β2-Adrenergic receptor agonist induced hepatic steatosis in mice: modeling nonalcoholic fatty liver disease in hyperadrenergic states

Abstract

Nonalcoholic fatty liver disease (NAFLD) is a spectrum of disorders ranging from hepatic steatosis [excessive accumulation of triglycerides (TG)] to nonalcoholic steatohepatitis, which can progress to cirrhosis and hepatocellular carcinoma. The molecular pathogenesis of steatosis and progression to more severe NAFLD remains unclear. Obesity and aging, two principal risk factors for NAFLD, are associated with a hyperadrenergic state. β-Adrenergic responsiveness in liver increases in animal models of obesity and aging, and in both is linked to increased hepatic expression of β₂-adrenergic receptors (β₂-ARs). We previously showed that in aging rodents intracellular signaling from elevated hepatic levels of β₂-ARs may contribute to liver steatosis. In this study we demonstrate that injection of formoterol, a highly selective β₂-AR agonist, to mice acutely results in hepatic TG accumulation. Further, we have sought to define the intrahepatic mechanisms underlying β₂-AR mediated steatosis by investigating changes in hepatic expression and cellular localization of enzymes, transcription factors, and coactivators involved in processes of lipid accrual and disposition-and also functional aspects thereof-in livers of formoterol-treated animals. Our results suggest that β₂-AR activation by formoterol leads to increased hepatic TG synthesis and de novo lipogenesis, increased but incomplete β-oxidation of fatty acids with accumulation of potentially toxic long-chain acylcarnitine intermediates, and reduced TG secretion-all previously invoked as contributors to fatty liver disease. Experiments are ongoing to determine whether sustained activation of hepatic β₂-AR signaling by formoterol might be utilized to model fatty liver changes occurring in hyperadrenergic states of obesity and aging, and thereby identify novel molecular targets for the prevention or treatment of NAFLD.NEW & NOTEWORTHY Results of our study suggest that β₂-adrenergic receptor (β₂-AR) activation by agonist formoterol leads to increased hepatic TG synthesis and de novo lipogenesis, incomplete β-oxidation of fatty acids with accumulation of long-chain acylcarnitine intermediates, and reduced TG secretion. These findings may, for the first time, implicate a role for β₂-AR responsive dysregulation of hepatic lipid metabolism in the pathogenetic processes underlying NAFLD in hyperadrenergic states such as obesity and aging.

Keywords: aging; lipogenesis; liver; obesity; triglycerides.

Graphical abstract

Figure 1.

Intraperitoneal injection of formoterol (Form) induces hepatic steatosis and increases gene expression of lipid droplet-associated proteins in mice. A: liver triglyceride (TG) content was measured and normalized to tissue weight in young (3.6–5.4 mo old) male mice injected with Form (20 µg/g) or saline (Control, Ctr). n = 7 mice/group. B: representative Oil Red O (ORO)-stained liver sections of individual saline (Ctr) and Form-treated mice. Scale bar, 50 µm. C: quantitation of lipid levels in mouse liver determined by ORO staining. n = 6 mice/group. D: mRNA levels of lipid droplet-associated proteins in livers of mice injected with saline (Ctr) or Form measured by quantitative real time PCR. n = 5–7 mice/group. E: hepatic glycogen levels were measured using a glycogen assay kit (Cat. No. MAK016-1KT; Sigma-Aldrich) after Form treatment of mice and compared to levels from saline treated (Ctr) animals. n = 7 mice/group. Data are presented as means ± SE in all studies; *P < 0.05 vs. Ctr; **P < 0.01; ***P < 0.001.

Figure 2.

Expression of enzymes involved in hepatic triglyceride synthesis is increased upon formoterol (Form) treatment. A: hepatic mRNA expression of glycerol-3-phosphate acyltransferase (Gpat), lipin-1, and diacylglycerol acyltransferase 1 (Dgat1) was measured by quantitative real-time PCR in saline- (Control, Ctr) and Form-treated animals. n = 5 mice/group. B: protein levels of lipin-1 in endoplasmic reticulum (ER) and cytosolic (Cyto) compartments of liver were measured by western blot analysis. n = 7 mice/group. Inset: representative blots of lipin-1 protein levels in ER and Cyto fractions. Calnexin and tubulin were used as loading controls for ER and Cyto, respectively. Numbers represent molecular weight markers, kD. Data are presented as means ± SE; *P < 0.05 vs. Ctr; ***P < 0.001; #0.05<P < 0.1.

Figure 3.

Formoterol (Form) treatment upregulates mRNA and protein levels of factors involved in hepatic de novo lipogenesis. A: quantitative real-time PCR of liver samples from mice treated with Form or saline (Control, Ctr) was used to measure mRNA levels of sterol regulatory element binding protein-1c (SREBP-1c), acetyl-CoA carboxylase (ACC), and fatty acid synthase (FAS) involved in de novo lipogenesis. n = 5 mice/group. B: Western blot analysis was used to measure protein levels of SREBP-1c upon Form or saline (Ctr) treatment of mice. Precursor (p)-SREBP-1c; mature (m)-SREBP-1c. n = 7 mice/group. Inset: representative blots of p-SREBP-1c and m-SREBP-1c protein levels; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. Numbers represent molecular weight markers, kD. C: protein levels of FAS were measured by western blot analysis. n = 4 mice/group. Inset: Immunoblot of FAS levels; actin was used as loading control. Numbers represent molecular weight markers, kD. Data are presented as means ± SE; * P <0.05 vs. Ctr; **P < 0.01.

Figure 4.

Formoterol (Form) treatment of mice upregulates lipin-1 nuclear protein levels and peroxisome proliferator activated receptor α (PPARα) and PPARγ coactivator 1α (PGC1α) mRNA and protein levels in liver. A: Western blot analysis was performed to measure lipin-1 protein levels in hepatic nuclear fractions from mice injected with saline (Ctr) or Form. n = 7 mice/group. Inset: representative Western blot depicting lipin-1 protein levels; histone was used as loading control. Numbers represent molecular weight markers, kD. B: quantitative RT-PCR was used to measure mRNA levels of PPARα and PGC1α in livers from Ctr and Form treated mice. n = 5 mice/group. PPARα and PGC1α protein levels in liver homogenates (C) and hepatic nuclear fractions (D) were measured by Western blot analysis. n = 4–7 mice/group. Insets to (C) and (D): representative blots of PPARα and PGC1α, with actin and histone as respective loading controls. Numbers represent molecular weight markers, kD. Data are expressed as means ± SE; *P < 0.05 vs. Ctr; **P < 0.01.

Figure 5.

Hepatic levels of long-chain (14–20 carbon atoms) acylcarnitines (AcCa) are increased in response to formoterol (Form) treatment of mice. AcCa levels in lipid extracts of livers from Form- and saline (Control, Ctr)-treated animals were measured by HPLC-electrospray ionization-mass spectrometry as described in materials and methods. Numbers in parentheses designate the number of carbon atoms:number of double bonds. Data are expressed as means ± SE; n = 5 mice/group; *P < 0.05 vs. Ctr; **P < 0.01; ***P < 0.001.

Figure 6.

Formoterol (Form) treatment reduces VLDL-TG secretion from liver. A: hepatic VLDL-TG secretion was determined by measuring plasma TG concentrations at indicated times after Triton WR-1339 injection (time 0); Triton was administered 22 h after initial injection of saline (Control, Ctr) or Form. n = 4 mice/group. B: protein levels of microsomal triglyceride transfer protein (MTTP) were measured in the liver ER fraction by western blot analysis. n = 7 mice/group. Inset: representative western blot image depicting MTTP protein levels; calnexin was used as loading control. Numbers represent molecular weight markers, kD. C: lipid transfer activity of MTTP was measured in liver homogenates by fluorescence assay as described in materials and methods. n = 4 mice/group. Data are presented as means ± SE; *P < 0.05 vs Ctr. TG, triglyceride; VLDL, very low density lipoprotein.