Preventing High Fat Diet-induced Obesity and Improving Insulin Sensitivity through Neuregulin 4 Gene Transfer

Abstract

Neuregulin 4 (NRG4), an epidermal growth factor-like signaling molecule, plays an important role in cell-to-cell communication during tissue development. Its function to regulate energy metabolism has recently been reported. This current study was designed to assess the preventive and therapeutic effects of NRG4 overexpression on high fat diet (HFD)-induced obesity. Using the hydrodynamic gene transfer method, we demonstrate that Nrg4 gene transfer in mice suppressed the development of diet-induced obesity, but did not affect pre-existing adiposity and body weight in obese mice. Nrg4 gene transfer curbed HFD-induced hepatic steatosis by inhibiting lipogenesis and PPARγ-mediated lipid storage. Concurrently, overexpression of NRG4 reduced chronic inflammation in both preventive and treatment studies, evidenced by lower mRNA levels of macrophage marker genes including F4/80, Cd68, Cd11b, Cd11c, and macrophage chemokine Mcp1, resulting in improved insulin sensitivity. Collectively, these results demonstrate that overexpression of the Nrg4 gene by hydrodynamic gene delivery prevents HFD-induced weight gain and fatty liver, alleviates obesity-induced chronic inflammation and insulin resistance, and supports the health benefits of NRG4 in managing obesity and obesity-associated metabolic disorders.

Figure 1. Nrg4 gene expression was reduced in liver and EWAT of obese mice.

Eight-week-old male C57BL/6 mice were fed a chow or HFD for total 12 weeks, Total RNA was extracted and relative mRNA levels of Nrg4 was determined in liver (a), EWAT (b), SubWAT (c), and BAT (d) by real-time PCR. *P < 0.05 compared to chow-fed mice (n = 3).

Figure 2. Nrg4 gene transfer prevented high fat diet-induced weight gain.

Eight-week-old C57BL/6 male mice were hydrodynamically injected via tail vein of 20 μg of pLIVE-NRG4 or pLIVE-SEAP control plasmid DNA (on days 1 and 28) and fed an HFD for 9 weeks. At the end of the experiment, total RNA was extracted from liver, EWAT, SubWAT, BAT, pancreatic tissues, and the relative mRNA levels of Nrg4 gene were determined by real-time or regular PCR. (a) Body weight-time curve; (b) Fat mass and lean mass at the end of 9-week feeding; (c) Representative images of mice at the end of the experiment; (d) Average food intake; (e) Detection of Nrg4 gene in the liver by regular PCR; and (f) Nrg4 mRNA level in different tissues determined by real time PCR. Each data point represents the mean ± SD of 5 animals. *P < 0.05, **P < 0.01 compared to control animals injected with pLIVE-SEAP plasmid DNA.

Figure 3. NRG4 suppressed chronic inflammation in EWAT and stimulated expression of thermogenic genes in BAT.

Mice were sacrificed after 9 weeks of HFD feeding. Fat pads including EWAT, SubWAT and BAT were collected and weighed. Adipose tissues were fixed in 10% neutrally buffered formalin and H&E staining was performed. Total RNA was extracted from EWAT and BAT and relative mRNA levels of selected genes were determined by real-time PCR. (a) Representative images of H&E staining of EWAT, SubWAT and BAT (100×); (b) Average size of adipocytes in EWAT and SubWAT (calculated from measurements of 200 adipocytes from 5 separate slides); (c) Weight of different adipose pads; (d) Relative mRNA levels of selected macrophage marker genes including F4/80, Cd68, Cd11c, chemotactic factor gene Mcp1, Adiponectin and Atgl; (e) Relative mRNA levels of thermogenic genes including Ucp1, Ucp3, Pgc1α, Cidea and Dio2. *P < 0.05, **P < 0.01 compared to that of control animals injected with pLIVE-SEAP (n = 5).

Figure 4. Nrg4 gene transfer improved hyperinsulinemia and insulin resistance of animals fed an HFD.

(a) Time-dependent blood glucose level followed by IP injection of glucose (1.5 g/kg); (b) Area under the curve from glucose tolerance test (a); (c) Relative ratio of glucose concentration upon IP injection of insulin (0.75 U/kg); (d) Serum insulin levels at the end of the 9-week HFD feeding; (e) Relative mRNA levels of Insulin1 and Insulin2 in pancreas; (f) mRNA levels of G6p and Pepck gene involved in glucogenesis. Each data point represents the mean ± SD of 5 animals. *P < 0.05, **P < 0.01 compared to control animals.

Figure 5. Nrg4 gene transfer inhibited lipogenesis and lipid accumulation in mouse liver.

At the end of the 9-week HFD feeding, mice were sacrificed and livers were collected and fixed for histochemistry. (a) Liver weight at the end of experiment; (b) Representative images of mouse livers; Liver sections were stained with H&E (c) or Oil Red O (d) (original magnifications 100×); (e) Relative mRNA levels of genes involved in hepatic lipid metabolism. Each data point represents the mean ± SD of 5 animals. *P < 0.05, **P < 0.01 compared to control animals.

Figure 6. NRG4 improved insulin resistance in obese mice.

C57BL/6 obese mice fed an HFD were hydrodynamically injected via tail vein with 20 μg of pLIVE-NRG4 or pLIVE-SEAP plasmid for 3 weeks. (a) Change of body weight; (b) Fat and lean mass measured 3 weeks after gene transfer; (c) Total food intake; (d) Time-dependent blood glucose level in glucose tolerance test (1.5 g/kg); (e) Area under the curve from glucose tolerance test; (f) Relative ratio of glucose concentration in insulin tolerance test (0.75 U/kg). *P < 0.05, **P < 0.01 compared to control animals (n = 5).

Figure 7. Effects of NRG4 overexpression on lipid storage in obese mice.

(a–c) Representative images of H&E staining of the liver, EWAT and SubWAT (original magnifications 100×); (d) mRNA levels of genes involved in lipogenesis in the liver; (e) mRNA levels of genes involved in hepatic lipid accumulation. *P < 0.05, **P < 0.01 compared to control animals (n = 5).

Figure 8. NRG4 overexpression reduced chronic inflammation in obese mice.

At the end of the 3-week. after gene transfer, obese animals were sacrificed. (a) Images of EWAT stained with H&E; (b–d) Relative mRNA levels of macrophage marker genes in EWAT, liver, and BAT including F4/80, Cd68, Cd11b, Cd11c and inflammatory factor Mcp1 and Tnfα. *P < 0.05, **P < 0.01 compared to control animals (n = 5).