Exendin-4, a glucagon-like protein-1 (GLP-1) receptor agonist, reverses hepatic steatosis in ob/ob mice

Abstract

Nonalcoholic fatty liver disease (NAFLD) represents a burgeoning problem in hepatology, and is associated with insulin resistance. Exendin-4 is a peptide agonist of the glucagon-like peptide (GLP) receptor that promotes insulin secretion. The aim of this study was to determine whether administration of Exendin-4 would reverse hepatic steatosis in ob/ob mice. Ob/ob mice, or their lean littermates, were treated with Exendin-4 [10 microg/kg or 20 microg/kg] for 60 days. Serum was collected for measurement of insulin, adiponectin, fasting glucose, lipids, and aminotransferase concentrations. Liver tissue was procured for histological examination, real-time RT-PCR analysis and assay for oxidative stress. Rat hepatocytes were isolated and treated with GLP-1. Ob/ob mice sustained a reduction in the net weight gained during Exendin-4 treatment. Serum glucose and hepatic steatosis was significantly reduced in Exendin-4 treated ob/ob mice. Exendin-4 improved insulin sensitivity in ob/ob mice, as calculated by the homeostasis model assessment. The measurement of thiobarbituric reactive substances as a marker of oxidative stress was significantly reduced in ob/ob-treated mice with Exendin-4. Finally, GLP-1-treated hepatocytes resulted in a significant increase in cAMP production as well as reduction in mRNA expression of stearoyl-CoA desaturase 1 and genes associated with fatty acid synthesis; the converse was true for genes associated with fatty acid oxidation. In conclusion, Exendin-4 appears to effectively reverse hepatic steatosis in ob/ob mice by improving insulin sensitivity. Our data suggest that GLP-1 proteins in liver have a novel direct effect on hepatocyte fat metabolism.

Figures

Fig. 1

The effect of Exendin-4 administration on the rate of net weight gain in ob/ob and their lean littermates. (A) All mice were weighed daily and administration of Exendin-4 is as described in the text for a total of 8 weeks. The data are mean weight in grams (g) for animals in each group (8 animals/group) outlined ± SE. (B) represents the percent reduction of weight gained at the completion of the experiment and compares mean body weight of treated ob/ob mice to their respective treated saline controls. (C) Effect of Exendin-4 administration on the ratio of liver weight to body weight for the respective animals at the time of sacrifice.

Fig. 2

Assessment of lipid content and hepatic histology in the liver of ob/ob mice and their lean littermates after Exendin-4 treatment. (A) Representative hepatic histology of saline-treated ob/ob mice, mice treated with low-dose and high-dose Exendin-4, and lean littermates treated with saline. Liver sections were stained with Oil Red O and Giemsa stain for nuclei. Original magnification: 40×. (B) Quantitative histomorphometric analysis for total lipid content of all hepatic histology for each treatment group; statistical analysis is with respect to saline-treated ob/ob histology. Histomorphometric analysis employed ImageProPlus as described in Materials and Methods. (C) Quantitation of lipid content per gram (wet weight) of liver from ob/ob mice was performed as described in Materials and Methods. Data are mean values performed in triplicate on eight specimens and reveal a significant decrease following high-dose Exendin-4 treatment. (D) Hematoxylin and Eosin staining of liver sections from ob/ob mice and their lean littermates following Exendin-4 treatment; panels are displayed exactly as in (A). Original magnification: 40×.

Fig. 3

TBAR measurements following Exendin-4 treatment reveals that high-dose therapy resulted in significant reduction in oxidative stress. The experiment was designed as described in Materials and Methods and Figs. 1 and 2. The data displayed are mean values ± SE for ob/ob mice treated with low-dose and high-dose Exendin-4 and saline. Values are compared with those of saline-treated ob/ob mice.

Fig. 4

Glucagon-like protein-1 receptor (GLP-1) detection and signaling through cAMP. (A) Immunoblot for rat hepatocyte lysates was performed as described in Materials and Methods. Anti-GLP-1R was a kind gift of Dr. Joel Hebener, Howard Hughes Institute; antibody titer (1:500). Pancreatic βcells were used as a positive control. The representative immunoblot is from three independent experiments. (B) Hepatocyte treatment with either GLP-1 or Exendin-4 significantly increased cAMP production. Shown are the mean cAMP production in pM/106 cells ± SE and compared with untreated hepatocytes. Pretreatment with the GLP-1 antagonist, Exendin fragment 9-39, abolished cAMP production by either GLP-1 or Exendin-4 in rat hepatocytes. Statistical analysis compares mean cAMP values ± SE vs. respective treatments alone. Forskolin served as a positive control. These experiments were performed three times in triplicate.

Fig. 5

The effect of Exendin-4 on mRNA expression of genes encoding SCD-1, SREBP-1c, and PPARα from whole livers in lean and ob/ob mice. (A) Total RNA extracted from liver tissues was used for mRNA expression analysis of SCD-1, SREBP-1c, and PPARα by RT-qPCR as described in Materials and Methods. Level of mRNA expression observed in lean mice treated with saline was set as 100% control; ob/ob mice treated with low-dose Exendin-4 and high-dose Exendin-4 were compared with the ob/ob mice that were treated with saline. Results are expressed as mean ± SE; n = 4 for each group. Each experiment was performed on three separate occasions in triplicate, *P < .01, #P < .05. (B) GLP-1– or Exendin-4–treated cultured rat hepatocytes were harvested for total RNA as described in Materials and Methods. cDNA primers were employed as detailed in Table 1. RT-qPCR was performed for each experiment three times in triplicate for AOX, PPARα, SCD1, SREBP-1c, and ACC. Data represent significant increases in genes associated with oxidation of fatty acids (*P < .05) with concomitant decreases in genes associated with fatty acid and triglyceride synthesis (*P < .05). Data presented are mean values ± SE. Data for GLP-1 and Exendin-4 are compared to untreated rat hepatocytes in serum free-media; data for GLP-1 or Exendin-4 pretreated with Exendin-9-39 are compared with data from respective treatments alone. Pretreatment with Exendin-9-39 abolished the positive effects of either GLP-1 or Exendin-4 on the mRNA for the genes outlined in (B) when compared with their respective treatments alone, §P < .05.