|Overview of Rare Diseases Research Activities
The National Institute on Alcohol Abuse and Alcoholism (NIAAA) conducts and supports research on the causes, consequences, prevention, and treatment of alcoholism and alcohol abuse. In addition to fetal alcohol spectrum disorders and various liver diseases, alcoholism is associated with and can be the cause of diseases targeting the lung, pancreas, heart, and other organs. The consequences of alcohol abuse and alcoholism are far reaching, so knowledge gained in related research programs will have broad application to other areas of human health and disease.
Recent Scientific Advances in Rare Diseases Research
Long-term heavy alcohol consumption is associated with both acute and chronic pancreatitis. Progression of pancreatitis may lead to multiple co-morbidities including maldigestion, diabetes, and pancreatic cancer. NIAAA-funded researchers have made significant progress in understanding the underlying mechanisms by which alcohol intake leads to the development of pancreatitis.
Role of Angiotensin II in Alcohol-Induced Pancreatic Fibrosis
This study was undertaken to investigate the effects of angiotensin-converting enzyme inhibitor (captopril) and angiotensin II receptor antagonist (losartan) on alcohol-induced pancreatic fibrosis using an intragastric ethanol-feeding model of male rats. Dietary alcohol caused diffuse gland atrophy and interlobular and intralobular fibrosis with mild structural distortion in the pancreas, an effect that was blunted by captopril or losartan treatment. Alcohol also increased the number of alpha-smooth muscle actin-positive cells and transforming growth factor-beta (TGF-β) mRNA expression in the pancreas. These increases were blunted significantly by captopril or losartan treatment. These data suggest that angiotensin II contributes to the development of chronic alcohol-induced pancreatic fibrosis through its stimulation of TGF-β expression.
Alcohol-Induced Alterations in the Pancreatic Exocrine Secretion
Chronic alcohol ingestion appears to increase susceptibility of the pancreas to pancreatitis through modulating pancreatic exocrine secretion. This study was undertaken to determine the effect of chronic low- and high-dose alcohol consumption on the neurohormonal control of the exocrine pancreas in rats. Chronic alcohol ingestion was associated with a dose-related inhibition of basal pancreatic protein secretion, which was reversed upon alcohol withdrawal.
Low-dose alcohol feeding had no effect on bethanechol-stimulated pancreatic secretion but altered 2-deoxy-D-glucose (2-DG)-stimulated pancreatic secretion. In rats given chronic high doses of alcohol, meal- and bethanechol-stimulated protein secretion was significantly potentiated during early and late phases. The response to CCK appeared to be disinhibited, whereas the response to 2-DG was uniformly blunted. Upon withdrawal of low-dose alcohol, the response to 2-DG was potentiated, whereas with the withdrawal of high-dose alcohol, the response to CCK was potentiated. Adaptation to chronic alcohol consumption differs depending on the alcohol dose. The most significant effects were seen after high-dose alcohol withdrawal, with apparent loss of central inhibitory regulation combined with exaggerated response at the acinar cell level. This combination of factors could increase susceptibility to acute alcoholic pancreatitis through a hyperstimulation mechanism.
Fatty Acid Ethyl Esters Increase Extracellular Matrix Protein Levels in Pancreas
Chronic alcoholic pancreatitis is characterized by fibrosis, which results from an excess of deposition of extracellular matrix (ECM) proteins in the pancreas; however, the mechanisms involved in ethanol-induced pancreas fibrosis are poorly understood. This study was designed to investigate the role of fatty acid ethyl esters (FAEE), nonoxidative ethanol metabolites, on ECM protein content in rat pancreas. The results demonstrated the FAEEs increased collagen, laminin, and fibronectin levels in pancreatic acini without affecting messenger RNA (mRNA) expression for these proteins. On the other hand, FAEEs reduced the activity of the serine protease, plasmin, and that of the urokinase-type plasminogen activator (uPA). Consistent with these results, the serine protease inhibitor aprotinin reproduced the effects of FAEEs and prevented the further increase in ECM proteins induced by FAEEs. In vivo, administration of FAEEs reduced plasmin and uPA activities and increased ECM protein levels in pancreas. These results suggest that FAEE-induced increases in ECM proteins in the pancreas are caused primarily by an inhibition in ECM degradation via serine proteases, including the plasminogen system.
Alcohol-induced Hepatic Fibrosis
Chronic heavy alcohol consumption is a major cause of liver cirrhosis, which ultimately results in death. Liver cirrhosis is a progression of fibrosis, which results from excessive deposition of ECM components, especially collagen, in the liver. Although various hepatic cells are involved in the development of fibrosis, hepatic stellate cells (HSCs) are the primary source of excessive ECM components. A major feature of fibrosis is the activation of HSCs, consisting of an early initiation phase followed by a perpetual phase. NIAAA-funded researchers have made the following progress in terms of understanding the mechanisms of HSC activation.
TGF-β-mediated Reciprocal Expression of Type I Collagen and Matrix Metalloproteinase-13 Genes in Hepatic Stellate Cells
TGF-β1 is a main cytokine involved in alcohol-induced liver fibrosis. It induces expression of the type I collagen genes in HSCs by a transcriptional mechanism, which is hydrogen peroxide dependent. In the present study, researchers investigated whether this cytokine is able to modulate the expression of collagen-degrading matrix metalloproteinase-13 (MMP-13) mRNA. The results showed that TGF-β1 induces a rapid decline in steady-state levels of MMP-13 mRNA at the time that it induces the expression of α1 (I) collagen mRNA. In addition, TGF-β1-mediated effect is de novo protein synthesis dependent and requires the activity of p38MAPK, phosphatidylinositol 3-kinase, AKT, and p70 (S6k). These results suggest that TGF-β1 modulates the expression of type I collagen and MMP-13 mRNAs in HSCs in a reciprocal manner (increasing collagen but decreasing MMP-13) that results in excess of collagen deposition in the liver—an important step in liver fibrosis.
Role of Connective Tissue Growth Factor in Hepatic Stellate Cells Activation
HSCs are the main source of increased collagen production that may result in hepatic fibrosis. Connective tissue growth factor (CCN2) is expressed during activation of HSCs and it promotes HSC proliferation, adhesion, and collagen production. This study was designed to investigate CCN2 signaling pathways in HSC. The results showed that CCN2 stimulated DNA synthesis and phosphorylation of FAK, Elk-1, and ERK1/2, the latter of which was blocked by heparin. The serum response element binding activity and luciferase reporter activity of the c-fos promoter, together with expression of c-fos, were enhanced by CCN2. CCN2-induced c-fos gene activation, expression, and cell proliferation were blocked by inhibiting ERK1/2 with PD98059. CCN2 promoter activity was enhanced by TGF-β1 or PDGF via a Smad7-dependent pathway. It was concluded that CCN2-stimulated HSC DNA synthesis is associated with transient induction of c-fos gene activation and expression as well as activation of the ERK1/2 signal pathway.
Adipogenic Transcriptional Regulation of Hepatic Stellate Cells
A major feature of fibrosis is the activation of hepatic stellate cells (HSCs) in which quiescent HSCs are transformed into collagen-producing activated HSCs, known as myofibroblast-like cells. The mechanisms of this transformation are not clear. In this study, researchers have demonstrated higher expression of adipogenic transcription factors such as C/EBP alpha, beta, and delta; PPARgamma; LXRalpha; SREBP-1c; and of adipocyte-specific genes in the quiescent HSCs, whereas increased expression of PPARbeta (transcription factor known to promote fatty acid oxidation) in activated HSCs was demonstrated. Furthermore, treatment of activated HSCs with the adipocyte-differentiation cocktail (MDI: isobutylmethylxanthine, dexamethasone, and insulin) or ectopic expression of PPARgamma or SREBP-1c in these cells induced a panel of adipogenic transcription factors, reduced PPARbeta, and caused the phenotypic reversal to quiescent HSCs. These results support the importance of adipogenic transcriptional regulation in HSC quiescence and provide a new framework for identifying novel molecular targets for the treatment of liver cirrhosis.
Fetal Alcohol Syndrome and Other Alcohol-related Birth Defects
Fetal alcohol spectrum disorders (FASD) describes a spectrum of prenatal alcohol effects resulting from drinking by the mother during pregnancy. The most serious disorder arising from prenatal alcohol exposure is fetal alcohol syndrome (FAS), a cluster of defects that includes mild craniofacial abnormalities, growth retardation, and central nervous system impairments manifested by deficits in executive function, memory and learning, and motor activity. FAS is considered the most common nonhereditary form of mental retardation. Alcohol-related neurodevelopmental disorder (ARND) is more variable in phenotype but can be equally debilitating. A variety of alcohol-related organ system birth defects have also been reported among children with FAS or ARND, including congenital heart defects, ocular abnormalities, and increased susceptibility to infections.
Choline Supplementation and ARND
Administering choline in the diet of postnatal rats that had been exposed to alcohol in the early neonatal period (equivalent to the third trimester of human pregnancy) was previously shown to enhance performance on a learning and memory task. Thus, choline supplementation appears to be a candidate for ameliorating deficits associated with FASD. Recent studies demonstrated that ethanol-induced motor deficits are not affected by choline supplementation. This highlights the fact that the effects of alcohol are multifaceted and that a single drug or supplement is not likely to be a panacea for the spectrum of FASD deficits. Drug discovery efforts may need to focus on the most debilitating behavioral deficits.
Ethanol and Cell Death in the Developing Brain
Ethanol has been shown to reduce the number of serotonin neurons during fetal development. Results from this study in cultured cells from rats strongly suggest that the neuronal loss is due to increased apoptosis, or programmed cell death. Apoptosis occurs in the course of the normal development of the nervous system. In addition to finding that ethanol increases apoptosis; the study also found that a serotonin-1A receptor agonist, which binds to and activates the receptor, protects the neurons by reducing apoptosis. This finding adds to the growing body of evidence that ethanol-induced cell death in a variety of developing neurons is mediated through apoptotic signaling pathways.
Promising Compounds to Prevent FAS
The NIAAA continues to support research activities to find ways to prevent FASD. Scientists previously found that NAP and SAL, the active peptides from two brain proteins known to protect nerve cells against a variety of toxins, potently antagonize ethanol’s teratogenic effects in a mouse model. Current studies revealed that the ethanol antagonist properties are not stereospecific; i.e., the synthetic D-stereoisomers were as effective as the L forms in preventing ethanol’s effects. This was surprising, since most synthetic D-ligands and D-substrates are inactive. The D-stereoisomers are potentially attractive candidates for preventing FAS because of their relative resistance to proteases. Building on earlier findings that ethanol withdrawal causes neurotoxicity via the NMDA site on the glutamate neurotransmitter receptor during the human third trimester equivalent of development in rats, scientists treated ethanol-exposed animals during alcohol withdrawal with eliprodil, an NMDA receptor antagonist selective for the polyamine modulatory site. Subsequently, the animals showed improved performance on a learning test relative to alcohol-exposed animals that did not receive the drug. Thus, not only does this study support the hypothesis that ethanol withdrawal contributes to ethanol teratogenesis via NMDA receptor interaction, but it also suggests that eliprodil may be a promising compound for therapeutic development to prevent adverse ethanol effects during fetal development when it is administered during alcohol withdrawal.
Significant Ongoing Rare Diseases Research Initiatives
Perinatal Alcohol, SIDS, and Stillbirth Initiative
In collaboration with the NICHD, the NIAAA has launched an initiative to develop multi-disciplinary research projects in communities, both nationally and internationally, where prenatal maternal alcohol consumption is high to determine the relationship between prenatal alcohol exposure and other variables in the risk for sudden infant death syndrome (SIDS) and adverse pregnancy outcomes such as stillbirth and FAS. Pilot studies will be implemented in early 2005.
Collaborative Initiative on FASD
The NIAAA continues to support the CIFASD, a collaborative, multi-disciplinary, and cross-cultural research program aimed at developing effective interventions and treatment for FASD. The consortium coordinates basic, behavioral, and clinical investigations that utilize novel and cutting edge techniques. One of the first steps will be to definitively outline a diagnostic schema so that the full range of effects from exposure to moderate or large amounts of alcohol can be determined. The goal of the CIFASD is to bring together researchers from around the world who are conducting research on FASD or are interested in the global problem of FASD and who have the capabilities and resources to utilize international subject populations to further knowledge in this area. Advances in science often require the appropriate technological, social, and cultural climates to foster those advances. Studies that could not be conducted in any one site due to lack of subject numbers or given expertise will become possible through this collaborative initiative
Rare Disease-specific Conferences, Symposia, and Meetings
Mechanisms of Alcohol-associated Cancers, Bethesda, Maryland, October 6–7, 2004. NIAAA organized this symposium in collaboration with ORD, ODS, NCI, NIDDK, and NIDA. The following topics were covered by 16 speakers: (1) general mechanisms of cancers; (2) epidemiology of alcohol-associated cancers; (3) alcohol and oral cancer; (4) cancers of upper aerodigestive tract and the large intestine; (5) acetaldehyde, microbes, and cancers of the digestive tract; (6) mechanisms of acetaldehyde-induced DNA damage; (7) alcohol and aldehyde dehydrogenase polymorphisms and cancer; (8) alcohol and hepatocellular carcinoma; (9) alcohol and pancreatic cancer; (10) role of alcohol in breast cancer; (11) marijuana and cancer; (12) nicotine and gastric cancer; (13) role of MAT and SAMe in alcohol-associated liver cancer; (14) alcohol, vitamin A, and cancer; (15) alcohol, iron-associated oxidative stress, and cancer; and (16) alcohol, folate, and cancer. The proceedings of the symposium will be submitted to a journal for publication.
Mechanisms of Alcohol-induced Hepatic Fibrosis
NIAAA has submitted a proposal to ORD for funding to support this symposium:
Fetal Alcohol Syndrome and Other Alcohol-related Birth Defects, Heidelberg/Mannheim, Germany, September 28–October 2, 2004. Dr. Kenneth Warren, Director of the NIAAA Office of Scientific Affairs, co-chaired this symposium as part of the 12th World Conference of the International Society for Biomedical Research on Alcoholism. Papers were presented on: Ophthalmological Involvement in the Fetal Alcohol Syndrome; Comparison of FAS in Moscow, Russia and San Diego, California; Neurobehavioral and Interventions for FASD in South Africa; and Brain Imaging Studies on Children with Fetal Alcohol Spectrum Disorders.
Publications Related to ORD-sponsored Symposiums
Purohit V, Russo D, Salin M, and Brown R (November 2003). Mechanisms of Alcoholic Pancreatitis: Introduction and Summary of the Symposium. Pancreas 27 (4): 281-285.Proceedings of the symposium (10 chapters), Mechanisms of Alcoholic Pancreatitis (November 2003). Pancreas 27 (4): 281-331.
Veech, Richard L., (March 2004). The therapeutic implications of ketone bodies: the effects of ketone bodies in pathological conditions: ketosis, ketogenic diet, redox states, insulin resistance, and mitochondrial metabolism. Prostaglandins, Leukotrienes and Essential Fatty Acids 70(2004) 309-319.