Research Article: Uniting Epidemiology and Experimental Disease Models for Alcohol-Related Pancreatic Disease

Date Published: , 2017

Publisher: National Institute on Alcohol Abuse and Alcoholism

Author(s): Veronica Wendy Setiawan, Kristine Monroe, Aurelia Lugea, Dhiraj Yadav, Stephen Pandol.

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Abstract

Findings from epidemiologic studies and research with experimental animal models provide insights into alcohol-related disease pathogeneses. Epidemiologic data indicate that heavy drinking and smoking are associated with high rates of pancreatic disease. Less clear is the association between lower levels of drinking and pancreatitis. Intriguingly, a very low percentage of drinkers develop clinical pancreatitis. Experimental models demonstrate that alcohol administration alone does not initiate pancreatitis but does sensitize the pancreas to disease. Understanding the effects of alcohol use on the pancreas may prove beneficial in the prevention of both pancreatitis and pancreatic cancer.

Partial Text

Acute pancreatitis is among the most common gastrointestinal causes of inpatient admission to U.S. hospitals. The annual incidence of acute pancreatitis ranges from 13 to 45 per 100,000 people, and chronic pancreatitis from 2 to 14 per 100,000 (Machicado et al. 2016; Yadav and Lowenfels 2013). The incidence of chronic pancreatitis in European countries varies from 1.8 cases per 100,000 people in the Netherlands (Spanier et al. 2013) to 13.4 cases per 100,000 in Finland (Jaakkola and Nordback 1993). A population-based U.S. study noted little change in the incidence of chronic pancreatitis between two time periods (from 3.3 in 1940–1969 to 4.0 per 100,000 in 1977–2006). In Japan, however, a progressive increase in incidence from 5.4 in 1994 to 11.9 in 2007 and 14.0 in 2014 has been noted (Machicado et al. 2016).

The risk of progression from acute to chronic pancreatitis is higher among alcoholics and smokers, and higher in men than in women. A meta-analysis of 14 studies on this progression concluded that 10 percent of patients with a first episode of acute pancreatitis and 36 percent of patients with recurrent acute pancreatitis develop chronic pancreatitis (Sankaran et al. 2015). Other research found that, following an episode of alcohol-related acute pancreatitis, the risk of progression to chronic pancreatitis was approximately 14 percent with complete abstinence or only occasional drinking, 23 percent with decreased but daily drinking, and 41 percent with drinking at the same level as before the acute episode (Takeyama 2009).

A meta-analysis of 51 international population-based studies concluded that heavy alcohol use was an impor-tant risk factor for pancreatic disease (Alsamarrai et al. 2014). Overall, the studies demonstrated an estimated 40 percent increased risk of pancreatic disease in heavy drinkers (i.e., those reporting more than 20 drinks per week). The prevalence of pancreatitis is approximately four times higher among people with a history of alcoholism (Yadav et al. 2007). Historically, an estimated 60 to 90 percent of chronic pancreatitis cases were attributed to alcohol use (Coté et al. 2011). However, more recent research suggests a lower prevalence of heavy drinking among chronic pancreatitis patients than previously estimated (Frulloni et al. 2009; Yadav et al. 2009). One recent study estimating the prevalence of alcohol-related pancreatitis used data from 539 patients and 695 unaffected study participants enrolled in a study of pancreatic disease at U.S. treatment centers (Coté et al. 2011). An estimated 44.5 percent of chronic pancreatitis cases were classified as alcohol related, based on physician assessment. The authors acknowledge that the lower-than-expected rate of alcohol-related disease may be due to the specialized nature of the treatment centers, the fact that alcohol users may be less likely to seek care, or because physicians who attribute a patient’s disease to alcohol use would be less likely to refer them to a specialist’s care. In Japan, a questionnaire to assess alcohol use among patients with alcoholic pancreatitis found that women developed pancreatitis at a younger age, with shorter duration of alcohol use, and after smaller cumulative amounts of alcohol consumption compared with male patients (Masamune et al. 2013). In this study, continued drinking led to the recurrence of pancreatitis.

Cigarette smoking and heavy alcohol use, commonly co-occurring behaviors, increase risk for pancreatitis and pancreatic cancer (Yadav and Whitcomb 2010). A study of 108 smokers with alcohol-related chronic pancreatitis examined disease outcomes in relation to tobacco dose. The researchers concluded that smoking accelerates the course of pancreatic disease in a dose-dependent fashion, separate from the level of alcohol consumption (Rebours et al. 2012). A meta-analysis of 12 studies reported that while smoking increases the risk of chronic pancreatitis independently from alcohol, the effects of smoking are stronger for alcohol-related pancreatitis (Andriulli et al. 2010). In a recent study, Setiawan and colleagues (2016) found that smoking was significantly associated with nongallstone acute and chronic pancreatitis. The risk associated with current smoking was highest among men who consumed more than 4 drinks per day. For pancreatic cancer, among current smokers, heavy alcohol consumption was associated with a significantly increased pancreatic cancer risk. Risk was increased insignificantly among light and moderate drinkers who were smokers (Rahman et al. 2015).

Although alcohol abuse and smoking are major environmental risk factors for pancreatic disease, only a small percentage of drinkers and smokers develop pancreatic disease (Yadav and Lowenfels 2013). This has led to a search for a role of genetic differences that could explain the susceptibility of some individuals to the effects of alcohol on the pancreas. Whitcomb and colleagues (2012) identified an association between genetic variants of Claudin-2 (CLDN2) and the risk of alcoholic pancreatitis. CLDN2 is an X-linked gene involved in tight junction permeability and is expressed by pancreatic acinar cells. Alterations in the function of tight junctions in the pancreas or possibly in the intestinal epithelium could inappropriately expose the pancreas to toxins that could interact with the direct effects of alcohol in the pancreas. A recent study (Koziel et al. 2015) concluded that genetic mutations in SPINK1, a protein that inhibits activation of trypsinogens within the pancreas, may predispose individuals to severe acute pancreatitis, especially in patients that abuse alcohol.

The general concepts that have been followed in developing animal models for alcohol research are based on observations originally described by Comfort and colleagues (1946). They found histological changes consistent with acute pancreatitis in patients with chronic pancreatitis. When followed longitudinally, these patients had greater amounts of necrosis indicative of acute pancreatitis early in the disease course and fibrosis in later stages, suggesting that chronic pancreatitis developed from repeated attacks of acute pancreatitis.

Research into the molecular mechanisms of alcohol-related pancreatitis has largely focused on the pancreatic acinar cell, the component of the pancreas devoted to synthesis, storage, and secretion of digestive enzymes. These studies suggest that alcohol does not directly damage acinar cells but may make cells susceptible to other factors that trigger cell damage. For example, in vitro and in vivo studies that focus on the effects of CCK on the transcription factor NF-κB, an intracellular signaling pathway involved in the inflammatory response of pancreatitis, show that alcohol treatments augment CCK-induced NF-κB activation (Pandol et al. 1999). Another study suggested that alcohol activates a specific isoform of the signaling molecule known as protein kinase C (i.e., protein kinase C epsilon, PKCɛ), which, in turn, is involved in NF-κB activation and the initiation of pancreatitis (Satoh et al. 2006). Further research using experimental models of acute pancreatitis examined the mechanisms through which PKCɛ regulates cell death. The researchers found that PKCɛ knockout mice (in which PKCɛ is genetically deleted) had decreased inflammation and necrosis and less severe acute pancreatitis in response to high doses of CCK analogues (Liu et al. 2014). In addition, alcohol has been found to promote secretion of digestive enzymes from the basolateral aspect of the acinar cell via mechanisms involving protein kinase C (Cosen-Binker 2007). Basolateral enzyme secretion would inject the digestive enzymes into the tissue of the pancreas where they can cause injury to the pancreas and pancreatitis.

Alcohol-related pancreatitis has been linked to the activation of pancreatic stellate cells (PaSC) (Apte et al. 1999, 2000; Vonlaufen et al. 2007, 2011). PaSC are normal resident cells in the exocrine pancreas. They are present in the periacinar space and have long cytoplasmic processes that surround the acinar structures and ducts of the exocrine pancreas (Omary et al. 2007).

Metabolism of ethanol by the exocrine pancreas occurs by both oxidative and nonoxidative routes (Gukovskaya et al. 2002; Haber et al. 2004). The oxidative pathway is the predominant pathway for ethanol elimination in the body, occurring mostly in the liver. In the oxidative pathway, ethanol is converted to acetaldehyde by alcohol dehydrogenases (ADH), and then acetaldehyde is converted to acetate by mitochondrial aldehyde dehydrogenases (ALDH). Both enzymes are functional and present in the exocrine pancreas. The nonoxidative route of ethanol metabolism involves covalent coupling of ethanol with fatty acids to yield lipophilic fatty acid ethyl esters (FAEEs). This pathway provides the transient storage of ethanol while it awaits oxidative metabolism for removal from the body. The importance of the nonoxidative pathway comes from observations that humans dying from alcohol intoxication have high levels of FAEEs in the pancreas (Laposata and Lange 1986) and the finding that the FAEEs are formed using the enzyme carboxylester lipase, a highly expressed digestive enzyme made in the pancreas and secreted during lipid digestion (Huang et al. 2014).

The neurotransmitter acetylcholine may play a role in alcohol-induced pancreatic damage. Lugea and colleagues (2010) found that atropine dramatically reduced cerulein-induced pancreatitis in alcohol-fed rats, indicating that alcohol-ensitizing effects are mediated at least in part through activation of cholinergic pathways. This effect is independent of the effects of smoking on nicotinic receptors present on the PaSC, described below.

Mitochondrial membrane permeabilization (MMP) triggers mitochondrial dysfunction and cell death and leads to tissue damage. The mitochondrial permeability transition pore (MPTP) plays a critical role in MMP. Research with pancreatic cells from mice found that oxidative metabolism of ethanol sensitizes mitochondria to activate MPTP, making them more sensitive to the toxicity by low concentrations of Ca2+ in the cell. This leads to mitochondrial failure and ATP depletion, making the pancreas susceptible to pancreatitis (Huang et al. 2014; Shalbueva et al. 2013).

Autophagy is a natural and regulated process for the cell to disassemble unnecessary or dysfunctional components. This disassembly allows for an orderly recycling of cellular components. The process of autophagy involves isolating targeted cellular constituents within a double-membrane vesicle known as the autophagosome. The autophagosome eventually fuses with the cell’s lysosomes to form a compartment where lysosomal enzymes carry out the disassembly. Recent studies have shown the importance of normal autophagy and lysosomal function in the mechanism of pancreatitis (Gukovskaya et al. 2016). That is, animal models created with genetic inhibition of key autophagic mediators (i.e., autophagy protein 5, Atg5, or Atg7) or the glycoprotein required for lysosomal integrity (i.e., lysosomal-associated membrane protein-2, LAMP2) lack normal autophagic processing, resulting in inappropriate processing of digestive enzymes in the acinar cells and spontaneous pancreatitis. Further, in nonalcoholic models of pancreatitis, findings of disordered fusion and function of the lysosomal-autophagic system have been described (Gukovskaya et al. 2016).

Despite the increased risk for pancreatic damage among heavy drinkers, the incidence of clinical pancreatitis in heavy drinkers is low (~5 percent) (Yadav et al. 2007). One potential explanation for the low rate of pancreatitis among heavy drinkers is that alcohol induces adaptive systems that serve to protect the pancreas from the damaging effects of alcohol. This theory holds that disease progresses when the damaging effects are stronger than the protective effects, or when the protective systems are impaired. Thus, the combination of alcohol use and another risk factor could represent an overwhelming burden and therefore lead to disease progression.

Most genetically engineered mouse models of pancreatic cancer are based on genetic mutations in the Kras gene. Mice expressing mutant Kras develop early and advanced forms of the most common pancreatic cancers in humans. However, Kras mutations alone are not sufficient to induce progression to the invasive stage of pancreatic cancer. Rather, different transgenes have been used to create models that progress to invasive cancer. For example, one common model based on Kras mutations is the PDX1-Cre;LSL-KrasG12D model. Xu and colleagues (2015) reported using this model in mice exposed to alcohol and given injections of cerulein. The mice developed fibrosis and had an increased level of cancerous lesions. The authors concluded that alcohol independently increased pancreatic-cancer risk associated with fibrosis. Another animal model induces pancreatic cancer through the implantation of dimethylbenzanthracene (DMBA) in the pancreas. Research using this method in mice resulted in the development of both precursor lesions and invasive tumors. There was a higher relative frequency of tumors in mice receiving alcohol compared with the control group (Wendt et al. 2007).

The combination of epidemiologic and experimental animal-model observations continues to reveal insights into both disease pathogenesis and potential adaptive protective mechanisms of alcohol use. The relationship between heavy alcohol consumption and acute and chronic pancreatitis is well established (Yadav 2016). The highest rates of nongallstone-related pancreatitis are observed in those who drink the greatest amount of alcohol. A recent epidemiological observation of a potential protective effect of moderate alcohol use should be considered preliminary, encourage further research to confirm and determine generalizability of these findings, and elucidate the potential mechanism. Further, smoking is associated with significant risk for non-gallstone-related pancreatitis and may add to the risk of pancreatitis with heavy drinking. A very low percentage of drinkers develop pancreatitis. Experimental models demonstrate that alcohol administration alone may not initiate pancreatitis, but it sensitizes the pancreas to pancreatitis by other insults.

 

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