FAM13A Represses AMPK Activity and Regulates Hepatic Glucose and Lipid Metabolism

Abstract

Obesity commonly co-exists with fatty liver disease with increasing health burden worldwide. Family with Sequence Similarity 13, Member A (FAM13A) has been associated with lipid levels and fat mass by genome-wide association studies (GWAS). However, the function of FAM13A in maintaining metabolic homeostasis in vivo remains unclear. Here, we demonstrated that rs2276936 in this locus has allelic-enhancer activity in massively parallel reporter assays (MPRA) and reporter assay. The DNA region containing rs2276936 regulates expression of endogenous FAM13A in HepG2 cells. In vivo, Fam13a^-/- mice are protected from high-fat diet (HFD)-induced fatty liver accompanied by increased insulin sensitivity and reduced glucose production in liver. Mechanistically, loss of Fam13a led to the activation of AMP-activated protein kinase (AMPK) and increased mitochondrial respiration in primary hepatocytes. These findings demonstrate that FAM13A mediates obesity-related dysregulation of lipid and glucose homeostasis. Targeting FAM13A might be a promising treatment of obesity and fatty liver disease.

Keywords: Biological Sciences; Cell Biology; Functional Aspects of Cell Biology.

Graphical abstract

Figure 1

Identification of Functional Variants at the FAM13A Locus Associated with Lipid Levels (A)LD matrix of SNPs (rs2276936, rs2167750, rs7695177) with previously frequently reported top GWAS SNPs (rs3822072 and rs9991328). The first two columns represent the major (red frame) and minor (blue frame) haplotypes of these five SNPs. (B–D) (B) Top: the diagram shows the ATAC-Seq and DNase-Seq signals around SNP rs2276936, rs2167750, and rs7695177 in liver-relevant cell and tissues profiled by Roadmap epigenomics project and ENCODE project. Bottom: reporter assays of FAM13A promoter with or without DNA regions spanning rs2276936, rs2167750, and rs7695177 in HepG2 cells. For: forward orientation; Rev: reverse orientations. Data are presented as mean ± SEM from three biological replicates with triplicate wells in each repeat. ∗p < 0.05 by unpaired Student's t test. Expression of FAM13A was assessed in HepG2 cells transfected with gRNA targeting rs2276936 for regional deletion (C) or indel deletion (D). Con: Control. Rs227-1 and rs227-2 are from two independent repeats. Data are presented as mean ± SEM from two biological replicates with triplicate wells in each repeat. ∗p < 0.05 by unpaired Student's t test.

Figure 2

Body Weight Gain in Mice after High-Fat diet (HFD) Treatment for Four Months (A) Gain of body weight measured during HFD treatment for four months in Fam13a^+/+ (n = 7) and Fam13a^−/− mice (n = 5). (B–D) (B) Fat and lean mass of mice (A) measured by MRI after four months of HFD treatment. Inguinal fat (C) and liver (D) mass normalized to body weight in mice treated with HFD (n = 6 for Fam13a^+/+; n = 5 for Fam13a^−/−). Data are presented as mean ± SEM. ∗p < 0.05 or ∗∗p < 0.01 by unpaired Student's t test.

Figure 3

Fam13a^−/− Mice Showed Improved Systemic and Hepatic Insulin Sensitivity after HFD Treatment (A and B) (A) Glucose tolerance test (GTT) and (B) insulin tolerance test (ITT) were performed after overnight fasting or 6-h fasting in female Fam13a^+/+ and Fam13a^−/− mice fed with HFD for 14 weeks, respectively. (C) Serum insulin was measured in female Fam13a^+/+ and Fam13a^−/− mice fed with HFD for four months. (D) Immunoblotting of phosphorylation of Akt (Thr308) in liver from HFD-fed Fam13a^+/+ and Fam13a^−/− mice for four months with or without insulin injection via portal vein. Mice were harvested five minutes after insulin treatment. Mean ± SEM shown represent the densitometry of bands averaged from 3 to 4 mice/group. ∗p < 0.05 or ∗∗p < 0.01 by unpaired Student's t test. (E) Akt phosphorylation at Thr308 was measured in primary hepatocytes cultured in the presence or absence of insulin at the concentration of 100 nM for 6 h. INS, insulin. (F) Hepatic glucose production was measured and normalized to total cellular protein amount in hepatocytes isolated from Fam13a^+/+ and Fam13a^−/− mice. (Mean ± SEM from three mice/genotypes). ∗p < 0.05, unpaired Student's t test.

Figure 4

HFD-fed Fam13a^−/− Mice Showed Improved Hepatic Steatosis (A) Levels of total cholesterol, HDL, and LDL/VLDL were determined in serum from Fam13a^+/+ and Fam13a^−/− mice after HFD treatment for four months. (B–E) (B) Expression of Apoa1 was measured in primary hepatocytes from Fam13a^+/+ and Fam13a^−/− mice using q-PCR. Free fatty acid (C), triglycerides (D), and cholesterol (E) were measured in murine liver samples. Data are presented as mean ± SEM. ∗p < 0.05 or ∗∗p < 0.01, unpaired Student's t test. (F) Representative images of HE staining of livers are shown. (n = 6 for Fam13a^+/+; n = 5 for Fam13a^−/− mice). Scale bar: 20 μm.

Figure 5

FAM13A Regulates Mitochondrial Function in Primary Hepatocytes (A) Mitochondrial respiration was measured by Seahorse assay in hepatocytes isolated from Fam13a^+/+ and Fam13a^−/− mice. Arrows indicate the sequential addition of oligomycin, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and rotenone/antimycin treatments. Representative results of one pair of mice from two biological replicates. (B–E) (B) The basal oxygen consumption rate (OCR), maximal mitochondrial respiration, ATP production, and non-mitochondrial respiration from A are shown. Data are presented as mean ± SD. ∗∗p < 0.01 by unpaired Student's t test. Phosphorylation of AMPK (Thr172) was measured in the liver samples from HFD-fed Fam13a^+/+ and Fam13a^−/− mice (C, n = 4–5 mice/genotype), in primary hepatocytes (D) and HepG2 cells transfected with small interfering RNA (siRNA) targeting FAM13A (E). Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01 by unpaired Student's t test.

Figure 6

FAM13A Regulates Mitochondrial Respiration through AMPK Signaling (A) The basal mitochondrial respiration, maximal mitochondrial respiration, ATP production, and non-mitochondrial respiration were shown in HepG2 cells transfected with the plasmid expressing FAM13A or empty vector. Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01 by unpaired Student's t test. (B) Phosphorylation of AMPK (Thr172) was determined in HepG2 cells with overexpression of Myc-tagged FAM13A. V: Vector control; F: Fam13a. (C) Immunoblotting showing the knockdown efficiency. Combo shRNA: combination of FAM13A shRNA and AMPK shRNA. (D) Bioenergetics assays measured by Seahorse in HepG2 cells with stable knockdown of FAM13A and/or AMPK by shRNA. Arrows indicated sequential addition of oligomycin, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and rotenone/antimycin. (E) The basal oxygen consumption rate (OCR), maximal mitochondrial respiration, ATP production, and non-mitochondrial respiration were shown. Data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01 by unpaired Student's t test.

Figure 7

Schematic Illustration of FAM13A Regulating lipid and Glucose Metabolism (A) Mechanism of FAM13A regulating Hepatic steatosis and insulin resistance through AMPK. TG: triglycerides. HFD: high-fat diet. (B) Metabolic phenotypes observed in human subjects with opposing genotypes for SNP rs2276936 and Fam13a^−/− mice (Ji et al., 2019).