Potential of incretin-based therapies for non-alcoholic fatty liver disease

Journal of Diabetes and Its Complications 27 (2013) 401–406

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Journal of Diabetes and Its Complications j o u r n a l h o m e p a g e : W W W. J D C J O U R N A L . C O M

Potential of incretin-based therapies for non-alcoholic fatty liver disease☆ Susan L. Samson ⁎, Mandeep Bajaj ⁎ Baylor College of Medicine, St. Luke's Episcopal Hospital, Houston, TX, 77030 USA

a r t i c l e

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Article history: Received 21 October 2012 Received in revised form 5 December 2012 Accepted 17 December 2012 Available online 24 January 2013 Keywords: Glucagon like peptide-1 GLP-1 Exenatide DPP4 Hepatic steatosis NAFLD Fatty liver AMP kinase GLP-1 receptor Gliptin Obesity

a b s t r a c t Non-alcoholic fatty liver disease (NAFLD) is becoming an epidemic, paralleling the increased prevalence of obesity and diabetes, which are risk factors. In this review, we present the current pre-clinical evidence showing that GLP-1 analogues and DPP4 inhibitors can improve hepatic steatosis. Although some of the effects could be due to overall improvement in metabolic parameters, there are data to support improvements independent of weight loss, as well as direct effects on the hepatocyte in vitro. Multiple hepatocyte signal transduction pathways appear to be activated by GLP-1 and its analogues, with both AMP-activated protein kinase and Akt proposed to be key players in improving hepatic steatosis. However, it is controversial as to whether the pancreatic-type GLP-1 receptor is present or responsible for conferring the GLP-1 signal in the hepatocyte. In total, the data support the need for more rigorous prospective clinical trials to further investigate the potential of incretin therapies for treatment of NAFLD. © 2013 Elsevier Inc. All rights reserved.

1. Introduction The prevalence of non-alcoholic fatty liver disease (NAFLD) has paralleled the rise in obesity and diabetes, which are major risk factors (Krawczyk, Bonfrate, & Portincasa, 2010). NAFLD is now the most common cause of chronic liver disease (Starley, Calcagno, & Harrison, 2010), and is present in one quarter to half of diabetes patients (Mazza et al., 2012). NAFLD encompasses a continuum of pathological changes to the liver. Initially, triglycerides accumulate in hepatocytes causing steatosis, which then can lead to steatohepatitis (NASH), characterized by chronic inflammation, fibrosis and necrosis (Starley et al., 2010). Finally, cirrhosis will develop in 20% of patients with NASH (Krawczyk et al., 2010). Cirrhosis is a risk factor for hepatocellular carcinoma (HCC), which is the third cause of cancer

☆ Conflict of interest statement: SLS has no potential conflicts of interest to disclose. MB has received research grants from Takeda, Amylin, Eli Lilly, Bristol-Myers Squibb, and Astra Zenica; lecture fees from Takeda, Eli Lilly, and Boehringer Ingelheim and Sanofi-Aventis; and has served as a consultant to Takeda and Sanofi-Aventis. ⁎ Corresponding authors. Susan L. Samson is to be contacted at Department of Medicine, Diabetes Research Center, Baylor College of Medicine, One Baylor Plaza, ABBR R615, Houston TX 77030. Tel.: +1 713 798 3076; fax: +1 713 798 8764. Mandeep Bajaj, Departments of Medicine and Molecular and Cellular Biology, Division of Endocrinology, Diabetes and Metabolism, Baylor College of Medicine, 1709 Dryden Street, Houston TX 77030. Tel.: +1 713 798 1712; fax: +1 713 798 5214. E-mail addresses: [email protected] (S.L. Samson), [email protected] (M. Bajaj). 1056-8727/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jdiacomp.2012.12.005

death worldwide (Krawczyk et al., 2010; Zhang et al., 2012). Disturbingly, HCC also can form in the setting of NASH without a background of cirrhosis (Baffy, Brunt, & Caldwell, 2012). Patients with type 2 diabetes have double or triple the risk of developing HCC, which is further exacerbated when combined with another risk factor such as viral hepatitis or alcohol abuse (Baffy et al., 2012; Lai et al., 2012). Because lifestyle changes and weight loss are exceedingly difficult to achieve by most patients, an effective pharmacologic treatment for NAFLD is desperately needed. There are accumulating pre-clinical data showing that incretinbased therapies have the potential to improve hepatic steatosis in the setting of obesity and diabetes. The incretin hormones are Glucagonlike peptide-1 (GLP-1) and Glucose-dependent insulinotropic polypeptide (GIP) (Meier, 2012). Both are secreted from intestinal endocrine cells in response to a meal and give rise to the “incretin effect”, i.e. insulin secretion is augmented following oral glucose intake (McIntyre, Holdsworth, & Turner, 1965). In type 2 diabetes, the β-cell response to GIP is blunted, but the response to GLP-1 is partially intact, so that administration of supraphysiologic amounts of GLP-1 can still improve first phase and late phase insulin secretion (Quddusi, Vahl, Hanson, Prigeon, & D'Alessio, 2003). As a result, focus has been on development of GLP-1-based therapies for type 2 diabetes. Endogenous GLP-1 undergoes proteolytic cleavage within minutes of secretion by the ubiquitous dipeptidyl peptidase 4 (DPP4) (Nauck, 2011). This enzyme recognizes the alanine at the second amino acid (a.a.) position of GLP-1 (as 7–37 or 7–36 amide), leaving GLP-1 as 9–

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37 or 9–36 amide (GLP9). In order to harness the potential of GLP-1 for clinical use, the development of incretin-based treatments for diabetes has taken two approaches: 1) modifications of GLP-1 or an analogous peptide to avoid DPP4 mediated degradation and clearance or, 2) inhibition of DPP4 activity to increase the duration of action of endogenous GLP-1 (Ahren & Schmitz, 2004). There are multiple GLP-1 receptor (GLP-1R) agonists in development, with exenatide and liraglutide currently approved for clinical use. As peptides, these agents require delivery by injection. On the other hand, the DPP4 inhibitors or “gliptins” are oral medications. Because DPP4 inhibitors protect endogenous GLP-1 from degradation, they do not achieve pharmacologic levels of GLP-1. As a result, there are improvements in glycemic control but without the associated weight loss which is observed with the GLP-1R agonists (Drucker & Nauck, 2006). DPP4 inhibitors currently in clinical use worldwide are sitagliptin, saxagliptin, linagliptin, and vildagliptin. 2. Incretin based therapies in models of hepatic steatosis Animal models of diabetes and obesity provide a means to investigate the effects of GLP-1 based therapies in the pre-clinical setting (Table 1). To find all relevant publications, our search strategy was to pair the terms incretin, GLP-1, GLP-1 receptor agonist, exenatide, or liraglutide with NAFLD or hepatic steatosis. Long-term exenatide administration has been associated with decreases liver triglyceride content in obese mouse models, including leptin-deficient Ob/Ob mice, leptin receptor deficient Db/Db mice, and high fat diet fed mice (Ding, Saxena, Lin, Gupta, & Anania, 2006; Samson et al., 2008; Samson et al., 2011). In recent years, researchers have worked to refine the composition of diets for rodent studies of NAFLD to induce histologic changes comparable to those seen in humans, such as a combination of high trans-fat (45% of calories from fat, with 30% trans-fat) with a high fructose corn syrup equivalent in the drinking water, termed the “American Lifestyle-Induced Obesity Syndrome” (ALIOS) diet (Tetri, Basaranoglu, Brunt, Yerian, & Neuschwander-Tetri, 2008). Mice on this diet develop necroinflammatory and fibrotic changes in the liver, with elevated TNFα and procollagen αI expression (Tetri et al., 2008). Liraglutide treatment reduces hepatic steatosis in mice on the ALIOS diet (Sharma, Mells, Fu, Saxena, & Anania, 2011). Treatment of mice with a modified ALIOS diet, with added cholesterol, using the Ex4 analogue, AC3174 (Met14Leu), significantly reduces steatosis and immunostaining for collagen I, as a marker of hepatic fibrosis (Trevaskis et al., 2012).

From animal data, benefits are not limited to GLP-1R agonists, which are given at pharmacologic doses, but also in models with DPP4 deficiency or inhibition, where endogenous GLP-1 is moderately elevated from baseline. Administration of the DPP4 inhibitor desfluoro-sitagliptin to mice on a linoleic acid and sucrose diet decreases liver triglycerides and the histologic grade of hepatic steatosis (Shirakawa et al., 2011). DPP4-deficient rats have 3-fold higher basal GLP-1 levels and significantly reduced serum and hepatic triglycerides levels compared with controls (Ben-Shlomo et al., 2011). Biliary secretory dysfunction caused by high fat diet was improved in the DPP4-deficient rats as indicated by lowered aminotransferase levels, decreased bile acid production, and increased synthesis of bile acid transporters (Shlomo et al., 2012). Do these findings in rodent models of obesity and diabetes translate to humans? Case reports of improvements in hepatic steatosis following exenatide and liraglutide treatment have been published (D'Amico, 2011; Tushuizen et al., 2006). Furthermore, in a cohort of patients treated with exenatide for more than three years, significant reductions in alanine aminotransferase (ALT) were observed, as an indirect marker of steatosis (Klonoff et al., 2008). The subjects with elevated baseline ALTs and with the most substantial weight loss during the study also had the most significant decline in ALT. A meta-analysis of clinical data from 12 trials also showed that ALT levels decrease with liraglutide therapy, although not with exenatide (Vilsboll, Christensen, Junker, Knop, & Gluud, 2012). In a retrospective study of type 2 diabetes patients with NAFLD, as determined by ultrasonography and elevated ALT, treatment with liraglutide but not sitagliptin reduced ALT and the AST-to-platelet counts ratio index (APRI), suggested to reflect hepatic fibrosis (Ohki et al., 2012). All of these results are in the context of significant weight loss among the subjects. In the only prospective study in humans, we treated patients with type 2 diabetes with either pioglitazone or combined pioglitazone and exenatide for one year (Sathyanarayana et al., 2011), along with nutritional counseling for a weight maintaining diet. After 12 months, both groups had significant reductions in liver fat and ALT levels, but the combination therapy of exenatide and pioglitazone (− 60%) was superior to pioglitazone alone (− 40%). Importantly, the combination treatment group in our study did not have a significant change in weight, suggesting that there could be a direct effect of exenatide on liver steatosis independent of metabolic improvements from weight loss. Although we cannot rule out that the reduction in hepatic steatosis with exenatide could require concomitant treatment with

Table 1 Evidence for decreased hepatosteatosis in animal models using incretin-based therapies. Species

Diet to induce NAFLD

GLP-1R Obese Ob/Ob mice N.A. agonists (leptin deficient) C57BL/6 mice and Db/Db HFD (42 kcal% fat) (leptin receptor deficient) C57BL/6 mice HFD (60 kcal% fat)

DPP4

C57BL/6 mice

ALIOS

C57BL/6 mice

HFD (60 kcal% fat)

Ob/Ob (leptin deficient) and C57BL/6 mice C57BL/6 DPP4-deficient rats C57BL/6 mice

Treatment

Outcomes

Associated with Reference Weight loss?

Ex4 injection

↓ Hepatic lipid

Yes

Ding, 2006

Helper dependent Adenovirus expressing Ex4 Osmotic pump administration of Ex4 Liraglutide injection

↓ Hepatic TG

Yes

Samson, 2008

↓ Hepatic TG content

No

Samson, 2011

↓ Hepatic lipid staining (Oil Red O) ↓ Hepatic TG

No Yes

Sharma, 2011; Mells, 2012 Tomas, 2011

↓ Hepatic Lipid; ↓ Collagen-I; ↓ ALT, not AST ↓ Hepatic TG ↓ Hepatic TG; ↓ ALT, AST ↓ Hepatic TG; ↓ Histologic grade of steatosis (Masson-Goldner staining)

No if pair-fed

Trevaskis, 2012

Yes No No

Lee, 2012 Ben-Shlomo, 2011 Shirakawa, 2011

Osmotic pump infusion of GLP9 (GLP-1 a.a. 7-36-amide) ALIOS with cholesterol (2 wt%) Osmotic pump infusion of and HTF or HLF (40 kcal% fat) AC3174 (Ex4 analogue) HFD (45 kcal% fat) Ex4 injections Chow and HFD (40 kcal% fat) N.A. High sucrose (36%) with Des-fluoro-sitagliptin linoleic or oleic acid

ALIOS = American Lifestyle-Induced Obesity Syndrome diet containing trans-fat and high fructose corn syrup equivalent; ALT = Alanine aminotransferase; AST = Aspartate aminotransferase; Ex4 = exendin 4 or exenatide; HFD = High fat diet; HTF = high trans-fat/fructose/cholesterol; HLF = high lard fat/ fructose/cholesterol; kcal% = fat% of diet determined by calories; MRS = Magnetic Resonance Spectroscopy; N.A. = Not applicable; TG = triglyceride; wt% = fat% of diet determined by weight.

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pioglitazone to achieve the results seen in this study, the findings are biologically plausible considering the pre-clinical animal data that has been discussed above. Some of the experimental data in rodents support the possibility of weight-independent effects of incretin therapies on hepatic steatosis. In DPP4 deficient rats, weight is not significantly affected compared to controls while the improvements in steatosis are notable (BenShlomo et al., 2011; Shirakawa et al., 2011). We have previously infused Ex4 by osmotic pump for 4 weeks in mice on a high fat diet (Samson et al., 2011). With this short period of treatment, there were no significant effects on weight or body fat, and yet liver triglycerides were markedly reduced. In another study with pair-feeding to equalize weight loss, the Ex4 analogue AC3174 was still able to elicit significant reductions in liver weight and lipid content (Trevaskis et al., 2012). There are in vitro data pointing to a direct effect of GLP-1 and its analogues on hepatocyte fatty acid metabolism. Treatment of Huh7 and HepG2 hepatoma cell lines with Ex4 is able to decrease intracellular lipid accumulation in the presence of free fatty acids in the media (Gupta et al., 2010; Lee et al., 2012). There are significant changes in hepatocyte gene expression with potential to enhance fatty acid oxidation while reducing lipogenesis (Ben-Shlomo et al., 2011; Ding et al., 2006; Samson et al., 2008; Shirakawa et al., 2011). There are consistent data among different studies and laboratories that treatment of mice with GLP-1 and analogues causes a decrease in hepatic transcript levels of fatty acid synthase, stearoyl CoA desaturase I, and sterol regulatory element-binding protein-1c (SREBP-1c), with the latter regulating the transcription of key enzymes involved in de novo fatty acid synthesis (Ben-Shlomo et al., 2011; Ding et al., 2006; Lee et al., 2012; Samson et al., 2008; Shirakawa et al., 2011). In some studies, transcript levels are increased for peroxisome proliferator-activated receptor (PPAR)α, which regulates gene expression of important enzymes for fatty acid oxidation (Ben-Shlomo et al., 2011; Ding et al., 2006; Samson et al., 2008; Shirakawa et al., 2011), and carnitine palmitoyl transferase I (CPT1), which regulates fatty acid transport to the mitochondria for beta oxidation (Ben-Shlomo et al., 2011; Shlomo et al., 2012). Others have shown that there are increases in expression of fatty acid binding protein, for uptake into the liver, ACOX2, which is responsible for peroxisomal β-oxidation, and the microsomal triglyceride transfer protein (MTTP), to improve lipid packaging and VLDL secretion (Mells et al., 2012). Some of the hepatic gene expression changes observed in vivo in rodent models can be recapitulated by treatment of hepatocytes in vitro with GLP-1 or another analogue confirming that the effects of incretin therapies on liver steatosis could in part be due to a direct effect of the peptides on hepatocytes, in addition to metabolic improvements (Ding et al., 2006; Shirakawa et al., 2011; Svegliati-Baroni et al., 2011). GLP-1R agonists reduce endoplasmic reticulum (ER) stress in the beta cell, including in the setting of lipotoxicity, thereby increasing cell survival (Cnop, Ladriere, Igoillo-Esteve, Moura, & Cunha, 2010; Cunha et al., 2009; Tsunekawa et al., 2007; Yusta et al., 2006). NAFLD also is a state in which there is a component of ER stress and dysregulation of the unfolded protein response (UPR) leading to the pathologic findings (Puri et al., 2008). To reproduce this state in vitro, primary human hepatoctyes will take up free fatty acids from the culture media causing decreased cell viability due to ER stress and the UPR, ending in apoptosis (Sharma et al., 2011). Treatment of the fat-laden hepatocytes with Ex4 in culture results in decreased fat content with decreased cleaved caspase 3 and improved cell survival (Sharma et al., 2011). Accompanying these observations are reduced ER stress markers and the activation of cell autophagy as a mechanism to reduce intracellular free fatty acids and protect the hepatocytes. GLP-1 also may decrease the production of inflammatory mediators, such as TNFα, which cause progression to hepatic necrosis and fibrosis (Ben-Shlomo et al., 2011), and oxidative stress markers in the setting of steatosis (Ding et al., 2006).

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3. Mechanisms of action There are accumulating data regarding GLP-1 mediated effects on hepatocyte signal transduction pathways. Direct activation of phosphoinositol-3-kinase, p70 S6 kinase, p44 and p42 MAP kinases with in vitro treatment with GLP-1 was reported more than a decade ago (Lopez-Delgado, Morales, Villanueva-Penacarrillo, Malaisse, & Valverde, 1998; Marquez, Gonzalez, Puente, Valverde, & Villanueva-Penacarrillo, 2001; Marquez, Trapote, Luque, Valverde, & Villanueva-Penacarrillo, 1998; Redondo, Trigo, Acitores, Valverde, & Villanueva-Penacarrillo, 2003; Trapote et al., 1996). Other investigators have independently confirmed many of these findings in more recent reports (Aviv et al., 2009; Gupta et al., 2010; SvegliatiBaroni et al., 2011). From these reports, GLP-1 and analogues can directly activate signal transduction pathways in hepatoctyes, but which pathways are responsible for the effects on steatosis? A central molecule may be AMP-activated protein kinase (AMPK) ( Fig. 1). AMPK is the energy sensor of the cell and is regulated by the relative levels of AMP and ATP in the cell (Viollet et al., 2009). Binding of AMP to the regulatory subunit modulates the stability of AMPK phosphorylation and the kinase activity of its catalytic subunit (Viollet et al., 2009). AMPK activation results in promotion of energy generating cellular processes while inhibiting energy consuming processes (Foretz & Viollet, 2011). AMPK phosphorylates and inactivates acetyl CoA carboxylases (ACC1 and 2). When ACC1 is inactivated, there is a decrease in the levels of malonyl CoA, the precursor for fatty acid chain elongation. Furthermore, with the decrease in malonyl CoA, there is release in the inhibition of carnitine palmitoyl transferase 1 (CPT1), which is then able to shuttle fatty acids into the mitochondria for β-oxidation (Schreurs, Kuipers, & van der Leij, 2010). In addition to these processes, AMPK also inhibits the activity of sterol regulatory element-binding protein-1c (SREBP-1c), which regulates the transcription of key enzymes involved in de novo fatty acid synthesis, such as fatty acid synthase (Li et al., 2011; Zhou et al., 2001). This could tip the balance from increased fatty acid synthesis and storage as triglyceride to the increased utilization of fatty acids. The overall consequence is a decrease in steatosis. We have reported increased phosphorylation of liver AMPK and its downstream target ACC by in vivo Ex4 treatment in a high fat diet mouse model of obesity and hepatic steatosis (Samson et al., 2011).

Fig. 1. A schematic diagram of the signal transduction pathways proposed to have a role in decreasing hepatocyte steatosis with incretin-based therapies. (Abbreviations: AMPK, AMPactivated protein kinase; cAMP, cyclic AMP; PDK-1, 3-phosphoinositide-dependent kinase1; PI3K, Phosphoinositide-3-kinase; PKA, Protein Kinase A; PKB/Akt, Protein Kinase B).

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Similarly, AMPK is activated in the hepatic tissue of DPP4-deficient rats, which have elevated endogenous GLP-1 levels, and expression of dominant negative AMPK abrogates the effects on steatosis (BenShlomo et al., 2011). The investigators also confirm the ability of GLP1 to activate AMPK phosphorylation with in vitro treatment of cultured hepatocytes (Ben-Shlomo et al., 2011). The upstream activators which mediate GLP-1 effects on AMPK phosphorylation and activity remain to be clarified. AMPK is phosphorylated by serine-threonine liver kinase B1 (LKB1), which responds to cellular energy status (Viollet et al., 2009). In the case of metformin, AMPK also is activated by an increase in the AMP/ATP ratio induced by inhibition of complex I of the mitochondrial electron transport chain (Foretz et al., 2010), and it is unknown whether GLP-1 and its analogues could affect mitochondrial function as well. However, it is intriguing that a smaller nine a.a. fragment of GLP-1, GLP-1(28–36) can localize to mitochondria (Tomas, Wood, Stanojevic, & Habener, 2011) and further experiments will be needed to determine the functional significance of these observations. In HepG2 and Huh7 cell lines, Ex4 treatment increases transcript levels of Sirtuin 1 (SIRT1) (Lee et al., 2012), a nutrient sensor which activates the LKB1-AMPK pathway (Ruderman et al., 2010). Moreover, nicotinamide, a SIRTI inhibitor, decreases AMPK phosphorylation, confirming that SIRT1 is upstream of AMPK in this particular pathway. A key consequence of SIRT1 and AMPK activation is the activation of peroxisome proliferator-activated receptor-γ (PPARγ) co-activator 1-α (PGC1α), which promotes mitochondrial biogenesis and fatty acid oxidation (Ruderman et al., 2010). However, these findings are from in vitro studies with cell lines and require further confirmation. In vivo, an upstream candidate is Fibroblast Growth Factor 21 (FGF21), which is a circulating factor which activates AMPK and SIRT1 (Chau, Gao, Yang, Wu, & Gromada, 2010). Hepatic steatosis is a state of FGF21 resistance, as reported for obese mice (Fisher et al., 2010) as well as humans (Dushay et al., 2010), and is associated with elevation of FGF21 levels in an attempt to overcome this resistance. In a high fat diet mouse model, Ex4 infusion decreased FGF21 resistance resulting in increased AMPK activation, and decreased hepatic steatosis (Samson et al., 2011). In addition to AMPK, there is a body of work implicating Akt signaling in the actions of GLP-1 at the level of the hepatocytes (Fig. 1). Redondo et al. (Redondo et al., 2003) first showed that Akt signaling is directly increased in hepatocytes in response to GLP-1 agonists, and this was confirmed more recently by other groups (Aviv et al., 2009; Gupta et al., 2010; Redondo et al., 2003; Svegliati-Baroni et al., 2011). Gupta et al. have focused on Akt, rather than AMPK, as the key player in the reduction of steatosis with a model that proposes that 3-phosphoinositide dependent kinase 1 (PDK1) is the upstream activator of Akt to decrease steatosis. However, further studies need to be done to provide the molecular mechanisms by which Akt mediates a reduction in cellular lipid accumulation.

How the effects of these peptides are translated from plasma membrane to the intracellular environment remains somewhat contentious. Whether the pancreatic-type GLP-1R is present on hepatocytes is controversial and far from settled, in our opinion. Detection of expression of the known GLP-1R in liver or hepatocytes is inconsistent among different labs (Table 2). In the earliest reports, the majority of experiments did not detect GLP-1R RNA expression in the liver using various techniques including RNase protection, in situ hybridization, and reverse transcriptase-polymerase chain reaction (RT-PCR) with Southern blotting (Bullock, Heller, & Habener, 1996; Dunphy, Taylor, & Fuller, 1998; Villanueva-Penacarrillo et al., 1995; Wei & Mojsov, 1995; Yamato et al., 1997). In the more recent studies, many of the experiments are performed with cell lines, which could have a different gene expression profile. When primary cells are used, it is unclear how long they have been cultured, which also could alter gene expression patterns. The pancreatic type GLP-1R is a seven-transmembrane G-protein coupled receptor (GPCR), and activation by GLP-1 and its analogues activates adenylate cyclase to significantly increase cAMP production in highly expressing tissues, such as beta cells, or when GLP-1R is ectopically expressed in non-expressing cell lines (Fehmann et al., 1994; Moens et al., 1996; Thorens, 1992; Thorens et al., 1993; Wheeler et al., 1995). In the context of hepatocytes, the results of most in vitro experiments have not supported significant GLP-1 mediated activation of adenylate cyclase in hepatocytes (Blackmore, Mojsov, Exton, & Habener, 1991; Flock, Baggio, Longuet, & Drucker, 2007; Ghiglione et al., 1985; Redondo et al., 2003; Villanueva-Penacarrillo et al., 1995). When cAMP levels are increased, the fold increase in cAMP is well below that of a known physiologic GPCR activator, such as glucagon (Aviv et al., 2009; Ding et al., 2006). In one study, activation of cAMP signaling occurred, but there was no GLP-1R receptor expression detected (Aviv et al., 2009), so there is still a possibility that another GPCR exists which can be activated by GLP-1 and its analogues. For some in the field, these results have motivated a new hypothesis about a second possible GLP-1R, or alternative mechanisms for peptide entry into the cell (Tomas & Habener, 2010). The DPP4 product of GLP1, GLP9, has the ability to decrease hepatic steatosis in high fat diet mouse model even though it has low affinity for the GLP-1R (Tomas et al., 2011). However, although knockout of the GLP-1R abrogates most of the metabolic effects of the Ex4 analogue AC3174, there was some protection of the liver from the effects of a high fat diet (Trevaskis et al., 2012). Perhaps, some of these controversies could be best settled by key in vitro experiments using hepatocytes from the GLP-1R-knockout mouse (Ayala et al., 2008). 4. Conclusions There is accumulating and convincing pre-clinical evidence that GLP-1 and its analogues have the ability to decrease hepatic steatosis

Table 2 Evidence for and against hepatocyte expression and activity of the pancreatic-type G-protein coupled GLP-1 receptor. Receptor Present

Receptor Absent

Source

Detection method

Reference

Source

Detection method

Reference

Mouse Liver Rat liver Rat hepatocytes Mouse hepatocytes

Northern of mRNA RT-PCR Western blot; cAMP production cAMP production (Receptor not detected) Western blot; RT-PCR

Campos, 1994 Egan, 1994 Ding, 2006 Aviv, 2009

Primary rat hepatocytes Primary rat hepatocytes Primary rat hepatocytes Human liver

cAMP production cAMP production cAMP production RNase protection

Valverde, 1994 Ghiglione, 1985 Blackmore, 1991 Wei, 1995

Gupta, 2010

Rat liver

Bullock, 1996

Western blot; RT-PCR

Svegliati-Baroni, 2011

Rat liver

cAMP production Western blot; RT-PCR

Ben-Shlomo, 2011 Lee, 2012

Primary mouse hepatocytes Primary mouse hepatocytes

RT-PCR; RNase Protection; In situ hybridization RT-PCR with Southern blotting; Nuclease protection RT-PCR; cAMP production RT-PCR; Western blot

HuH7 cells; HepG2 cells; Primary human hepatocytes Human liver biopsies; Rat liver; Primary rat hepatocytes Primary rat hepatocytes HuH7 cells; HepG2 cells

cAMP = cyclic adenosine monophosphate; RT-PCR = reverse transcriptase polymerase chain reaction.

Dunphy, 1998 Flock. 2007 Tomas, 2010

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