High-fat diet-induced adipose tissue expansion occurs prior to insulin resistance in C57BL/6J mice

Abstract

Background: To date, there is only scare evidence characterizing the temporal features and progression of metabolic dysfunction in high-fat diet (HFD)-fed obese mice. Hence, its specific pathogenesis remains unclear.

Methods: Sixty 6-week-old male C57BL/6J mice were randomly divided into HFD and control diet (CD) groups and sacrificed at 1, 5, 9, 13, 17, and 21 weeks, respectively. At weekly intervals, intraperitoneal glucose tolerance testing (IPGTT) and intraperitoneal insulin tolerance testing (IPITT) were performed in both groups. A detailed time course in HFD-fed mice was investigated by evaluating the initiation of glucose homeostasis impairment, dyslipidemia, systemic insulin sensitivity, monocyte chemoattractant protein-1 (MCP-1) levels, epididymal white adipose tissue (eWAT) expansion, macrophage content changes, pro-inflammatory (M1)/anti-inflammatory (M2) macrophage imbalance, lipid accumulation in the liver, and β-cell morphometry in the pancreas.

Results: In the HFD group, progressive weight gain and impairments in glucose metabolism (elevated fasting blood glucose and area under the curve (AUC) of IPGTT) were observed from the 3rd week, and a significantly elevated AUC of IPITT was first detected after week 7 of HFD feeding. As for dyslipidemia, after 9 weeks of feeding, the low-density lipoprotein cholesterol level and total cholesterol level in HFD group were significantly higher than those in the CD group (all P < 0.05), whereas no significant differences were shown in triglyceride level. Adipocyte size increased significantly in the HFD group in the 1st week, a phenotypic switch in eWAT from anti-inflammatory (M2) to pro-inflammatory (M1) macrophages was observed in the 5th week, and the metabolic inflammation was distinct in eWAT in the 9th week. Additionally, liver steatosis was considerably obvious at the 17th week and pancreatic β-cell morphometry did not change during 21 weeks of HFD feeding.

Conclusion: The eWAT expansion was detected early in HFD-induced obese mice, which occurred prior to obvious insulin resistance.

Keywords: High-fat diet; Metabolic dysfunction; Metabolic inflammation; Obesity.

Fig. 1

High-fat diet (HFD) led to progressive hyperglycemia and weight gain. (A) Weekly morning fasting body weight (FWt) measured for 21 weeks in control diet (CD)-fed and HFD-fed mice (n = 10/group). (B) Weekly morning fasting blood glucose (FBG) concentrations measured through 21 weeks in CD-fed and HFD-fed mice (n = 10/group). All data are presented as means ± SD (n = 5). Significance was determined by independent samples t-test. ^aP < 0.05; ^bP < 0.01.

Fig. 2

Progressive glucose intolerance and insulin sensitivity assessed with intraperitoneal glucose tolerance testing (IPGTT) and intraperitoneal insulin tolerance testing (IPITT) in control diet (CD)-fed and High-fat diet (HFD)-fed mice. (A–C) IPGTT (2 g/kg) results (A: blood glucose values, B: the corresponding insulin secretion curves, C: Area under the curve (AUC) for the blood glucose levels) for CD and HFD-fed mice at multiple time points through 3 and 7 weeks of both diets. (D–F) IPITT (0.5 unit/kg) results (D: blood glucose values, E: glucose values expressed as percentages of the time 0 value, F: AUC for the blood glucose levels) for CD and HFD-fed mice at multiple time points through 3 and 7 weeks of both diets. All data are presented as means ± SD (n = 5). Significance was determined by independent samples t-test. ^aP < 0.05; ^bP < 0.01.

Fig. 3

High-fat diet (HFD) led to progressive hyperlipidemia. The comparative serum lipid levels in two group after 5-week (A) and 9-week (B) feeding. All data are presented as means ± SD (n = 5). Significance was determined by independent samples t-test. ^aP < 0.05; ^bP < 0.01. LDL-C: low-density lipoprotein TC: total cholesterol; TG: Triglyceride; CD: control diet.

Fig. 4

Dynamic changes in epididymal white adipose tissue (eWAT) over 21-week high-fat diet (HFD) feeding. Histologically quantified number of (A) adipocytes and (B) crown-like structures per mm². In the 1st week of HFD, adipocyte size increased significantly. (C) The serum concentrations of MCP-1 in two groups after 9-week feeding. (D–L) mRNA expression levels of macrophage marker (E: F4/80), pro-inflammatory (M1) macrophage markers (D: MCP-1; F: IL6; G: TNF-α; H: iNOS), anti-inflammatory (M2) macrophage markers (I: CD206; J: Chi3l3), and genes responsible for insulin signaling (K: Resistin; L: Glut4). (M) Western blots of insulin signaling molecules (pAkt and Glut4) in eWAT. All mRNA expression data were normalized to control diet (CD) group and are presented as means ± SD (n = 5). Significance was determined by independent samples t-test. ^aP < 0.05; ^bP < 0.01.

Fig. 5

Prolonged high-fat diet (HFD) feeding resulted in lipid accumulation in the liver. A significant elevation in triglyceride (TG) and cholesterol accumulation in the liver was detected at the 17th week (A) and 21st week (B). Lipid levels are presented as means ± SD (n = 5), and significance was determined by independent samples t-test. Density and integrated optical density (IOD) of red oil-stained liver sections are presented as medians with interquartile ranges, and significance was determined by Wilcoxon's rank sum test. ^aP < 0.05; ^bP < 0.01. TC: total cholesterol; CD: control diet.