ErbB4 deletion predisposes to development of metabolic syndrome in mice

Abstract

ErbB4, a member of the EGF receptor family, plays a variety of roles in physiological and pathological states. Genetic studies have indicated a link between ErbB4 and type 2 diabetes and obesity, but its role in metabolic syndrome (MetS) has not been reported. In the current study we found that mice with ErbB4 deletion developed MetS after 24 wk on a medium-fat diet (MFD), as indicated by development of obesity, dyslipidemia, hepatic steatosis, hyperglycemia, hyperinsulinemia, and insulin resistance, compared with wild-type mice. ErbB4 deletion mice also exhibited increased amounts of subcutaneous and visceral fat, with increased serum leptin levels, compared with wild-type mice, whereas levels of adiponectin were not significantly different. Histologically, severe inflammation, indicated by F4/80 immunostaining and M1 macrophage polarization, was detected in inguinal and epididymal white adipose tissue in ErbB4 deletion mice. ErbB4 expression decreased during 3T3-L1 preadipocyte differentiation. Administration of neuroregulin 4, a specific ligand for ErbB4, to 3T3-L1 adipocytes had no effect on adipogenesis and lipolysis but significantly inhibited lipogenesis, promoted browning, induced GLUT4 redistribution to the cell membrane, and increased glucose uptake. Neuroregulin 4 also significantly increased glucose uptake in adipocytes isolated from wild-type mice, while these effects were significantly decreased in adipocytes isolated from ErbB4 deletion mice. In conclusion, our results indicate that ErbB4 may play an important role in glucose homeostasis and lipogenesis. ErbB4 deficiency-related obesity and adipose tissue inflammation may contribute to the development of MetS.

Keywords: ErbB4; inflammation; insulin resistance; lipogenesis; neuregulin 4.

Fig. 1.

ErbB4 deletion induced obesity in mice fed the medium-fat diet (MFD). A: body weight during the MFD feeding period. Values are means ± SE (n = 9–10 in each group). #P < 0.05, *P < 0.001 vs. wild-type. B: representative images of 28-wk-old mice fed the MFD for 24 wk. C: weight of liver, heart, kidney, brain, and adipose tissue (AT) at 28 wk of age. D: organ weights in C expressed as percentage of body weight. E: weight of total AT, brown AT (BAT), subcutaneous AT (SAT), and visceral AT (VAT). F: representative image of ErbB4 expression levels in BAT, inguinal white AT (iWAT), and epididymal white AT (eWAT) in wild-type and ErbB4 deletion mice. GAPDH was used as loading control. Values are means ± SE (n = 9–10 in each group). *P < 0.001 vs. corresponding wild-type.

Fig. 2.

ErbB4 deletion induced hyperglycemia and affected glucose homeostasis. A and B: blood glucose and insulin levels in ErbB4 deletion and wild-type mice after a 16-h fast. C: homeostatic model assessment of insulin resistance (HOMA-IR), calculated as follows: HOMA-IR = [glucose (mg/dl)] * [insulin (mU/l)]/405. D: blood glucose levels of ErbB4 deletion and wild-type mice during intraperitoneal glucose tolerance test (ipGTT). E: total area under the curve (AUC) for relative blood glucose levels during ipGTT. F: blood glucose levels during intraperitoneal insulin tolerance test (ipITT) expressed as percentage of initial (baseline) value. Values are means ± SE (n = 9–10 in each group). *P < 0.01 vs. wild-type (in A, B, C, and E) or vs. wild-type at the corresponding time point (D and F).

Fig. 3.

Mice with ErbB4 deletion developed dyslipidemia and hepatic steatosis. A: serum leptin and adiponectin levels in ErbB4 deletion and wild-type mice fed the medium-fat diet for 24 wk. B: blood lipid profile indicating levels of cholesterol (Chol), high-density lipoprotein cholesterol (HDL), triglyceride (TG), and low-density lipoprotein cholesterol (LDL). C: plasma free fatty acid (FFA) levels. D: plasma alanine aminotransferase (ALT) levels. Values are means ± SE (n = 9–10 in each group). *P < 0.01 vs. wild-type. E: representative hematoxylin-eosin- and oil red O-stained sections of liver from wild-type and ErbB4 deletion mice. Scale bars = 100 µm. F: relative hepatic lipogenic gene [sterol regulatory element-binding transcription factor 1 (Srebf1), fatty acid synthase (Fasn), and glucokinase (Gck)] expression levels from real-time PCR assay. Values are means ± SE (n = 6 in each group). *P < 0.05 vs. wild-type.

Fig. 4.

ErbB4 deletion resulted in adipocyte hypertrophy with inflammation. A: hematoxylin-eosin staining of brown adipose tissue (BAT), inguinal white adipose tissue (iWAT), and epididymal white adipose tissue (eWAT) from wild-type and ErbB4 deletion mice indicating hypertrophy with lipid accumulation in all fat depots with ErbB4 deletion compared with wild-type. Scale bars = 100 µm. B: representative image of F4/80 immunofluorescence staining. Red, F4/80; blue, DAPI. Scale bars = 100 µm. C: F4/80-positive cell quantification using ImageJ. HP, high-power. D–F: relative mRNA levels of inducible nitric oxide synthase (iNOS) and arginase 1 (Arg1), preinflammatory cytokines [TNF-α, IL-6, IL-1β, C-C motif chemokine ligand 2 (CCL2), and chemokine (C-X-C motif) ligand 1 (CXCL1)], and anti-inflammatory cytokines (IL-10 and IL-4) in iWAT and eWAT from ErbB4 deletion and wild-type (dashed line) mice. Values are means ± SE. n = 9–10. *P < 0.01 vs. wild-type.

Fig. 5.

EGFR family member expression during 3T3-L1 differentiation and neuroregulin 4 (NRG4) effects on lipogenesis. A: 3T3-L1 differentiation protocol and time line. M, medium. B: oil red O staining of 3T3-L1 preadipocytes (no induction) and adipocytes (with induction) on differentiation day 10. C: representative Western blots of EGFR family member expression levels during 3T3-L1 preadipocyte differentiation. FABP4, marker for adipocyte maturation (fatty acid-binding protein 4). GAPDH was used as loading control. D: NRG4 treatment did not affect cell proliferation. E: NRG4 treatment significantly reduced lipid content in 3T3-L1 adipocytes. CTR, control; OD, optical density. Values are means ± SE. n = 5. *P < 0.05.

Fig. 6.

Effect of ErbB4 signaling on glucose uptake. A: GLUT4 immunofluorescence in 3T3-L1 adipocytes treated for 15 min with insulin or neuroregulin 4 (NRG4) compared with vehicle-only controls (CTR). Secondary antibody (2nd Ab) only was used as negative control for immunofluorescent staining. Scale bars = 100 µm. B: glucose uptake of 3T3-L1 cells treated with NGR4, insulin, or NGR4 + insulin for 30 min. NRG4 treatment significantly stimulated glucose uptake by 3T3-L1 adipocytes, although not as potently as insulin. NRG4 did not show an additive effect on insulin-stimulated glucose uptake. RLU, relative light units; NS, not significant. Values are means ± SE. *P < 0.05. C: glucose uptake of isolated adipocytes from ErbB4 deletion or wild-type mice treated with NRG4 or insulin. Values are means ± SE. n = 5. *P < 0.01 vs. wild-type.

Fig. 7.

Effect of neuroregulin 4 (NRG4) on 3T3-L1 adipocyte browning and lipolysis. A: relative mRNA expression levels of markers for beige or brown adipocytes after treatment of 3T3-L1 cells with NRG4 or isoproterenol (Iso). PAT2, proton/amino acid transporter 2; CD137, cluster of differentiation 37; UCP1, uncoupling protein 1; PRDM16, PR domain zinc finger protein 16; NS, not significant. Values are means ± SE. *P < 0.05 vs. control. B: lipolysis assay of 3T3-L1 adipocytes treated with NRG4, Iso, or Iso + NGR4. Values are means ± SE. n = 5. *P < 0.01.