Fig 2: Adenoviral overexpression of dominant-negative (DN)-KLHL3 augmented mitochondrial function in primary hepatocytes isolated from C57BL/6 mice.a The oxygen consumption rates (OCR) and b extracellular acidification ratio (ECAR) in primary hepatocytes (1 × 104 cells/well) infected with Ad-GFP, Ad-KLHL3, or Ad-DN-KLHL3 were measured with a Seahorse XF analyzer. Cells were treated with oligomycin, FCCP, and rotenone + antimycin A at the indicated time points. c Statistical analysis of basal respiration in the OCR curve. d Statistical analysis of ATP production levels in the OCR curve. e Statistical analysis of nonmitochondrial respiration in the OCR curve. f Statistical analysis of the proton leak level in the OCR curve. g Statistical analysis of the maximal respiratory capacity in the OCR curve. h Energy map showing the increased energy status of primary hepatocytes overexpressing Ad-DN-KLHL3. Data represent three independent experiments. Data are presented as the mean ± standard error of the mean *P < 0.05, **P < 0.01, n.s., not significant (one-way ANOVA test for c–g).

Fig 3: KLHL3 deficiency mitigates high-fat (HF) diet-induced obesity and decreases energy expenditure.a Body weight changes in Klhl3+/+ and Klhl3-/- mice fed a HF diet for 12 weeks (n = 10). b Representative dual-energy X-ray absorptiometry imaging of mice (left) and quantification of body weight, body fat percentage, fat mass, and bone area (right) of Klhl3+/+ and Klhl3-/- mice after 12 weeks on a HF diet (n = 5). c, d Representative macroscopic images and H&E-stained dissection images and adipocyte area in epididymal-white adipose tissue (eWAT) (c, n = 6) and inguinal-white adipose tissue (iWAT) (d, n = 6) of Klhl3+/+ and Klhl3-/- mice fed a HF diet for 12 weeks. Scale bar, 500 µm. e Food intake of Klhl3+/+ and Klhl3-/- mice during HF diet feeding (n = 4). f–i Energy expenditure (f, EE), VO2 g, VCO2 h, and physical activity levels i of Klhl3+/+ and Klhl3-/- mice after 6 weeks of HF diet feeding (n = 4). EE, VO2, and VCO2 were normalized by lean body mass. Data are presented as the mean ± standard error of the mean *P < 0.05, **P < 0.01, n.s., not significant (Mann–Whitney U test for a–e, two-way ANOVA for f–i).

Fig 4: KLHL3 deficiency in mice prevents methionine- and choline-deficient (MCD) diet-induced nonalcoholic steatohepatitis.a Representative images and b quantification graphs of the NAFLD activity score, steatosis, lobular inflammation, and hepatocyte ballooning of liver sections stained with H&E from Klhl3+/+ and Klhl3-/- mice fed a normal chow (NC) or MCD diet (n = 5–6). Scale bar, 200 µm. c Representative images of liver sections stained with oil red O from Klhl3+/+ and Klhl3-/- mice fed a NC or MCD diet (n = 5–6). Scale bar, 200 µm. d Hepatic triglyceride (TG) quantification in Klhl3+/+ and Klhl3-/- mice fed a NC or MCD diet (n = 6–9). e Plasma aspartate aminotransferase (AST) and f alanine aminotransferase (ALT) levels of Klhl3+/+ and Klhl3-/- mice fed a NC or MCD diet (n = 4–6). g Representative images of CD45 (red) immunostained liver sections of Klhl3+/+ and Klhl3-/- mice fed a NC or MCD diet (n = 5–6). Scale bar, 100 µm. h mRNA levels of the F4/80, Cd11c, Tnfa, and Il-6 genes in the livers of Klhl3+/+ and Klhl3-/- mice fed a NC or MCD diet (n = 4–5). The qRT–PCR results were normalized to 18 S. i Representative images and the quantification graph of liver sections stained with Picrosirius red of Klhl3+/+ and Klhl3-/- mice fed a NC or MCD diet (n = 3–6). Scale bar = 200 µm. j mRNA levels of Col1a1 and Acta2 in the livers of Klhl3+/+ and Klhl3-/- mice fed a NC or MCD diet (n = 4–5). The qRT–PCR results were normalized to 18 S. Data are presented as the mean ± standard error of the mean *P < 0.05, **P < 0.01, n.s., not significant (two-way ANOVA for b, d–f, and h–j).

Fig 5: Adeno-associated virus (AAV) dominant-negative (DN)-Klhl3 ameliorates high-fat (HF) diet-induced obesity by augmenting energy expenditure.a Body weight changes in AAV-Control- and AAV-DN-Klhl3-injected mice fed a HF diet for 10 weeks (n = 12). b Representative dual-energy X-ray absorptiometry imaging of mice (left) and quantification of body weight, body fat percentage, fat mass, and bone area (right) of AAV-Control- and AAV-DN-Klhl3-injected mice after 10 weeks on a HF diet (n = 4). c, d Representative macroscopic and H&E-stained dissection images and adipocyte area in epididymal-white adipose tissue (eWAT) (c n = 6) and inguinal-white adipose tissue (iWAT) (d n = 6) of AAV-Control- and AAV-DN-Klhl3-injected mice fed a HF diet for 10 weeks. Scale bar, 200 µm. e Food intake of AAV-Control- and AAV-DN-Klhl3-injected mice during HF diet feeding (n = 4). f–i Energy expenditure (f EE), VO2 g, VCO2 h, and physical activity i of AAV-Control- and AAV-DN-Klhl3-injected mice after 6 weeks of HF diet feeding (n = 4). EE, VO2, and VCO2 were normalized by lean body mass. Data are presented as the mean ± standard error of the mean *P < 0.05, **P < 0.01, n.s., not significant (Mann–Whitney U test for a–e, two-way ANOVA for f–i).