The glucagon-like peptide-1 receptor agonist Exendin 4 has a protective role in ischemic injury of lean and steatotic liver by inhibiting cell death and stimulating lipolysis

Abstract

Nonalcoholic fatty liver disease is an increasingly prevalent spectrum of conditions characterized by excess fat deposition within hepatocytes. Affected hepatocytes are known to be highly susceptible to ischemic insults, responding to injury with increased cell death, and commensurate liver dysfunction. Numerous clinical circumstances lead to hepatic ischemia. Mechanistically, specific means of reducing hepatic vulnerability to ischemia are of increasing clinical importance. In this study, we demonstrate that the glucagon-like peptide-1 receptor agonist Exendin 4 (Ex4) protects hepatocytes from ischemia reperfusion injury by mitigating necrosis and apoptosis. Importantly, this effect is more pronounced in steatotic livers, with significantly reducing cell death and facilitating the initiation of lipolysis. Ex4 treatment leads to increased lipid droplet fission, and phosphorylation of perilipin and hormone sensitive lipase - all hallmarks of lipolysis. Importantly, the protective effects of Ex4 are seen after a short course of perioperative treatment, potentially making this clinically relevant. Thus, we conclude that Ex4 has a role in protecting lean and fatty livers from ischemic injury. The rapidity of the effect and the clinical availability of Ex4 make this an attractive new therapeutic approach for treating fatty livers at the time of an ischemic insult.

Figure 1

Exendin 4 (Ex4) mitigates necrosis after ischemia reperfusion injury. A: Body weights of mice fed a high fat diet and regular chow were monitored for 12 weeks. B: Oil Red O staining showing fat globules (as depicted by a red color) in the livers of mice fed HFD (left) and regular chow (right). C–H: Light microscopic images of H&E staining showing percent necrosis in liver tissue specimens. Representative image of lean control mice (C), lean mice subjected to hepatic ischemia reperfusion injury (IRI) without Ex4 (D) and with Ex4 (20 μg/kg) (E), control mice fed HFD diet (F), HFD fed mice subjected to hepatic IRI without Ex4 (G) and with Ex4 (H). Arrows indicate the areas of necrosis. Insets represent higher magnification of lipid droplets. I: Graphical representation of percent necrosis calculated out of total area per field using Metamorph software (n = 8 per group; P < 0.0001). Open bars represent lean and HFD controls; gray bars represents lean and HFD IRI+; and black bars represent lean and HFD IRI+/Ex4+. J: Serum alanine aminotransferase levels in lean and HFD mice subjected to IRI with and without Ex4 treatment. Open bars represent lean and HFD controls; gray bars represent lean and HFD IRI+; and black bars represent lean and HFD IRI+/Ex4+. Data are mean ± SD; n = 8 per group; lean IRI+ versus HFD IRI+; *P < 0.05; ^†P < 0.01; HFD IRI versus HFD IRI+Ex4+; ^‡P < 0.008.

Figure 2

Exendin 4 (Ex4) decreases apoptosis induced by ischemia reperfusion injury (IRI). High fat diet (HFD)-fed mice were subjected to hepatic IRI with or without Ex4 treatment. A–F: Light microscopic images of H&E stain showing apoptotic bodies in lean and HFD mice with or without Ex4 treatment. The number of apoptotic bodies was counted relative to live cells in four random areas per slide, eight slides per group. Representative images of lean control (A), lean IRI+ (B), lean IRI+/Ex4+ (C) HFD control (D) HFD IRI+(E), HFD IRI+ Ex4 +(F) are shown. Arrows indicate apoptotic bodies. Inset shows higher magnification of apoptotic bodies. G: Graphical representation of the number of apoptotic bodies with or without Ex4 treatment (n = 8 per group; one-way analysis of variance; *P < 0.01). In vivo terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining showing TUNEL-positive cells (green florescence) in lean and HFD mice controls (H and K) subjected to IRI (I and L), and lean and HFD IRI+/Ex4+ (J and M), graphical representation of TUNEL-positive cells (N). Open bars lean and HFD controls, gray bars lean and HFD IRI+, and black bars represent lean and HFD IRI+/Ex4+, respectively. Data are mean ± SD; n = 8; *P < 0.001.

Figure 3

Exendin 4 (Ex) decreases apoptosis induced by ischemia reperfusion injury in hepatocytes in vitro. HuH7 cells were plated on chamber slides and made steatotic by addition of free fatty acids. Cells were then subjected to hypoxia (Hy+) and reperfusion with or without Ex4 treatment, before and after hypoxia. Shown here are representative confocal images of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive stain [green fluorescence (A–F)]. Lean and steatotic hepatocyte controls (A and D), respectively. Lean and steatotic hepatocytes exposed to hypoxia and reperfusion is shown in (B and E), respectively. Lean and steatotic hepatocytes exposed to hypoxia and treated with Ex4 are shown in (C and F), respectively. Arrows represent TUNEL positive cells. Graphical representation of TUNEL-positive cells is shown in (G). These data are representative of three independent experiments done in triplicate (*P < 0.001).

Figure 4

Exendin 4 (Ex4) decreases fat content and causes lipid droplet fission in ischemia reperfusion injury (IRI) of a steatotic liver. Steatotic mice fed a high fat diet (HFD) were sacrificed 24 hours after IRI, liver tissues were fixed in formalin, paraffin sections were processed for H&E, and frozen sections for Oil Red O staining, respectively. Representative image of fat droplets by H&E stain in HFD control mice (A), HFD IRI+ (B) and HFD IRI+/Ex4+ (C). Panel below represents Oil Red O stain in HFD control mice (D), HFD IRI+ (E), and HFD IRI+/Ex4+ (F). Electron microscopic image of liver tissues of steatotic control mice (G and J) and steatotic mice subjected to IRI without Ex4 (H and K) and with Ex4 treatment (I and J). Lipid droplet fusion and fission are indicated by short arrows. Representative image of steatotic mice subjected to IRI showing fusion of lipid droplet (H) and fission of lipid droplets after Ex4 treatment (I) are shown. Higher magnifications of the previously described images (K and L, respectively) are also shown. Graphical representation of triglyceride levels in steatotic mice (M). Open bars represent mice fed HFD control, gray bar represent HFD IRI without Ex4, and the black bar represents HFD IRI+/Ex4+. These data are representative of n = 8 per group; *P < 0.009.

Figure 5

Exendin 4 (Ex4) decreases fat content in steatotic HuH7 cells undergoing ischemia-reperfusion in vitro. Representative images for Oil Red O (ORO) staining of control, non-steatotic HuH7 cells (A), HuH7 free fatty acids (FFA) + (B), HuH7 FFA+/hypoxia (Hy+) (C), and HuH7 FFA+ Hy+/Ex4+ (D). Graphical representation of quantification of ORO stain by image pro software. Open bars are lean and FFA+ controls, gray bar is FFA+/Hy+, and black bar represents FFA+Hy+ Ex4+; *P < 0.006. Shown is a representative figure from three independent experiments done in triplicate.

Figure 6

Exendin 4 (Ex4) upregulates phosphorylation of perilipin and hormone sensitive lipase; and initiates lipolysis in adipocytes and steatotic HuH7 cells. A–F: Confocal immunofluorescence images of adipocytes representing lipolysis. Adipocytes were grown and differentiated and treated with or without Ex4 and subjected to immunofluorescence with phospho-perilipin and phospho-hormone sensitive lipases (HSL) antibodies. Green fluorescence represents fat staining, blue represents nuclear staining, and red represents phospho-perilipin (A–C), and phospho-HSL (D–F). Untreated adipocytes are shown in A and D, adipocytes treated with Ex4 and stained for phospho-perilipin (B) and stained for phospho-HSL (E). Positive control for lipolysis, forskolin (FSK) is shown in C and F. Graphical representation of co-localization intensity of fat and phospho-perilipin (G) fat and phospho-HSL (H) are shown. Open bar represents control, gray bar represents Ex4 treatment, black bar represents FSK-treated cells. Similar immunofluorescence staining for phospho-HSL was performed in HuH7 cells. Nuclear stain (blue), fat stain (green), and phospho-HSL (red) (arrow) and merged images of HuH7 control (I), HuH7 treated with Ex4 (J), and graphical representation of mean staining intensity of phospho-HSL (K). Open bar represents untreated cells and black bar represents Ex4-treated HuH7 cells. This is representative from three independent experiments done in triplicate; *P < 0.04.

Figure 7

Schematic representation of GLP-1R signaling. We have previously shown that activation of GLP-1R by its agonist Exendin 4 (Ex4) increases intracellular cyclic adenosine monophophate (cAMP) levels. Here, we propose that the activation GLP-1R by Ex4 mediates phosphorylation of perilipin and hormone sensitive lipases (HSL) leading to initiation of lipolysis and increased cell survival in steatotic hepatocytes.