Research Objectives - Background

Alcoholic Liver Disease (ALD) and Nonalcoholic Fatty Liver Disease (NAFLD) are major cause of illness and death in the United States. In the initial stage of the disease, fat accumulation in hepatocytes leads to the development of fatty liver (steatosis) which is characterized by an excess triglyceride deposition. NAFLD is often associated with elements of the metabolic syndrome, a clinical constellation of obesity, hypertension, insulin resistance, glucose intolerance and hyperlipidemia and encompasses a spectrum of liver disorders from the simple hepatic steatosis to the more ominous condition known as nonalcoholic steatohepatitis (NASH). Several mechanisms that underlie the accumulation of liver steatosis in association with obesity and the metabolic syndrome appear to be in common with alcoholic fatty liver. Thus, if alcohol consumption is continued, steatosis may progress to hepatitis and fibrosis, which may eventually lead to liver cirrhosis. Similarly, steatosis in NAFLD may ultimately lead to clinically significant progressive liver injury, fibrosis and cirrhosis. Simple fatty liver has long been considered benign; however, increasing evidence suggests that it is a potentially pathologic condition and precedes the development of alcoholic or nonalcoholic steatohepatitis and cirrhosis. In alcoholic liver disease, this assumption is based on features reported to be associated with fatty liver developed as a result of chronic administration of Lieber-DeCarli liquid diet (36% alcohol calories) to rats including: 1) induction of hepatic cytochrome P4502E1 (CYP2E1); 2) increased hepatic levels of 4-hydroxynonenal (4-HNE), a marker of lipid peroxidation; 3) increased deposition of iron in the liver; 4) selective hepatic mitochondrial glutathione (GSH) reduction and mitochondrial dysfunction; 5) hepatic S-adenosylmethionine (SAMe) reduction; 6) increased concentration of serum tumor necrosis factor-alpha (TNF-á) and increased hepatic expression of TNF-á mRNA levels; 7) elevated serum alanine aminotransferase (ALT) levels; and 8) increased hepatic levels of cellular fibronectin and alpha smooth muscle actin (á-SMA). Many of these factors may also play a role in the evolution of fatty liver of NAFLD to nonalcoholic steatohepatitis, providing the so-called “second hit” involved in the pathogenesis of NASH.

Induction of CYP2E1 activity, increased levels of 4-HNE, increased deposition of iron, and depletion of SAMe and GSH are markers of oxidative stress, which is known to play a pivotal role in the pathogenesis of steatosis to the more severe liver disorders, NASH and ALD. TNF-á has been implicated in the pathogenesis of alcoholic and nonalcoholic fatty liver injury in humans as well as in animals. An increased level of serum alanine aminotransferase ( ALT) is a marker of hepatic injury. Increased level of hepatic cellular fibronectin is an early response to liver injury, and increased levels of á-SMA suggest stellate cell activation, which may result in excess of collagen deposition and subsequent fibrosis. These findings clearly suggest that fatty liver is a potential pathologic condition and that accumulated fat can sensitize liver to further injury such as inflammation, fibrosis, and cirrhosis.

Preventing the accumulation of fat within liver or degradation of accumulated fat may block or delay the progression of fatty liver to steatohepatitis and fibrosis. In order to achieve this goal, it is important to understand the underlying biochemical and molecular mechanisms which lead to fat accumulation in the liver due to alcohol consumption as well as metabolic factors such as obesity and insulin resistance. Excess fat accumulation in the liver could result from increased transportation of fatty acids from the peripheral organs to the liver, increased hepatic fatty acid synthesis, impaired hepatic fatty acid oxidation, increased triglyceride synthesis in the liver, and/or diminished export of triglycerides from the liver.

Scope of Research

The purpose of this PA is to encourage research grant applications that will use an integrative approach of state-of-the-art technologies to gain insight into the molecular mechanisms of both alcoholic and nonalcoholic fatty liver. This includes investigating the mechanisms by which alcohol and nonalcoholic metabolic factors: a) accelerate import of free fatty acids into hepatocytes; b) impair mitochondrial â-oxidation of fatty acids; c) impede the entry of free fatty acids into mitochondria; d) promote de novo fatty acid synthesis; e) promote esterification of free fatty acids into triglycerides; and f) disrupt export of triglycerides from hepatocytes through very low density lipoprotein (VLDL).

NIAAA, NIDDK, and ODS also encourage research to develop non-invasive biomarkers for fatty liver, using genomic, proteomic, and metabolomic technologies. Proposals investigating the modulating effects of dietary fatty acids, obesity, diabetes, and insulin resistance on the development of alcoholic and nonalcoholic fatty liver are also encouraged.

a.  Increased Hepatic Uptake of Fatty Acids:

Fatty acid levels are significantly increased in the liver after alcohol consumption and in persons with obesity and the metabolic syndrome. Alcohol may increase hepatic fatty acid uptake by increasing hepatic blood flow. In addition, studies with isolated hepatocytes and perfused livers have suggested that alcohol can directly increase fatty acid uptake independent of blood flow. This could be due to a direct effect of alcohol on the physical properties of the hepatic plasma membrane consequent to altered lipid composition. Further studies are required to delineate the role of fatty acids and the modulatory effect of alcohol and the metabolic syndrome upon hepatic uptake of fatty acids.

b.  Impaired Fatty Acid Oxidation:

Alcohol and excess fatty acids have been shown to inhibit fatty acid oxidation in liver slices from rats and humans, in perfused rat liver, in rat hepatocytes, and in vivo in humans. Various mechanisms have been proposed for this effect. A redox shift resulting in increased ratio of NADH/NAD+ has been implicated in the development of fatty liver via inhibition of mitochondrial fatty acid beta oxidation and tricarboxylic acid cycle. However, normalization of redox state failed to attenuate alcohol-induced fatty liver, suggesting that other mechanisms may also be contributing to this condition.

Alcohol and excess fatty acids may also impair fatty acid oxidation by inhibiting the activities of enzymes involved in fatty acid oxidation. Peroxisome proliferators-activated receptor alpha (PPAR-á) is a member of the nuclear hormone receptor super family, which when dimerized with retinoid X receptor alpha (RXR-á) regulates transcription of a set of genes involved in the oxidation and transport of fatty acids. Chronic ethanol feeding has been shown to decrease RXR-á protein levels, inhibit DNA binding of the PPAR-á/RXR-á heterodimer, and decrease the levels of mRNA for several PPAR-á regulated genes in the liver of mice. These findings were associated with the development of fatty liver. Simultaneous administration of a PPAR-á agonist attenuated some of these effects, including progression of fatty liver, despite continued ethanol administration. These results suggest that chronic ethanol may inhibit fatty acid oxidation by inhibiting PPAR-á activity via decreasing RXR-á protein levels and subsequently impairing DNA binding of PPAR-á/RXR-á heterodimer. However, mechanisms by which chronic ethanol decreases RXR-á levels are not clear. Furthermore, the significance of RXR-á levels in the DNA binding of PPAR-á/RXR-á is not clear since treatment of ethanol-treated animals with PPAR-á agonist restored DNA binding ability without increasing RXR-á levels. Further research is required to investigate various fatty acid oxidation pathways that are impaired by excess fatty acids and by ethanol consumption.

c.  Impaired Transport of Fatty Acids into Mitochondria:

Transport of free fatty acids from cytosol to mitochondria is a required for mitochondrial beta oxidation of fatty acids. This transport is accomplished primarily through an enzyme, carnitine palmitoyltransferase-1 (CPT-1) located at the outer membrane of mitochondria. Chronic ethanol has been shown to reduce the activity of CPT-1, which may impair the transport of fatty acids into mitochondria that in turn may result in reduced fatty acid oxidation. Ethanol may inhibit CPT-1 activity by triggering a cascade of events starting from an activation of sterol regulatory element-binding protein (SREBP) to up-regulation of acetyl Co-A carboxylase (ACC), and subsequent increased production of malonyl Co-A that is known to inhibit CPT-1 activity. Alternatively, ethanol may inhibit CPT-1 activity by triggering another cascade of events starting from inhibition of PPAR-á activity to inhibition of malonyl Co-A decarboxylase (MCD) activity, and subsequent increased production of malonyl Co-A.

d.  Accelerated de novo Fatty Acid Synthesis:

Chronic ethanol use and metabolic stresses such as obesity and insulin resistance have been shown to stimulate lipogenesis in the liver through increased transcription of genes for lipogenic enzymes. The SREBPs are a family of transcription factors that are key regulators for cholesterol and fatty acid synthesis; they exert their effect by directly activating the expression of more than 30 genes in the liver. Recently, researchers have demonstrated that chronic ethanol administration can significantly increase the production of hepatic SREBP-1, which is associated with increased expression of lipogenic genes as well as accumulation of triglycerides in the liver. Using 4-methylpyrazole and cyanamide, it was shown that this effect of alcohol was dependent on its metabolism to acetaldehyde. Alcohol-induced lipogenesis appears to be modulated by the dietary concentration of carbohydrate and fat. While higher carbohydrate and low fat diet may promote lipogenesis by providing excess pyruvate for the synthesis of acetyl-CoA (precursor for fatty acid synthesis), higher fatty acid and low carbohydrate diet may inhibit lipogenesis by inhibiting the activities of lipogenic enzymes. Further studies are required to understand the role of SREBPs in hepatic fatty acid synthesis in human alcoholics administered with variable concentrations of fat and carbohydrate in their diet. In addition, role of acetate in the synthesis of fatty acids via acetyl Co-A needs to be evaluated.

e.  Increased Esterification of Free Fatty Acids into Triglycerides:

The increased supply of free fatty acids in the liver of alcoholics and persons with obesity, insulin resistance and the metabolic syndrome along with reduced ability of liver to oxidize these compounds can lead to their esterification and storage as triglycerides, resulting in fatty liver. Several animal studies suggest that alcohol intake can increase esterification of free fatty acids into triglycerides. This is primarily due to ethanol-induced up-regulation of phosphatidate phosphohydrolase (PAP), the rate limiting enzyme in triglyceride synthesis. Both acute and chronic alcohol administration have been shown to up-regulate hepatic PAP activity in rats, hamsters, and baboons. However, the mechanisms of this effect of ethanol are not clear. Studies are required to understand the relative role of PAP in the development of alcoholic fatty liver. Whether ethanol affects other enzymes of esterification pathway needs investigation.

f.  Decreased Export of Triglycerides from the Liver:

Triglycerides are generally exported from the liver by very low-density lipoproteins (VLDL) particles, which are assembled through a complex process and made of triglycerides, cholesterol, phosphatidylcholine, and apolipoproteins. Inhibition of this process at any of several levels may result in accumulation of triglycerides in hepatocytes and consequently development of fatty liver. Studies with perfused livers, isolated hepatocytes, and alcohol fed rats have shown that alcohol can inhibit secretion of VLDL and this may contribute to the development of fatty liver. Alcohol's metabolite acetaldehyde may impair VLDL secretion by reacting with lysine residue of tubulin (a cytoskeleton protein), resulting in acetaldehyde-tubulin adduct formation. This may impair microtubule formation and consequently VLDL secretion. Alcohol may also impair triglyceride export by inhibiting the synthesis of phosphatidylcholine (via inactivating phosphatidylethanolamine methyl transferase activity), which is an important component of VLDL formation. In addition, alcohol may impair transport by inhibiting apolipoprotein synthesis through inhibition of PPAR-á activity. Studies are required to understand the mechanisms which impair the formation, intracellular transport through the cytoskeleton, and secretion of VLDL.

g.  Role of Dietary Fatty Acids:

Dietary fat has been shown to play an important role in the development of steatosis and in the pathogenesis of ALD. While polyunsaturated fatty acids potentiate the severity of alcoholic liver injury, saturated fatty acids are protective. On the other hand, phospholipids such as phosphatidylcholine (soybean extract) have been shown to prevent alcohol-induced fibrosis and cirrhosis in baboons. The toxic effects of polyunsaturated fatty acids are thought to be mediated through increased oxidative stress (lipid peroxidation), whereas the mechanisms of the protective effects of saturated fatty acids and phospholipids are not clear. Understanding the underlying molecular mechanisms by which different types of fats potentiate or prevent hepatic steatosis may help to develop dietary interventions for the prevention or treatment of the disease.

h.  Modifying Factors:

The metabolic syndrome components individually or collectively may modulate the course of alcoholic fatty liver and vice versa. Heavy alcohol consumption is associated with insulin resistance and increased plasma levels of insulin, which is known to accelerate de novo fatty acid synthesis in the liver. Alcohol consumption has been reported to promote obesity in some individuals, and obesity has been reported to increase the risk of fatty liver, hepatitis, and cirrhosis caused by chronic alcohol consumption. Likewise, individuals with the metabolic syndrome may sensitize to the steatotic effects of alcohol. In cross sectional studies of the U.S. population, alcohol consumption was associated with a higher rate of liver enzyme abnormalities largely among persons who were overweight or obese. The combined effect of obesity and alcohol consumption on the development of hepatic steatosis needs further elucidation.

i.  Biomarkers of Alcoholic Fatty Liver:

There are about 8 million people with alcoholism in the USA, who may be affected with fatty liver. If these people continue drinking, at least 20% of them are likely to develop hepatitis and/or cirrhosis. In addition, there are about 42 million obese individuals who may also be affected with fatty liver. Among individuals who progress from steatosis to NASH, approximately 15% will develop cirrhosis. Therefore it is important to be able to identify individuals with fatty liver for intervention before this condition progresses to hepatitis or cirrhosis. Ultrasound is a very sensitive method for detecting fatty liver, but it is not specific for this condition and the procedure is costly. An AST:ALT ratio of >2 is considered by some investigators specific for alcoholic fatty liver; however, other investigators have not confirmed this finding. Studies are required to develop specific and sensitive non-invasive biomarkers – utilizing blood, urine, saliva, or hair – for the diagnosis of fatty liver. Advanced techniques such as genomics, proteomics, and metabolomics are encouraged for these studies.

j.  Use of Novel Approaches:

Recent advances in functional genomics, both conceptually and technologically, are providing us tremendous opportunities to decipher the interconnecting networks of genes, proteins, and metabolites. These global approaches can be generally carried out at four major levels: genome, transcriptome, proteome, and metabolome. The studies at genome level include sequencing, polymorphism (such as SNP), haplotype, genetic variation, QTL, genetic or epigenetic alterations. The studies at transciptome level include gene expression profiling (by DNA microarray, SAGE, RAGE, or other types of techniques), gene regulation, and alternative splicing. The studies at proteome level are to use systematic approaches to study proteins for their identity, quantity, and function. At metabolome level, metabolomics involves a detailed, quantitative analysis of low molecular weight metabolite over changing environmental conditions (e.g., alcohol administration or increasing levels of obesity) in a biological system (human patients or animal models). The determination of these metabolites can be achieved by using spectroscopic methods, most powerfully, mass spec, NMR, and HPLC. Metabolomics can be used to identify metabolite differences associated with alcohol or alcohol-related illnesses, to discover biomarkers for diagnosis or efficacy, to identify drug targets, to determine the safety of drugs, and so on. This metabolomic information can be integrated with genome, transcriptome, and proteome information to understand the mechanisms and provide new avenues for therapeutic treatments of fatty liver. To understand how fatty liver is developed, novel approaches using advanced technologies in the areas of genomics and functional genomics are strongly encouraged. Other novel approaches such as transgenic and knockout mouse models or RNA interference are also encouraged if a gene or genes are found to play a potential role in the development of alcoholic or nonalcoholic fatty liver.

k.  Areas of Research

Applications should focus on research that may contribute to understanding the role of alcohol and its metabolites acetaldehyde and acetate and nonalcoholic mechanisms in the development of fatty liver. Examples of research that might be supported under this PA include, but are not limited to, the following:

• Investigation of the mechanisms of increased import of fatty acids into the liver.
• Determination of the mechanisms of inhibition of fatty acid oxidation via regulating transcription of enzymes involved in fatty acid oxidation.
• Discerning the mechanisms which inhibit transport of fatty acids from cytosol to mitochondria.
• Understanding the mechanisms that accelerate de novo fatty acid synthesis in the liver via regulating transcription of lipogenic enzymes.
• Elucidation of the role of acetate in the de novo synthesis of fatty acids.
• Determination of the mechanisms of alcohol- and obesity-induced accelerated hepatic triglyceride synthesis, including the mechanisms of phosphotidate phosphohydrolase (PAP) activation.
• Elucidation of the mechanisms by which alcohol and obesity or insulin resistance impair synthesis, assembly, intracellular transport, and hepatic secretion of VLDL.
• Understanding the underlying mechanisms by which different types of fats potentiate or prevent alcoholic and nonalcoholic fatty liver injury.
• Investigation of the role of insulin resistance and obesity in the development of fatty liver.
• Development of specific and sensitive non-invasive biomarkers (utilizing blood, urine, saliva, or hair) for the diagnosis of alcoholic and nonalcoholic fatty liver.

Prepared:  March 9, 2006