β-Hydroxybutyrate Reduces Body Weight by Modulating Fatty Acid Oxidation and Beiging in the Subcutaneous Adipose Tissue of DIO Mice

Abstract

β-hydroxybutyrate (BHB) serves as an alternative cellular fuel during states of low glucose availability, such as fasting or carbohydrate restriction, when the body shifts to using fats and ketone bodies for energy. While BHB has shown potential metabolic benefits, its mechanisms of action in the context of obesity are not fully understood. In this study, we examined the effects of BHB supplementation on subcutaneous adipose tissue (SAT) metabolism in a diet-induced obesity (DIO) mouse model. Adult male mice were first fed a high-fat diet for six weeks, followed by a standard diet with or without BHB supplementation for an additional six weeks. BHB supplementation led to significant body weight loss independent of food intake. This weight reduction was associated with decreased adipocyte differentiation, reflected by reduced peroxisome proliferator-activated receptor gamma (PPARγ) protein levels and lower uncoupling protein 1 (UCP1) expression, indicating altered SAT function. Transcriptomic analysis of SAT revealed upregulation of genes involved in fatty acid activation and transport (e.g., Slc27a2, Plin5, Acot4, Acsm3, Rik). Functional enrichment highlighted the activation of the PPAR signaling pathway and enrichment of peroxisomal components in the BHB group. Together, these results suggest that BHB promotes lipid remodeling in SAT, enhancing fatty acid metabolism while suppressing thermogenic pathways, and thus may represent a novel mechanism contributing to adiposity reduction and metabolic improvement.

Keywords: SAT; adipose tissue; fatty acid oxidation; obesity; thermogenesis; weight loss; β-hydroxybutyrate.

Figure 1

BHB supplementation reduces body weight gain without affecting food intake and modulates serum metabolite levels in a DIO mouse model. Male C57BL/6J mice were fed a high-fat diet (HFD; 60% fat) for 6 weeks to induce obesity, creating a diet-induced obesity (DIO) model. Following this induction period, the mice received water supplemented with BHB) for an additional 6-weeks to assess the effects of ketone body supplementation. (A) Body weight (g) pre- and post-60% high-fat diet and body weight (g) pre- and post-treatment, showing a significant reduction in the BHB-supplemented group. To investigate the metabolic effects of BHB supplementation in obesity, we analysed the serum concentrations of key metabolites associated with BCAAs amino acids and energy metabolism. Serum concentration of (B) lysine, (C) isoleucine, (D) valine were significantly decreased, whereas (E) pyruvate and (F) citric acid concentrations were elevated in DIO mice following BHB supplementation. Data are expressed as the mean (±SEM). Student’s t-test was performed (n = 8–10). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

BHB modifies thermogenic markers in adipose tissue of DIO mice. The mRNA expression levels of key thermogenic genes were analysed in BAT, VAT, and SAT to better understand the effect of BHB on the regulation of thermogenic pathways during obesity. (A) mRNA expression level of Ucp1 in BAT was significantly reduced in BHB mice vs. controls, while mRNA expression levels of Ucp1 in VAT and SAT remained unchanged. (B) mRNA expression level of Pgc-1α in SAT was significantly higher in BHB mice vs. controls, while mRNA expression levels of Pparɣ in SAT of BHB group were not modified. (C) Protein levels of PGC-1α were not altered, while PPARɣ and UCP1 protein levels were diminished in SAT after BHB supplementation. (D) Immunoblot images are shown. Data are expressed as mean (±SEM). Student’s t-test was performed. (n = 4–8). (C: control; B: β-Hydroxybutyrate). * p < 0.05, ** p < 0.01.

Figure 3

Identification of differentially expressed genes related to fatty acid oxidation (FAO), thermogenesis in SAT of DIO mice supplemented with BHB compared to control group. RNA sequencing (RNA-seq) analysis was performed on SAT comparing the BHB-supplemented group with the control group to identify changes in gene expression (A) Volcano plot displaying differentially expressed genes between the two experimental groups. Pink dots represent significantly downregulated genes, while blue dots represent significantly upregulated genes in SAT. Genes in grey have no statistical significance. The horizontal dashed black line indicates an adjusted p-value threshold of 0.05. (B) Filtered count data for selected upregulated (blue squares) and downregulated (pink squares) genes compared to control group (white circles), derived from the volcano plot. The y-axis represents normalized expression counts, and the x-axis lists individual genes grouped by their regulation status (upregulated or downregulated). (C) Gene Ontology over-representation analysis in regards to Biological process (D), Cellular components and (E) Molecular function showing the most significantly enriched pathways ranked by adjusted p-value in ascending order. The x-axis represents the number of significant genes identified within each functional category. (F) KEGG pathway enrichment analysis highlighting the activation of the PPAR signalling pathway based on differentially expressed genes, performed using ExpHunter Suite (v. 1.16.0) (n = 4–8). Data are expressed as the mean (±SEM). Student’s t-test was performed. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 4

CPT2 levels were not significantly altered in SAT of DIO mice supplemented with BHB. (A) CPT2 protein expression was analysed in SAT from C57BL/6 DIO mice supplemented with BHB and control groups, with (B) Representative immunoblot images of CPT2 protein levels are shown. BHB supplementation did not significantly change the levels of CPT2 compared to controls. Data are presented as the mean (±SEM). Student’s t-test was performed. (n = 6). (C: control; B: β-Hydroxybutyrate).