple range test; microbiota comparisons used the nonparametric Wilcoxon signed rank test. P < 0.05 or P < 0.01 was considered significant.
**Key Results:** After 12 weeks, the HFD group weighed approximately 10 g more than the ND group. Both LCA and HCA significantly reduced body weight in a dose-dependent manner. Fasting blood glucose was highest in the HFD group; HCA significantly lowered it (specific values not reported in text; Fig. 1B referenced). HCA significantly reduced liver weight to levels not different from ND, and improved liver color (reduced lipid accumulation). Spleen weight was increased by HFD but not significantly reduced by calebin A; kidney weights did not differ among groups. HCA significantly reduced perigonadal and mesenteric white adipose tissue weights. BAT and inguinal WAT weights were lower with calebin A treatment. HFD caused gastrocnemius muscle loss, which calebin A did not reverse. In the cold tolerance test, HFD-fed mice showed the steepest temperature drop, while HCA-treated mice maintained rectal temperature most effectively; the area under the curve for HCA was significantly higher than for HFD. Gut microbiota analysis showed that the F/B ratio in HFD was not significantly higher than in ND, but HCA reduced the ratio from 1.24 ± 0.29 to 1.03 ± 0.42. Verrucomicrobia increased from 0.002 ± 0.001 to 0.022 ± 0.02 (p = 0.22) in HCA, and Proteobacteria trended upward from 0.03 ± 0.01 to 0.05 ± 0.01 (p = 0.10). ACE richness was significantly reduced by HCA, but Shannon diversity did not differ. PCA showed that ND and HCA groups clustered separately, while LCA and HCA were closer to each other. At the genus level, HFD decreased unidentified Ruminococcaceae and Butyricicoccus, and calebin A restored them. HCA significantly increased Akkermansia (Verrucomicrobia phylum) and Ruminiclostridium_9 compared with HFD.
**Clinical Implications:** This study provides preclinical evidence that calebin A acts through dual mechanisms—enhancing adaptive thermogenesis (as demonstrated by improved cold tolerance) and favorably modulating the gut microbiota (increasing Akkermansia, Butyricicoccus, Ruminiclostridium_9, and unidentified Ruminococcaceae)—to prevent HFD-induced obesity and hyperglycemia in mice. These findings are consistent with reported effects of curcumin, a structural analog. The observed dose-dependent reductions in body weight, adipose tissue mass, and liver steatosis, alongside improved glycemic control, suggest that calebin A may be developed as a novel gut microbiota modulator and thermogenesis-promoting agent for obesity prevention. However, these results are limited to a murine model; human studies are needed to confirm efficacy, safety, and translational relevance.