Genetic association-based functional analysis detects HOGA1 as a potential gene involved in fat accumulation

Abstract

Although there are a number of discoveries from genome-wide association studies (GWAS) for obesity, it has not been successful in linking GWAS results to biology. We sought to discover causal genes for obesity by conducting functional studies on genes detected from genetic association analysis. Gene-based association analysis of 917 individual exome sequences showed that HOGA1 attains exome-wide significance (p-value < 2.7 × 10^-6) for body mass index (BMI). The mRNA expression of HOGA1 is significantly increased in human adipose tissues from obese individuals in the Genotype-Tissue Expression (GTEx) dataset, which supports the genetic association of HOGA1 with BMI. Functional analyses employing cell- and animal model-based approaches were performed to gain insights into the functional relevance of Hoga1 in obesity. Adipogenesis was retarded when Hoga1 was knocked down by siRNA treatment in a mouse 3T3-L1 cell line and a similar inhibitory effect was confirmed in mice with down-regulated Hoga1. Hoga1 antisense oligonucleotide (ASO) treatment reduced body weight, blood lipid level, blood glucose, and adipocyte size in high-fat diet-induced mice. In addition, several lipogenic genes including Srebf1, Scd1, Lp1, and Acaca were down-regulated, while lipolytic genes Cpt1l, Ppara, and Ucp1 were up-regulated. Taken together, HOGA1 is a potential causal gene for obesity as it plays a role in excess body fat development.

Keywords: 3T3-L1; HOGA1; adipogenesis; association analysis; exome sequencing; functional study; obesity.

FIGURE 1

Manhattan plot of results from gene-based association analysis for BMI. The negative logarithm of the association p-value for genes distributed in the autosomal genome is represented as a dot. The red line represents the exome-wide significant p-value (p-value < 2.7 × 10^–6).

FIGURE 2

HOGA1 expression levels in adipocytes detected from the GTEx dataset. HOGA1 expression was compared between normal and obese individuals using RNA sequencing data of adipocyte samples. Group differences were assessed by the Wilcoxon rank-sum test (*p < 0.05, **p < 0.01, ***p < 0.001 vs normal).

FIGURE 3

The effect of Hoga1 knockdown on adipogenesis in mouse 3T3-L1 cells. (A) Relative expression levels of Hoga1 and Pparg during adipocyte differentiation. (B) Hoga1 knockdown efficiency of two independent siRNAs (si#1 and si#2) compared to scrambled control siRNA (scr). (C) Lipid accumulation during adipocyte differentiation in 3T3-L1 pre-adipocytes transfected with two independent Hoga1-targeting siRNAs or scr siRNA. Red dots indicate lipid droplets stained with Oil-Red-O. (D) Expression levels of Pparg, aP2, Cd36, Cebpa, and Adiponectin genes related to adipogenesis in 3T3-L1 pre-adipocytes transfected with two independent Hoga1-targeting siRNAs or scr siRNA. Notes: The mRNA level of each gene was measured on day 1 after treatment with each Hoga1-siRNA or scr siRNA by RT-qPCR analysis. Results are expressed as the mean ± SD of three independent experiments (*p < 0.05 and **p < 0.01 vs scrambled control).

FIGURE 4

Increased Hoga1 expression in adipose tissues of diet-induced obese mice. High-fat diet (HFD) C57BL/6J mice were treated with Hoga1 ASO (HFD Hoga1 ASO) or scrambled control ASO (HFD vehicle) twice per week (25 mg per kg body weight per dose) for 6 weeks. In addition, normocaloric diet (NCD) mice were treated with scrambled control ASO (NCD vehicle). The mRNA and protein levels of Hoga1 in epididymal white adipose tissue (eWAT) (A) and in the liver (B) of the NCD vehicle, HFD vehicle, and HFD Hoga1 ASO groups were measured by qRT-PCR (left panel) and Western blot (right panel) analyses, respectively. Five mice were used per group (^# p < 0.05 indicates a significant difference between the NCD vehicle and HFD vehicle; *p < 0.05 indicates a significant difference between the HFD vehicle and HFD Hoga1 ASO).

FIGURE 5

Reversible weight loss in diet-induced obese mice due to Hoga1 knockdown. Body weight gain (A) and food intake (B) in NCD and HFD mice treated with scrambled control ASO or Hoga1 ASO were measured from six mice per group (^## p < 0.01 indicates a significant difference between the NCD vehicle and HFD vehicle; *p < 0.05 indicates a significant difference between the HFD vehicle and HFD Hoga1 ASO).

FIGURE 6

The effect of Hoga1 knockdown on lipid properties in obese mice. Fat pad (A) and liver (B) masses of C57BL/6J mice from NCD or HFD mice treated with scrambled control ASO or Hoga1 ASO were measured from six mice per group. (C) Representative images of H&E-stained sections from eWAT of NCD or HFD mice treated with scrambled control ASO or Hoga1 ASO (×200 magnification). (D) Average adipocyte size per microscopic field was compared among the NCD vehicle, HFD vehicle, and HFD Hoga1 ASO groups (N = 6 per group). (E) Triglyceride, total cholesterol, and leptin levels of NCD and HFD mice treated with scrambled control ASO or Hoga1 ASO were measured from six mice per group. Results are expressed as the mean ± SD of three independent experiments (^## p < 0.01 indicates a significant difference between the NCD vehicle and HFD vehicle; *p < 0.05 and **p < 0.01 indicate significant differences between the HFD vehicle and HFD Hoga1 ASO, respectively).

FIGURE 7

The effect of Hoga1 knockdown on clinical indexes of insulin resistance in obese mice. Fasting blood glucose, serum insulin concentration, and HOMA-IR were compared among the NCD vehicle, HFD vehicle, and HFD Hoga1 ASO groups (N = 6 per group). Results are expressed as the mean ± SD of three independent experiments (^# p < 0.05 and ^## p < 0.01 indicate significant differences between the NCD vehicle and HFD vehicle, respectively; *p < 0.05 indicates a significant difference between the HFD vehicle and HFD Hoga1 ASO).

FIGURE 8

The effect of Hoga1 knockdown on adipose tissue inflammation in obese mice. (A) mRNA expression of inflammatory genes in eWAT was measured from NCD and HFD mice treated with scrambled control ASO or Hoga1 ASO (N = 5 per group). (B) Serum inflammatory cytokine concentrations compared among the NCD vehicle, HFD vehicle, and HFD Hoga1 ASO groups (N = 5 per group). Results are expressed as the mean ± SD of three independent experiments (^# p < 0.05 and ^## p < 0.01 indicate significant differences between the NCD vehicle and HFD vehicle; *p < 0.05 and **p < 0.01 indicate significant differences between the HFD vehicle and HFD Hoga1 ASO).

FIGURE 9

Lipogenic and lipolytic gene expression in mouse eWAT. The levels of proteins (A) and mRNAs (B) of lipogenic genes were compared in eWAT from NCD and HFD mice treated with scrambled control ASO or Hoga1 ASO. The levels of proteins (C) and mRNAs (D) of lipolytic genes were compared in eWAT from NCD and HFD mice treated with scrambled control ASO or Hoga1 ASO. Notes: N = 5 per group. Results are expressed as the mean ± SD of three independent experiments (^# p < 0.05 and ^## p < 0.01 indicate significant differences between the NCD vehicle and HFD vehicle; *p < 0.05 and **p < 0.01 indicate significant differences between the HFD vehicle and HFD Hoga1 ASO).