Motor protein KIF13B orchestrates hepatic metabolism to prevent metabolic dysfunction-associated fatty liver disease

Abstract

Background: Kinesin family member 13B (KIF13B), a crucial motor protein, exerts multiple cellular biological functions. However, the implication of KIF13B in metabolic dysfunction-associated fatty liver disease (MAFLD) has not been explored yet. This study aimed to investigate KIF13B's role and underlying mechanism in MAFLD and proposes it as a potential pharmacological target.

Methods: We assessed KIF13B expression in MAFLD patients and rodent models. The roles of Kif13b in lipid metabolism and MAFLD were investigated using whole-body Kif13b knockout mice, hepatocyte-specific Kif13b-deficient mice and hamsters exposed to different diets. The underlying mechanisms by which Kif13b governed hepatic lipid homeostasis and MAFLD progression were explored in vitro. Finally, the Kif13b's impact on atherosclerotic development was studied in the context of MAFLD.

Results: KIF13B expression was reduced in patients and murine models with MAFLD. Rodents with global or liver-specific knockout of the Kif13b gene exhibit spontaneous hepatic steatosis, which is further exacerbated by different overnutrition diets. Overexpression of human KIF13B by lentivirus effectively prevented metabolic dysfunction-associated steatohepatitis (MASH) in methionine-choline-deficient diet (MCD)-fed mice. Furthermore, Kif13b deficiency accelerates atherosclerosis in the context of MAFLD. Mechanistically, Kif13b depletion increases hepatic lipid synthesis and impairs mitochondrial oxidative phosphorylation. Further screening reveals that Kif13b interacts with AMP-activated catalytic subunit alpha 1 (AMPKα1) to regulate the phosphorylation of AMPKα1, governing mitochondrial homeostasis and suppressing sterol regulatory element binding protein 1 (Srebp1)-mediated de novo lipogenesis in the liver.

Conclusion: This work establishes a causal relationship between KIF13B deficiency and MAFLD, emphasizing KIF13B as a potential therapeutic target for treating MAFLD.

Keywords: AMP-activated catalytic subunit alpha 1; Kinesin family member 13B; Lipid metabolism; Metabolic dysfunction-associated fatty liver disease; Mitochondrial homeostasis.

Fig. 1

Reduced hepatic KIF13B expression in MAFLD patients and mice. a Volcano plot of hepatic KIF13B mRNA expressions in metabolic dysfunction-associated fatty liver (MAFL) vs. healthy controls (HC), and metabolic dysfunction-associated steatohepatitis (MASH) vs. HC from Gene Expression Omnibus (GEO) database (GSE126848). b Representative images of H&E, oil red O (ORO), and immunofluorescence staining of human KIF13B in liver tissues from HC (n = 3) and patients with metabolic dysfunction-associated fatty liver disease (MAFLD, n = 18) are presented. The red color in immunofluorescence staining represents the KIF13B protein. Scale bars = 100 μm. c Pearson correlation analysis of KIF13B expression related to glucose metabolism, lipid metabolism, inflammation, and fibrosis from the same dataset in (a). d Heat map showing the expression of hepatic Kif13b and other genes regulating lipid metabolism in wild-type (WT) mice fed on chow diet (CD) and high-fat diet (HFD) (n = 4), as well as WT and ob/ob mice (n = 6). e The hepatic mRNA and protein levels of Kif13b were analyzed in male WT mice on different diets, including a CD, a HFD, a Western diet (WD), a methionine-choline-deficient diet (MCD), and male ob/ob mice (n = 5). f After starvation for 12 h, HepG2 cells were treated with 0.2% bovine serum albumin (BSA) and palmitic acid (PA; 300 μmol/L) for 24 or 48 h. The mRNA and protein levels of KIF13B in the HepG2 cell line were measured. Data are means ± SEM. **P < 0.01, ***P < 0.001. P-values were calculated by unpaired two-tailed Student’s t-test. KIF13B kinesin family member 13B

Fig. 2

Kif13b deficiency elicits hepatic steatosis and worsens diet-induced steatohepatitis. Eight-week-old male Kif13b^+/+ and Kif13b^−/− mice were randomly assigned to two dietary groups: a 16-week chow diet (CD) group for spontaneously hepatic steatosis and an 8-week high-fat diet (HFD) group for diet-induced hepatic steatosis. a Body weight of mice fed with CD or HFD. b Plasma total cholesterol (TC) and triglyceride (TG) of mice fed with CD or HFD. c The ratio of liver weight to body weight (LW/BW) of mice fed with CD or HFD. d Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels of mice fed with CD or HFD. e Liver TC and TG contents of mice fed with CD or HFD. f Representative histological images of liver cryo-sections with H&E staining and oil red O (ORO) staining from Kif13b^+/+ and Kif13b^−/− mice fed with CD or HFD. Scale bars = 100 μm. n = 5. Eight-week-old male Kif13b^+/+ and Kif13b^−/− mice were randomly assigned to two dietary groups: a 4-week methionine-choline-deficient diet (MCD) group and a 20-week Western diet (WD) group. g Body weight and LW/BW ratio of mice fed with MCD or WD. h Plasma TC and TG of mice fed with MCD or WD. i Plasma ALT and AST levels of mice fed with MCD or WD. j Liver TC and TG contents of mice fed with MCD or WD. k Representative histological images of liver cryo-sections with H&E staining, ORO staining, immunofluorescence staining of CD68, and Sirius red staining from Kif13b^+/+ and Kif13b^−/− mice fed with MCD or WD. Scale bars = 100 μm. l Histological scoring of steatosis, lobular inflammation, hepatocyte ballooning, and fibrosis were calculated. **P < 0.01, ***P < 0.001, Kif13b^−/− vs. Kif13b^+/+. m CD68⁺ cell ratio was calculated. n = 6. Data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. P-values were calculated by unpaired two-tailed Student’s t-test. KIF13B kinesin family member 13B

Fig. 3

Targeting hepatic Kif13b leads to metabolic dysfunction-associated steatohepatitis (MASH) and fibrosis in mice and hamsters under overnutrient conditions. a Analysis of single-nucleus RNA sequencing of KIF13B mRNA in hepatocytes in human control liver and MASH liver from the Gene Expression Omnibus (GEO) database (GSE212837). b Analysis of single-cell RNA sequencing of Kif13b mRNA in hepatocytes in mouse control liver and MASH liver from the GEO database (GSE225381). P-values were calculated by Deseq2 (Wald’s test) in (a) and the one-sided Mann–Whitney U test in (b). Eight-week-old male Kif13b^f/f and Kif13b^LKO mice were fed with a Western diet (WD) for 20 weeks. c Body weight of indicated mice. d Plasma total cholesterol (TC) and triglyceride (TG). e The ratio of LW/BW. f Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. g Liver TC and TG contents. h Representative histological images of liver cryo-sections with H&E staining, oil red O (ORO) staining, immunofluorescence staining of CD68, and Sirius red staining from Kif13b^f/f and Kif13b^LKO mice fed with WD. Scale bars = 100 μm. Histological scoring of MAFLD activity score, CD68⁺ cell ratio, and hepatic collagen areas were presented also. n = 6. Eight-week-old WT hamsters received jugular vein injections of AAV8-shKif13b to deplete Kif13b from their livers, with AAV8-negative control (NC) as a control. All hamsters were then placed on a CD for 4 weeks, followed by a high-fat and high-cholesterol diet (HFHCD) for another 4 weeks. i Body weight and the ratio of LW/BW. j Plasma TC, TG, ALT, and AST levels. k Liver TC and TG contents. l Representative histological images of liver cryo-sections with H&E staining, ORO staining, immunofluorescence staining of CD68, and Sirius red staining. Scale bars = 100 μm. MAFLD activity score, CD68⁺ cells, and hepatic collagen areas were calculated. n = 8. Data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. P-values were calculated by unpaired two-tailed Student’s t test. KIF13B kinesin family member 13B, MAFLD metabolic dysfunction-associated fatty liver disease

Fig. 4

Kif13b regulates lipid synthesis and mitochondrial function. Gene set enrichment analysis (GSEA) (a) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (b) analysis based on the RNA sequencing in the livers of male Kif13b^+/+ and Kif13b^−/− mice fed with chow diet (CD) (n = 5). c Western blotting analysis of liver samples from CD-fed mice. d The mRNA expression profiles of genes associated with the Srebp signalling pathway, cholesterol biosynthesis, fatty acid biosynthesis, electron transport activity, and fatty acid oxidation in the liver of mice fed a CD. e Oil red O staining of primary mouse hepatocytes and HepG2 cells following KIF13B silencing by siRNA exposure to palmitic acid (PA; 300 μmol/L) after 12 h of starvation. Scale bars = 100 μm. f ATP production of primary mouse hepatocytes and HepG2 cells with KIF13B silence by siRNA or KIF13B overexpression by plasmid exposure with PA (300 μmol/L) after 12 h of starvation. g Oxygen consumption rates (OCR) were measured by Seahorse XF96 analyzer in KIF13B knockdown HepG2 cells (n = 6). OCR relating to mitochondrial basal respiration, maximal respiration, spare respiration capacity, and ATP production were respectively analyzed and normalized to the corresponding total protein content per well in KIF13B knockdown HepG2 cells. h Representative images demonstrating alterations in the morphology and number of mitochondria in HepG2 cells following KIF13B silencing were captured using a confocal microscope. Scale bars = 2 μm. The relative fluorescence quantification of mitochondria was shown also. Data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. P-values were calculated by unpaired two-tailed Student’s t-test. KIF13B kinesin family member 13B, NES normalized enrichment score, Srebp sterol regulatory element binding protein

Fig. 5

Kif13b orchestrates lipid metabolism and mitochondrial function in an AMPKα dependent manner. Primary mouse hepatocytes from Kif13b^+/+ and Kif13b^−/− mice were starved 12 h and then exposed to palmitic acid (PA) at a concentration of 300 μmol/L with or without metformin (Met) at 2 mmol/L for 24 h. a ATP production in primary mouse hepatocytes. b Representative images from oil red O (ORO) staining. c Representative images of Western blotting. In HepG2 cells, transfection with scrambled RNA (scrRNA) or siKIF13B occurred for 48 h, followed by exposure to 300 μmol/L PA and 2 mmol/L Met for a further 24 h, after 12 h of starvation. d ATP production in HepG2 cells. e Representative images from ORO staining. f Representative images of Western blotting. HepG2 cells were transfected with a negative control (NC) or KIF13B plasmid for 48 h and treated with 300 μmol/L PA and 110 nmol/L AMPK-IN3 (an AMPKα inhibitor) for a further 24 h, after 12 h of starvation. g ATP production in HepG2 cells. h Representative images from ORO staining. i Representative images of Western blotting. Each experiment was independently replicated three times. Scale bars = 100 μm. Data are means ± SEM. **P < 0.01, ***P < 0.001. P-values were calculated by one- or two-way ANOVA followed by Tukey’s test. KIF13B kinesin family member 13B

Fig. 6

Metformin improves Kif13b deficiency-induced MAFLD by activating AMPKα. a Eight-week-old male mice were subjected to a 20-week Western diet (WD) and received vehicle or metformin every 2 d via gavage for 8 weeks. b Body weight. c The ratio of liver weight to body weight (LW/BW). d Plasma total cholesterol (TC), triglyceride (TG), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels. e Liver TC and TG contents. f Representative histological images of liver cryo-sections with H&E staining, oil red O (ORO) staining, and immunofluorescence staining of CD68 and α smooth muscle actin (α-SMA) from mice in (a). Scale bars = 100 μm. Histological scoring of MAFLD activity score, CD68⁺ cells ratio and α-SMA positive area were calculated. g Representative images and relative quantification of Western blotting of liver samples from mice in (a). h mRNA levels of proinflammatory cytokines and fibrosis markers of liver samples from mice in (a). n = 6. Data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. P-values were calculated using two-way ANOVA followed by Tukey’s test. KIF13B kinesin family member 13B, MAFLD metabolic dysfunction-associated fatty liver disease

Fig. 7

AMPKα1 deficiency inhibits the protective effect of KIF13B on MAFLD. a Co-immunoprecipitation (Co-IP) experiments investigate the interaction between Kif13b and AMPKα in WT mouse livers and HepG2 cells overexpressing KIF13B. b Following the overexpression of KIF13B-GFP in HepG2 cells, liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis of purified proteins co-immunoprecipitated with Kif13b-GFP employed GFP protein as a control, and the 1174 identified protein was observed exclusively in the Kif13b-GFP group. c Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis based on the proteins identified in (b). Pathways with P < 0.05 were chosen and sorted by -log₁₀ P-value in reverse order. d Protein secondary structure analysis of AMPKα1. e Eight-week-old male WT mice were subjected to a 1-week diet of methionine-choline-deficient (MCD) feeding. In addition to lentivirus (LV) or LV-KIF13B through the portal vein, they were administered either AAV8 or AAV8-shAMPKα1 via tail vein injection, followed by another 2-week MCD diet. f Western blotting analysis of liver samples from mice in (e). Relative quantification of Kif13b to GAPDH and the ratio of phosphorylated AMPKα1 to total AMPKα. g Body weight and the ratio of liver weight to body weight (LW/BW). h Plasma total cholesterol (TC), triglyceride (TG), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels. i Liver TC and TG contents. j Representative histological images of liver cryo-sections with H&E staining, oil red O (ORO) staining, and immunofluorescence staining of bodipy, CD68, and α smooth muscle actin (α-SMA) from mice in (e). Scale bars = 100 μm. Histological scoring of MAFLD activity score, CD68⁺ cells ratio and α-SMA positive area were calculated. n = 5. Data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. P-values were calculated by one-way ANOVA followed by Tukey’s test. KIF13B kinesin family member 13B, MAFLD metabolic dysfunction-associated fatty liver disease, AMPKα1 AMP-activated catalytic subunit alpha 1

Fig. 8

Deletion of Kif13b in the liver exacerbates MASH through suppression of AMPKα activity. In physiological settings, Kif13b plays a crucial role in enhancing AMPKα activity, inhibiting Srebp1 nuclear translocation, fostering fatty acid oxidation, and preserving mitochondrial homeostasis. Nonetheless, in the liver’s absence of Kif13b, the phosphorylation of AMPKα is impeded, resulting in a disturbance in lipid metabolism and mitochondrial function. Consequently, this disturbance contributes to the accumulation of excess lipids, thereby intensifying the progression of MASH. Conversely, the restoration of AMPKα phosphorylation through either KIF13B overexpression or the administration of metformin proves effective in mitigating MASH. KIF13B kinesin family member 13B, AMPKα1 AMP-activated catalytic subunit alpha 1, WT wild-type, FA fatty acid, KO knockout, MASH metabolic dysfunction-associated steatohepatitis, Srebp1 sterol regulatory element binding protein 1