Genetic Targeting of GRP78 in the VMH Improves Obesity Independently of Food Intake

Abstract

Recent data have demonstrated that the hypothalamic GRP78/BiP (glucose regulated protein 78 kDa/binding immunoglobulin protein) modulates brown adipose tissue (BAT) thermogenesis by acting downstream on AMP-activated protein kinase (AMPK). Herein, we aimed to investigate whether genetic over-expression of GRP78 in the ventromedial nucleus of the hypothalamus (VMH: a key site regulating thermogenesis) could ameliorate very high fat diet (vHFD)-induced obesity. Our data showed that stereotaxic treatment with adenoviruses harboring GRP78 in the VMH reduced hypothalamic endoplasmic reticulum ER stress and reversed vHFD-induced obesity. Herein, we also demonstrated that this body weight decrease was more likely associated with an increased BAT thermogenesis and browning of white adipose tissue (WAT) than to anorexia. Overall, these results indicate that the modulation of GRP78 in the VMH may be a target against obesity.

Keywords: ER stress; GRP78; brown adipose tissue; browning; hypothalamus; thermogenesis; white adipose tissue.

Figure 1

Effect of glucose regulated protein 78 kDa (GRP78) overexpression in the ventromedial nucleus of the hypothalamus (VMH) of very high fat diet (vHFD) rats on energy balance. (A,C) Body weight change, (B,D) average daily food intake (n = 9–10 animals per group) and (E,F) representative Western blot autoradiographic images and corresponding VMH protein levels of endoplasmic reticulum (ER) unfolding protein response (UPR) pathway (n = 7 animals per group) of standard laboratory diet (STD) or vHFD rats stereotaxically treated with green fluorescence protein (GFP) or GRP78 adenoviruses into the VMH. Statistical significance was determined by t-Student test. Error bars represent the SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. (STD or vHFD) Ad GFP. For the Western blot analysis, representative images for all proteins are shown; in the case of the loading controls a representative gel is displayed, although each protein was corrected by its own internal control (β-actin). In the gel images, all the bands for each picture come always from the same gel, but they may be spliced for clarity.

Figure 2

Effect of GRP78 overexpression in the VMH of vHFD rats on brown adipose tissue (BAT) thermogenesis. (A,C) Representative infrared thermal images (left panel) and temperature of the BAT area (right panels; n = 9–10 animals per group), (B,D) rectal temperature (n = 8–9 animals per group) and (E,F) representative Western blot autoradiographic images and corresponding BAT protein levels of UCP1 (n = 7 animals per group) of STD or vHFD rats stereotaxically treated with GFP or GRP78 adenoviruses into the VMH. Statistical significance was determined by t-Student test. Error bars represent the SEM. * p < 0.05, and *** p < 0.001 vs. (STD or vHFD) Ad GFP. For the Western blot analysis, representative images for all proteins are shown; in the case of the loading controls a representative gel is displayed, although UCP1 protein was corrected by its own internal control (α-tubulin). In the gel images, all the bands for each picture come always from the same gel, but they may be spliced for clarity.

Figure 3

Effect of GRP78 overexpression in the VMH of vHFD rats on gonadal white adipose tissue (gWAT) browning. (A) Representative immunohistochemistry with anti-UCP1 antibody showing UCP1 staining (left panels; original magnification 20×; scale bar 100 µm), UCP1 stained area (right upper panels; n = 8–10 animals per group), adipocyte area (right lower panels; n = 8–10 animals per group) and (B,C) relative messenger RNA (mRNA) levels of thermogenic markers (n = 8–10 animals per group) in the gWAT of the STD or vHFD rats stereotaxically treated with GFP or GRP78 adenoviruses into the VMH. Statistical significance was determined by t-Student test or ANOVA. Error bars represent the SEM. * p < 0.05 and *** p < 0.001 vs. (STD or vHFD) Ad GFP; # p < 0.05 and ## p < 0.01 vs. vHFD Ad GFP (panel A).

Figure 4

Effect of GRP78 overexpression in the VMH of vHFD rats on subcutaneous WAT (sWAT) browning. (A) Representative immunohistochemistry with anti-UCP1 antibody showing UCP1 staining (left panels; original magnification 20×; scale bar 100 µm), UCP1 stained area (right upper panels; n = 8–10 animals per group), adipocyte area (right lower panels; n = 8–10 animals per group) and (B,C) relative mRNA levels of thermogenic markers (n = 8–10 animals per group) in the sWAT of the STD or vHFD rats stereotaxically treated with GFP or GRP78 adenoviruses into the VMH. Statistical significance was determined by t-Student test or ANOVA. Error bars represent the SEM. * p < 0.05, and *** p < 0.001 vs. (STD or vHFD) Ad GFP; # p < 0.05 and ### p < 0.001 vs. vHFD Ad GFP (panel A).

Figure 5

Effect of GRP78 overexpression in the VMH of vHFD rats on hepatic steatosis, glucose homeostasis and insulin sensitivity. (A) Representative oil red O stained liver sections (left panel; scale bar 100 µm) and their quantification (right panel; n = 7–10 animals per group), (B) glucose tolerance tests (GTT) (n = 8–10 animals per group), (C) insulin tolerance tests (ITT) (n = 9–11 animals per group) and (D) the area under the curve (AUC) (n = 9–11 animals per group) from ITT of the STD or vHFD rats stereotaxically treated with GFP or GRP78 adenoviruses into the VMH. Statistical significance was determined by t-Student test or ANOVA. Error bars represent the SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. (STD or vHFD) Ad GFP; ### p < 0.001 and vs. vHFD Ad GFP (panel A).

Figure 6

Effect of GRP78 overexpression in the VMH of different rat models of obesity. Correlation analysis (linear regression) between obesity degree and body weight loss in rats fed a vHFD (60% fat; labelled as 1 in the graph; n = 8 animals per group), rats fed a HFD for 6 months (45% fat; labelled as 2 in the graph; n = 20 animals per group) [23], rats fed a HFD for 3 months (45% fat; labelled as 3 in the graph; n = 27 animals per group) [23], obese Zucker rats (labelled as 4 in the graph; OZR; n = 37 animals per group) [17,23], and rats receiving intracerebroventricular (ICV) injections of ceramide 6 (labelled as 5 in the graph; n = 23 animals per group) [17], stereotaxically treated with Ad GRP78 adenoviruses into the VMH; in all the cases the adenoviral treatment lasted 7 days. Error bars represent the SEM.