miR-379 mediates insulin resistance and obesity through impaired angiogenesis and adipogenesis regulated by ER stress

Abstract

We investigated the role of microRNA (miR-379) in the pathogenesis of obesity, adipose tissue dysfunction, and insulin resistance (IR). We used miR-379 knockout (miR-379KO) mice to test whether loss of miR-379 affects high-fat diet (HFD)-induced obesity and IR via dysregulation of key miR-379 targets in adipose tissue. Increases in body weight, hyperinsulinemia, and IR in wild-type (WT)-HFD mice were significantly attenuated in miR-379KO-HFD mice with some sex differences. Relative to control chow-fed mice, in WT-HFD mice, expression of miR-379 and C/EBP homologous protein (Chop) (pro-endoplasmic reticulum [ER] stress) and inflammation in perigonadal white adipose tissue (gWAT) were increased, whereas adipogenic genes and miR-379 target genes (Vegfb and Edem3) were decreased. These changes, as well as key parameters of brown adipose tissue dysfunction (including mitochondrial defects), were significantly attenuated in miR-379KO-HFD mice. WAT from obese human subjects with and without type 2 diabetes showed increased miR-379 and decreased miR-379 target genes. In cultured 3T3L1 pre-adipocytes, miR-379 inhibitors increased miR-379 targets and adipogenic genes. These data suggest that miR-379 plays an important role in HFD-induced obesity through increased adipose inflammation, mitochondrial dysfunction, and ER stress as well as impaired adipogenesis and angiogenesis. miR-379 inhibitors may be developed as novel therapies for obesity and associated complications.

Keywords: Adipogenesis; Angiogenesis; Endoplasmic reticulum stress; GapmeRs; Inflammation; Insulin resistance; MicroRNAs; Obesity.

Graphical abstract

Figure 1

HFD-induced obesity and adipocyte hypertrophy are attenuated in miR-379KO mice (A) Representative image of male (M) and female (F) WT and miR-379KO control (Con) and HFD-fed mice after 24 weeks on the HFD. (B and D) Average body weight in (B) M and (D) F mice at 10 and 24 weeks of HFD feeding. (C and E) Total body fat in (C) M and (E) F mice. n = 5–8/group for 10 weeks of study and n = 11–14/group for 24 weeks of study in M mice. n = 5–8/group for 10 weeks of study and n = 8–9/group for 24 weeks of study in F mice. Each dot represents one mouse/group. (F) The size of adipocytes in gWAT sections (H&E staining), which are smaller in miR-379 KO-HFD mice versus WT-HFD mice at 10 and 24 weeks on the HFD in M and F mice, respectively. (G and H) Quantitative analysis of adipocyte size in (G) M and (H) F mice. n = 100 adipocytes/group. One-way ANOVA with post hoc Tukey’s test for multiple comparisons; ±SEM; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Scale bars, 50 μm; 40× magnification.

Figure 2

miR-379KO-HFD mice show improved insulin sensitivity (A and B) Insulin tolerance test (ITT) in M mice after (A) 10 and (B) 24 weeks of HFD feeding. (C and D) ITT in F mice at (C) 10 and (D) 24 weeks of HFD feeding. n = 4–8/group. (E and F) Plasma insulin levels in (E) M and (F) F mice after 24 weeks of HFD feeding. n = 7–11/group for M; n = 5/group for F. (G and H) Homeostatic model assessment of IR (HOMA-IR) in (G) M and (H) F mice. n = 7–11/group for M; n = 5/group for F. Each dot represents one mouse/group. (A–D) ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus WT-Con. ⁺p < 0.05, ⁺⁺p < 0.01versus WT-HFD, using two-way repeated measures ANOVA. One-way ANOVA with post hoc Tukey’s test for multiple comparisons; ±SEM; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 3

Lack of miR-379 promotes mitochondrial function markers in BAT (A) H&E staining shows increased lipid droplets and whitening of brown adipose tissue (BAT) in M and F WT-HFD mice, which are reduced in miR-379KO-HFD mice. The arrow indicates lipid droplets in BAT. (B and C) IHC staining of (B) FIS1 and (C) UCP-1 in BAT in M and F mice. (D and E) Quantitative analysis of FIS1 expression in (D) M and (E) F mice after 24 weeks of HFD feeding. n = 20 areas/group. (F and G) Quantitative analysis of UCP-1 expression in (F) M and (G) F mice after 24 weeks of HFD feeding. n = 20 areas/group/M. n = 40 areas/group/F. Scale bars, 50 μm; 20× magnification. (H and I) miR-379 gene expression in BAT of (H) M and (I) F mice. n = 5/group. (J and K) Fis1 gene expression in (J) M and (K) F mice. (L and M) Ucp-1 gene expression in (L) M and (M) F mice at 24 weeks of HFD feeding. n = 5–6/group. Each dot represents one mouse/group. One-way ANOVA with post hoc Tukey’s test for multiple comparisons; ±SEM; ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 4

Increased expression of miR-379 and Chop and decreased expression of adipogenic markers in gWAT of WT-HFD mice are reversed in samples from miR-379KO-HFD mice (A and B) miR-379 expression in (A) M and (B) F mice. n = 5–7/group. (C and D) Chop expression in (C) M and (D) F mice. n = 5–6/group. (E–J) expression of the adipogenesis-related genes Pparg, Cebpβ, and Foxo1 in (E–G) M and (H–J) F mice in gWAT. n = 4–6/group. (K and L) Expression of miR-379 target genes (Edem3, Fis1, and Vegfb) in (K) M and (L) F mice. (M and N) Expression of Flt1 (VEGF receptor 1) in (M) M and (N) F mice. 24 weeks of HFD experiments. n = 5–6/group. Each dot represents one mouse/group. One-way ANOVA with post hoc Tukey’s test for multiple comparisons; ±SEM; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 5

miR-379KO reverses HFD-mediated suppression of EDEM3 and the angiogenic markers VEGFB and CD31 in gWAT (A–C) Immunofluorescence (IF) staining of EDEM3 (A), IHC staining of VEGFB (B), and IF staining of CD31 (C) in gWAT sections of M and F mice. (D and E) Quantitative analysis of EDEM3 in (D) M and (E) F mice. n = 20 areas/group. Scale bars, 100 μm. 20× magnification. Green, EDEM3. (F and G) Quantitative analysis of VEGFB in (F) M and (G) F mice. n = 40 areas/group. Scale bars, 50 μm. 40× magnification. (H and I) Quantitative analysis of CD31 in (H) M and (I) F mice. n = 20 areas/group. Scale bars, 100 μm. 20× magnification Green, CD31; red, Perilipin; blue, nucleus, DAPI. 24 weeks of HFD experiments. One-way ANOVA with post hoc Tukey’s test for multiple comparisons, ±SEM; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 6

miR-379 regulates inflammatory gene expression and insulin signaling under HFD feeding (A–D) Expression of IL-1β in gWAT in (A) M and (B) F mice and Mcp-1 in (C) M and (D) F mice. n = 4–8/group. Each dot represents 1 mouse/group. (E) IF staining of F4/80 (red signals) in gWAT sections in M and F mice. (F and G) Quantitative analysis of F4/80 in (F) M and (G) F mice (n = 40–50 areas/group). (H) PAS (periodic acid-Schiff) staining in gWAT sections. 24 weeks of HFD experiments. Scale bars, 50 μm; 40× magnification. (I–N) miR-379 plays a role in HFD-mediated suppression of insulin signaling in gWAT. (I) Levels of p-AKT in M and F mice. (J and K) Quantitative analysis of pAKT in (J) M and (K) F mice. n = 15 area/group. (M and N) Quantitative analysis of p-INSR in (M) M and (N) F mice after 24 weeks of HFD feeding. n = 20–30 areas/group. Scale bars, 50 μm; 40× magnification. One-way ANOVA with post hoc Tukey’s test for multiple comparisons ±SEM; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 7

miR-379 is increased, and key targets of miR-379 are decreased, in adipose tissue from obese human subjects (A) Body mass index (BMI) in lean, obese, and obese individuals with T2D (obese/T2D). n = 10–11/group. (B) Quantitative analysis of adipocyte size (n = 100 adipocytes/group). (C) Adipose tissue miR-379 expression (n = 8–10/group). (D) Correlation of miR-379 gene expression with BMI (n = 18). (E) Expression of target genes in adipose tissue from lean, obese, and obese/T2D donors. (F–H) adipose tissue gene expression of (F) CHOP, (G) PPARG, and (H) TNF-α. n = 4–7/group. (I) IHC staining of VEGFB (scale bars, 50 μm; 40× magnification) and IF staining of VE-Cadherin in adipose tissue from lean, obese, and obese/T2D donors. Scale bars, 50 μm; 40× magnification. Green, VE-Cadherin; red, Perilipin; blue, nucleus, DAPI. (J and K) Quantitative analysis of (J) VEGFB (n = 20–30 areas/group) and (K) VE-Cadherin (n = 20 areas/group). Data were analyzed using Student’s t tests for comparisons between two groups. ±SEM; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 8

miR-379 regulates key factors related to adipogenesis in 3T3L1 adipocytes 3T3L1 pre-adipocytes were transfected with the miR-379 mimic oligo and miR-379 inhibitor (200 nmol/L). Cells transfected with negative Con (NC) oligos (200 nmol/L) were used as NCs. (A) miR-379 expression in cells transfected with the miR-379 mimic oligo. (B) miR-379 expression in cells transfected with the miR-379 inhibitor. n = 3–4/group. (C and D) Expression of the miR-379 target genes (C) Edem3 and (D) Vegfb. (E–H) Expression of (E) Chop and the adipogenesis-related genes (F) Cebpβ, (G) Foxo1, and (H) Pparg. n = 8–12/group. (I–R) 3T3L1 pre-adipocytes were transfected with miR-379 mimic or miR-379 inhibitor oligos or NC. (I) miR-379 expression in miR-379 mimic-treated cells. (J) miR-379 expression in miR-379 inhibitor-transfected cells. n = 4/group. (K) Differentiation was confirmed using oil red O staining. Cells transfected with NC oligos (200 nmol/L) were used as NCs. (L) Percentage of oil red O-positive area (n = 20 area/group). (M and N) Expression of the miR-379 target genes (M) Edem3 and (N) Vegfb. (O–R) Expression of (O) Chop and the adipogenesis-related genes (P) Cebpβ, (Q) Foxo1, and (R) Pparg. n = 4–8/group. (S–X) 3T3L1 pre-adipocytes were transfected with NC GapmeR or miR-379-GapmeR (1 μmol/L) as described under materials and methods. Non-transfected cells were used as a Con. (S) miR-379 expression (n = 8/group). (T and U) Expression of the miR-379 target genes (T) Edem3 and (U) Vegfb. (V–X) Expression of (V) Chop and the adipogenic related genes (W) Cebpβ and (X) Pparg. n = 6–8/group. Data were analyzed using Student’s t tests for comparisons between two groups. ±SEM; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.