Nonalcoholic fatty liver disease (NAFLD) has become the most common chronic liver with hepatocellular lipid deposition followed by inflammation. NAFLD has been considered as hepatic manifestation of metabolic syndrome and consists of progressive stages, ranging from simple steatosis to NASH, fibrosis, and cirrhosis. In the patients with a sedentary lifestyle, obesity, or IR, an increased influx of free fatty acid (FFA) to the hepatocyte was observed in the liver. While several factors, such as obesity, diabetes, and dyslipidemia, have been implicated in NAFLD, the pathogenesis of NAFLD and its progression to fibrosis and chronic liver disease are still unclear .
It has been proposed that NAFLD may be considered as a disease with a “two-hit” process of pathogenesis with lipid peroxidation-mediated liver injury. The “first hit” is excessive hepatocyte triglyceride accumulation which may results from insulin resistance. The second hit is unclear, but the presumed factors initiating second hits are suggested to be oxidative stress and subsequent lipid peroxidation and proinflammatory cytokines .
Several data supported the implication of cAMP response element-binding protein (CREB) in NAFLD progress. The aim of this study is to evaluate the role of hepatic cAMP/CREB pathway in the development of experimental nonalcoholic fatty liver to clarify its pathogenesis which could provide a therapeutic approach. In our study, we used a HFD rat model of insulin resistance and NASH because it is easy to establish and resemble the human condition.
Histological examination of the liver tissues in control group revealed better liver histology, with normal hepatic architecture and organization, when compared to NAFLD one. Few hepatocytes showed rarefaction of the cytoplasm and micro-vesicular steatosis at 6th week of HFD induction. At 10th week of feeding, hepatocytes showed rarefied cytoplasm with micro- and macro-vesicular steatosis and hepatocellular ballooning. Mallory’s bodies, hyaline eosinophilic irregular-shaped aggregates in the cytoplasm of hepatocytes, were also present. The liver tissues of the NAFLD group at 14th week of HFD induction demonstrated more disturbance of the hepatic architecture; marked Mallory’s bodies, macro-vesicular steatosis, periportal inflammatory cellular infiltrates, and bridging fibrosis were noticed. The majority of authors on this topic consider the presence of fat, ballooning, and hepatocyte injury to be the minimum histopathological changes required for establishing a diagnosis of NASH which evolves into advanced fibrosis . In accordance with our results, Lieber et al., who used the quietly similar type of high-fat diet for only 3 weeks in Sprague-Dawley rats, reproduced hepatic lesions of human NASH . Moreover, another study demonstrated that Wister rats seem to be more sensitive to developing steatosis when consuming diets with a higher fat content in comparison with Sprague-Dawley rats .
The AST, ALT, GGT, and levels of total and direct bilirubin among other markers of liver injury may be useful parameters in measuring NAFLD. The results of the present study revealed a remarkable increase in these parameters in NAFLD rats as early as 2nd week of induction compared to control group. Our results are in accordance with those reported by Hanafi et al., who showed that the activities of serum transaminases, AST, ALT, and GGT, were significantly increased in NAFLD rats. Also, NAFLD rats demonstrated higher bilirubin values . These significant abnormalities of liver function tests revealed a state of hepatocytes inflammation and slightly damage as indicated by the histological results.
In the current work, dyslipidemia was evidenced in the NAFLD group from the second week of HFD as indicated by a significant increase in triglycerides, cholesterol, and LDL-cholesterol levels. Moreover, HDL-cholesterol was significantly decreased in the NAFLD group from the 4th week of induction. Hyper-triglyceridemia and abnormal low level of HDL-cholesterol are evidenced in the course of induction from the 6th week of feeding while hypercholesterolemia and abnormal high level of LDL-cholesterol are detected later at the 10th week. Moreover, NAFLD rats revealed typical duration–dependent increase in serum lipid profile including TG, TC, and LDL-C and duration-dependent decline in the level of HDL-C. In agreement with these results, it was reported that dyslipidemias are common abnormalities observed in NAFLD and have been reported in up to 81% of patients . It is suggested that hyper-triglyceridemia is more likely to increase the risk of NAFLD than hypercholesterolemia [33, 34]. Furthermore, many evidences suggest that among different types of cholesterol, abnormality of HDL-C is the most frequent lipid profile in NASH patients, while LDL-C as well as total cholesterol are more likely to be within normal ranges .
An increase in intrahepatic fat content leads to an upregulation of oxidative mechanisms, or it can re-esterified and secreted again as hepatic VLDL-triglycerides (TG), the major source of circulating TG [36, 37], which is responsible for the increase in serum TG concentrations observed in our study. However, the liver capacity in NAFLD to export TG is limited by an inadequate increase of the secretion rate of apoB100. A reduction in apo B synthesis and secretion may impair hepatic lipid export and favor hepatic triglyceride accumulation , which was confirmed in histopathological assessment of our study from the 2nd week of NAFLD induction. The current work revealed a significant raise in the hepatic TG and TC content in the liver of the NAFLD group compared to control rats as early as 2nd week of feeding. Additionally, there was a significant duration–dependent elevation in the hepatic TG and TC liver contents in the NAFLD rats during 14 weeks of HFD feeding.
One of our promising findings is that hepatic TG accumulation proceeds or at least concomitant with hyperinsulinemia at 4th week and IR which also became significant from the 4th week of induction. In accordance with these results, it was reported that once the liver is fatty, the ability of insulin to inhibit hepatic glucose production is impaired, which leads to an increase in the fasting plasma glucose concentration . This in turn stimulates insulin secretion resulting in mild hyperinsulinemia and lowering of glucose to near-normal levels; also, the inhibitory action of insulin on VLDL production is impaired whereas VLDL clearance remains unchanged [40, 41].
The association between IR and NAFLD is an area of public health impact. IR and subsequent compensatory hyperinsulinemia have been shown to have a key role in the pathogenesis of NAFLD by causing an imbalance between factors that favor hepatic lipid accumulation (such as lipid influx and de novo lipid synthesis) and factors that ameliorate lipid build-up, such as lipid export or oxidation [42, 43].
Abnormally elevated insulin levels, under IR conditions, are required to metabolize glucose and inhibit hepatic glucose production effectively due to the reduced insulin sensitivity of the peripheral tissues. In this context, the pancreas is stimulated to increase insulin secretion into the portal vein, leading to higher insulin levels in the liver than in the periphery. High concentrations of hepatic glucose and plasma insulin are recognized as biomarkers of hepatic IR . Elevated fasting glucose results from hepatic IR, whereas increased FFA concentrations are caused by peripheral IR. The FFAs interact with insulin signaling, thereby contributing to IR. Hepatic IR contributes to steatosis of NAFLD by impairing insulin receptor substrate 1/2 tyrosine phosphorylation [45, 46].
As expected in our results, during NAFLD induction, a fasting glucose level is significantly higher in NAFLD group compared to control group from the 2nd week of induction, and its level increased directly with the duration of induction. Hyperglycemia was detected at 10th week of induction and thereafter. Also, fasting insulin and insulin resistance index (HOMA-IR) revealed higher degrees of IR from the 4th week of induction, and with increasing duration, the situation become worse. Furthermore, these abnormalities of glucose homoeostasis during HFD feeding are associated with serious derangements in lipid profile as discussed previously.
The cAMP is a key second messenger in numerous signal transduction pathways including cell growth, differentiation, gene transcription, protein expression, and in the regulation of cellular metabolism as well as hormonal action in the peripheral tissues. Subsequently, specific proteins are phosphorylated by PKA to evoke cellular reactions. The phosphorylation of CREB, a transcription factor, is important in the regulation of gene transcription. Extracellular signals activate the transcription of a variety of target genes via alterations in CREB phosphorylation, thereby, resulting in multiple physiological functions [47, 48].
CREB activity is tightly regulated by phosphorylation as well as by the level of ICER, a natural CREB antagonist. ICER is generated from an alternative CREM promoter and is the only inducible CRE-binding protein. ICER acts as a passive repressor that competes with CREB for binding to target gene promoters. In the normal situation, ICER activity is transiently induced by the same stimuli that induce CREB, but repression occurs only when ICER reaches certain levels [49, 50]. There is a strong evidence for the association between CREB and ICER, while CREB rapidly induces the expression of target genes in response to stimuli; the repressor ICER restores their initial expression levels and thereby permits transient induction .
Stimuli that trigger CREB and ICER activities include cell proliferation, cell cycle, metabolism, DNA repair, differentiation, inflammation, angiogenesis, immune responses, and survival response for instance, and this occurs in response to an elevation of cAMP levels. Thereby, as a passive repressor, ICER activity is directly correlated with its abundance [49, 52]. It is thus predictable that dysregulation in the levels of ICER impact to CREB activity, hence leading to cells dysfunction and eventually certain pathologies.
Our results indicated that, in NAFLD rats, the hepatic contents of cAMP and CREB are prominently elevated compared to control rats. Furthermore, the results showed that NAFLD rats feeding HFD revealed a typical duration–dependent increase in both cAMP and CREB levels in the liver tissue. Notably, the results of the present study also demonstrated that the expression of ICER gene in the liver tissue was downregulated in NAFLD rats from 6th week of induction compared to control rats. This is in close agreement with the results of Favre et al. who found that the expression of ICER was decreased in insulin-resistant obese mice indicating an impaired induction of ICER and, hence, leading to persistently increased CREB activity .
The significant high level of both cAMP and CREB levels in the liver could be explained by IR state, a prominent factor of NAFLD, which favors the lipolytic pathway as a consequence of increasing glucagon/insulin ratio; the generated FFAs and glycerol may further exaggerate the IR in tissues in a vicious cycle. Our study is in accordance with Erion et al. findings who used a CREB-specific antisense oligonucleotide (ASO) to knock down CREB expression in the liver. Interestingly, CREB ASO treatment dramatically reduced fasting plasma glucose concentrations in Zucker Diabetic Fatty rats and a streptozotocin-treated high-fat fed rat model of type II diabetes mellitus (T2DM) with its concomitant IR . Nonetheless, our results are not in accordance with the study of Zingg et al. who reported that, in the liver, the HFD significantly decreased liver cAMP levels compared to low fat diet, and curcumin increased it . Furthermore, a study investigating the therapeutic effects of resveratrol, a naturally occurring polyphenol, in NAFLD showed that its protective effects were mediated by the cAMP pathway. In particular, resveratrol improved hepatic steatosis in a HFD mouse model of NAFLD via improved fatty acid β-oxidation by increasing cAMP through inhibiting PDE4 .
Correlation studies revealed a direct positive correlation between the intracellular level of cAMP and CREB in the liver tissue. On the other hand, the levels of cAMP and CREB in the liver tissue were negatively correlated with the ICER gene expression. Moreover, the level of cAMP in the liver tissue was positively correlated with fasting blood glucose, insulin, HOMA, liver function tests (ALT, AST, GGT, total bilirubin, direct bilirubin), lipid profile tests (serum triglycerides, cholesterol, LDL-cholesterol, but not HDL-cholesterol) as well as hepatic triglycerides and cholesterol contents. Contrariwise, the level of cAMP was negatively correlated with the level of HDL-cholesterol.