Ethanol inhibits pancreatic acinar cell autophagy through upregulation of ATG4B, mediating pathological responses of alcoholic pancreatitis.

Excessive alcohol intake is a major risk factor for pancreatitis, sensitizing the exocrine pancreas to stressors by mechanisms that remain obscure. Impaired autophagy drives nonalcoholic pancreatitis, but the effects of ethanol (EtOH) and alcoholic pancreatitis on autophagy are poorly understood. Here, we find that ethanol reduces autophagosome formation in pancreatic acinar cells, both in a mouse model of alcoholic pancreatitis induced by a combination of EtOH diet and cerulein (a CCK ortholog) and in EtOH+CCK-treated acinar cells (ex vivo model). Ethanol treatments decreased pancreatic level of LC3-II, a key mediator of autophagosome formation. This was caused by ethanol-induced upregulation of ATG4B, a cysteine protease that, cell dependently, regulates the balance between cytosolic LC3-I and membrane-bound LC3-II. We show that ATG4B negatively regulates LC3-II in acinar cells subjected to EtOH treatments. Ethanol raised ATG4B level by inhibiting its degradation, enhanced ATG4B enzymatic activity, and strengthened its interaction with LC3-II. We also found an increase in ATG4B and impaired autophagy in a dissimilar, nonsecretagogue model of alcoholic pancreatitis induced by EtOH plus palmitoleic acid. Adenoviral ATG4B overexpression in acinar cells greatly reduced LC3-II and inhibited autophagy. Furthermore, it aggravated trypsinogen activation and necrosis, mimicking key responses of ex vivo alcoholic pancreatitis. Conversely, shRNA Atg4B knockdown enhanced autophagosome formation and alleviated ethanol-induced acinar cell damage. The results reveal a novel mechanism, whereby ethanol inhibits autophagosome formation and thus sensitizes pancreatitis, and a key role of ATG4B in ethanol's effects on autophagy. Enhancing pancreatic autophagy, particularly by downregulating ATG4B, could be beneficial in mitigating the severity of alcoholic pancreatitis.NEW & NOTEWORTHY Ethanol sensitizes mice and humans to pancreatitis, but the underlying mechanisms remain obscure. Autophagy is important for maintaining pancreatic acinar cell homeostasis, and its impairment drives pancreatitis. This study reveals a novel mechanism, whereby ethanol inhibits autophagosome formation through upregulating ATG4B, a key cysteine protease. ATG4B upregulation inhibits autophagy in acinar cells and aggravates pathological responses of experimental alcoholic pancreatitis. Enhancing pancreatic autophagy, particularly by down-regulating ATG4B, could be beneficial for treatment of alcoholic pancreatitis.

[Influence of environmental factors and polymorphic loci rs6580502 of the SPINK1 gene, rs10273639 of the PRSS1 gene, rs213950 of the CFTR gene on the risk of developing acute alcoholic-alimentary pancreatitis].

The study of genetic and environmental factors on the risk of acute alcoholic-alimentary pancreatitis (AАAР) is especially relevant to interpret individual links of pathogenesis, to reduce the incidence by eliminating the impact of harmful factors and improve the quality of life of the population through the introduction of optimal nutrition, and a healthy lifestyle, which is especially important for carriers of risk genotypes. The aim of the research was to study the influence of environmental factors and polymorphic loci rs6580502 of the SPINK1 gene, rs10273639 of the PRSS1 gene, rs213950 of the CFTR gene on the risk of АAР. Material and methods. Blood DNA samples obtained from 547 patients with AАAР and 573 healthy individuals were used as the material for the study. The groups were comparable by sex and age. All participants were assessed qualitatively and quantitatively for risk factors, smoking and alcohol consumption, the frequency, quantity and regularity of intake of various types of foods, as well as the size and number of portions eaten. Genomic DNA was isolated by the standard phenol-chloroform extraction method, multiplex genotyping of SNPs was performed on a MALDI-TOF MassARRAY-4 genetic analyzer. Results. It was found that the T/T genotype (p=0.0012) of the rs6580502 SPINK1 was associated with an increased risk of AAAP, and the T allele (p=0.0001) and C/T and T/T genotypes (p=0.0001) of the rs10273639 PRSS1, A allele (p=0.01) and A/G and A/A genotypes (p=0.0006) of the rs213950 CFTR were associated with an decreased risk of the disease. The revealed effects of polymorphic loci of candidate genes were enhanced by the effect of alcohol consumption. The risk of AAAP was reduced by fat intake of less than 89 g per day in carriers of the A/G-A/A CFTR genotypes (rs213950), consumption of fresh vegetables and fruits of more than 27 g per day in carriers of the T/C-T/T PRSS1 genotypes (rs10273639), protein intake of more 84 g per day in carriers of T/C-T/T PRSS1 rs10273639 and A/G-A/A CFTR rs213950. The most significant models of gene-environment interactions included risk factors: deficiency in the diet of protein, fresh vegetables and fruits, smoking, and polymorphic variants of the PRSS1 (rs10273639) and SPINK (rs6580502) genes. Conclusion. In order to prevent the development of AAAP, carriers of risk genotypes of candidate genes need not only to exclude or significantly reduce alcohol consumption (in terms of volume, frequency and duration); but also carriers of the A/G-A/A CFTR genotypes (rs213950) need to balance the diet by reducing fat intake to less than 89 g per day and increasing protein intake to more than 84 g per day; carriers of the T/C-T/T PRSS1 (rs10273639) genotypes should increase their intake of fresh vegetables and fruits over 27 g/day and protein over 84 g/day.

Genetic analysis of pancreatic phospholipase A2 (PLA2G1B) in patients with chronic pancreatitis.

BACKGROUND: Genetic mutations in various pancreatic enzymes or their counteracting proteins have been linked to chronic pancreatitis. In particular, variants in the genes encoding pancreatic lipase (PNLIP) and carboxyl ester lipase (CEL) have been associated with pancreatitis. Therefore, we investigated pancreatic phospholipase A2 (PLA2G1B) as a promising candidate gene in patients with chronic pancreatitis. METHODS: We analyzed all coding exons and adjacent intronic regions of PLA2G1B in 416 German patients with non-alcoholic chronic pancreatitis (NACP) and 186 control subjects by direct DNA sequencing. RESULTS: We detected 2 frequent synonymous variants in exon 3: c.222T>C (p.Y74 = ) and c.294G>A (p.S98 = ). The genotype and allele frequencies of these variants were similar between patients and controls (c.222 TC: 9.6% in NACP vs. 9.7% in controls; c.222CC: 0.2% in NACP vs. 0% in controls; c.294 GA: 31.3% in NACP vs. 28.0% in controls; c.294AA: 2.4% in NACP vs. 1.1% in controls). All p-values were non-significant. In addition, we found one synonymous variant, c.138C>T (p.N46 = ) and one non-synonymous variant, c.244A>G (p.S82G), in a single case each. CONCLUSIONS: Our results suggest that genetic alterations in PLA2G1B do not predispose to the development of non-alcoholic chronic pancreatitis.

Colocalization analysis of pancreas eQTLs with risk loci from alcoholic and novel non-alcoholic chronic pancreatitis GWAS suggests potential disease causing mechanisms.

BACKGROUND: Previous genome-wide association studies (GWAS) identified genome-wide significant risk loci in chronic pancreatitis and investigated underlying disease causing mechanisms by simple overlaps with expression quantitative trait loci (eQTLs), a procedure which may often result in false positive conclusions. METHODS: We conducted a GWAS in 584 non-alcoholic chronic pancreatitis (NACP) patients and 6040 healthy controls. Next, we applied Bayesian colocalization analysis of identified genome-wide significant risk loci from both, our recently published alcoholic chronic pancreatitis (ACP) and the novel NACP dataset, with pancreas eQTLs from the GTEx V8 European cohort to prioritize candidate causal genes and extracted credible sets of shared causal variants. RESULTS: Variants at the CTRC (p = 1.22 × 10-21) and SPINK1 (p = 6.59 × 10-47) risk loci reached genome-wide significance in NACP. CTRC risk variants colocalized with CTRC eQTLs in ACP (PP4 = 0.99, PP4/PP3 = 95.51) and NACP (PP4 = 0.99, PP4/PP3 = 95.46). For both diseases, the 95% credible set of shared causal variants consisted of rs497078 and rs545634. CLDN2-MORC4 risk variants colocalized with CLDN2 eQTLs in ACP (PP4 = 0.98, PP4/PP3 = 42.20) and NACP (PP4 = 0.67, PP4/PP3 = 7.18), probably driven by the shared causal variant rs12688220. CONCLUSIONS: A shared causal CTRC risk variant might unfold its pathogenic effect in ACP and NACP by reducing CTRC expression, while the CLDN2-MORC4 shared causal variant rs12688220 may modify ACP and NACP risk by increasing CLDN2 expression.

Homozygosity of short VNTR lengths in the CEL gene may confer susceptibility to idiopathic chronic pancreatitis.

OBJECTIVE: The carboxyl-ester lipase (CEL) gene contains a variable number of tandem repeats (VNTR) region. It remains unclear whether the number of repeats in the CEL VNTR is related to the risk of pancreatic diseases. The aim of this study was to investigate whether CEL VNTR length is associated with idiopathic chronic pancreatitis (ICP), alcoholic chronic pancreatitis (ACP), or pancreatic cancer in a cohort of Chinese patients. METHODS: CEL VNTRs were genotyped in patients diagnosed with ICP (n = 771), ACP (n = 222), or pancreatic cancer (n = 263), and in healthy controls (n = 927). CEL VNTR lengths were determined using a screening method combining PCR and DNA fragment analysis. RESULTS: Overall, the CEL VNTR lengths ranged from 5 to 22 repeats, with the 16-repeat allele ('normal' size, N) accounting for 73.82% of all observed alleles. The VNTR allele frequencies and genotype distributions were not significantly different between healthy controls and patients with ACP or pancreatic cancer. For the ICP group, allele frequencies did not differ significantly from the controls, while the frequency of the SS genotype (homozygosity for 5-15 repeats) was significantly higher in the patients (4.67%) than in the controls (1.94%) (p = 0.0014; OR = 2.47; 95% CI = 1.39-4.39). CONCLUSIONS: There were no associations between the CEL VNTR length and ACP or pancreatic cancer. However, homozygosity for short VNTR lengths may confer susceptibility to ICP.

Ethanol feeding accelerates pancreatitis progression in CPA1 N256K mutant mice.

Alcoholic pancreatitis is a multifactorial, progressive, inflammatory disorder of the pancreas. Alcohol initiates pancreatitis and promotes its progression in the context of genetic susceptibility and/or other environmental risk factors such as smoking. Genetic mutations can cause digestive enzyme misfolding, which induces endoplasmic reticulum (ER) stress and elicits pancreatitis. Here, we tested the hypothesis that alcohol synergizes with misfolding in promoting ER stress and thereby accelerates chronic pancreatitis progression. To this end, we fed an ethanol-containing diet to CPA1 N256K mice, which carry the human p.N256K CPA1 mutation and develop spontaneous chronic pancreatitis. Inexplicably, CPA1 N256K mice suffered generalized seizures after 2-3 wk of ethanol feeding, which resulted in high mortality and the early termination of the study. Analysis of CPA1 N256K mice euthanized after 3-3.5 wk of ethanol feeding revealed more severe chronic pancreatitis associated with significantly increased Hspa5 [ER chaperone immunoglobulin heavy chain-binding protein (BiP)] mRNA levels when compared with CPA1 N256K mice on a control liquid diet. In contrast, ethanol feeding of C57BL/6N mice for 4 wk increased Hspa5 levels to a lesser degree and caused no pancreatitis. We conclude that ethanol feeding synergizes with the misfolding CPA1 mutant in promoting ER stress and thereby accelerates progression of chronic pancreatitis in CPA1 N256K mice.NEW & NOTEWORTHY Alcoholic pancreatitis is a multifactorial, progressive, inflammatory disorder of the pancreas. This study demonstrates that alcohol synergizes with digestive enzyme misfolding in promoting endoplasmic reticulum stress and thereby accelerates progression of chronic pancreatitis.

Analysis of GPRC6A variants in different pancreatitis etiologies.

BACKGROUND: The G-protein-coupled receptor Class C Group 6 Member A (GPRC6A) is activated by multiple ligands and is important for the regulation of calcium homeostasis. Extracellular calcium is capable to increase NLRP3 inflammasome activity of the innate immune system and deletion of this proinflammatory pathway mitigated pancreatitis severity in vivo. As such this pathway and the GPRC6A receptor is a reasonable candidate gene for pancreatitis. Here we investigated the prevalence of sequence variants in the GPRC6A locus in different pancreatitis aetiologies. METHODS: We selected 6 tagging SNPs with the SNPinfo LD TAG SNP Selection tool and the functional relevant SNP rs6907580 for genotyping. Cohorts from Germany, further European countries and China with up to 1,124 patients and 1,999 controls were screened for single SNPs with melting curve analysis. RESULTS: We identified an association of rs1606365(G) with alcoholic chronic pancreatitis in a German (odds ratio (OR) 0.76, 95% confidence interval (CI) 0.65-0.89, p = 8 × 10-5) and a Chinese cohort (OR 0.78, 95% CI 0.64-0.96, p = 0.02). However, this association was not replicated in a combined cohort of European patients (OR 1.18, 95% CI 0.99-1.41, p = 0.07). Finally, no association was found with acute and non-alcoholic chronic pancreatitis. CONCLUSIONS: Our results support a potential role of calcium sensing receptors and inflammasome activation in alcoholic chronic pancreatitis development. As the functional consequence of the associated variant is unclear, further investigations might elucidate the relevant mechanisms.

Characterization of CEL-DUP2: Complete duplication of the carboxyl ester lipase gene is unlikely to influence risk of chronic pancreatitis.

BACKGROUND/OBJECTIVES: Carboxyl ester lipase is a pancreatic enzyme encoded by CEL, an extremely polymorphic human gene. Pathogenic variants of CEL either increases the risk for chronic pancreatitis (CP) or cause MODY8, a syndrome of pancreatic exocrine and endocrine dysfunction. Here, we aimed to characterize a novel duplication allele of CEL (CEL-DUP2) and to investigate whether it associates with CP or pancreatic cancer. METHODS: The structure of CEL-DUP2 was determined by a combination of Sanger sequencing, DNA fragment analysis, multiplex ligation-dependent probe amplification and whole-genome sequencing. We developed assays for screening of CEL-DUP2 and analyzed cohorts of idiopathic CP, alcoholic CP and pancreatic cancer. CEL protein expression was analyzed by immunohistochemistry. RESULTS: CEL-DUP2 consists of an extra copy of the complete CEL gene. The allele has probably arisen from non-allelic, homologous recombination involving the adjacent pseudogene of CEL. We found no association between CEL-DUP2 carrier frequency and CP in cohorts from France (cases/controls: 2.5%/2.4%; P = 1.0), China (10.3%/8.1%; P = 0.08) or Germany (1.6%/2.3%; P = 0.62). Similarly, no association with disease was observed in alcohol-induced pancreatitis (Germany: 3.2%/2.3%; P = 0.51) or pancreatic cancer (Norway; 2.5%/3.2%; P = 0.77). Notably, the carrier frequency of CEL-DUP2 was more than three-fold higher in Chinese compared with Europeans. CEL protein expression was similar in tissues from CEL-DUP2 carriers and controls. CONCLUSIONS: Our results support the contention that the number of CEL alleles does not influence the risk of pancreatic exocrine disease. Rather, the pathogenic CEL variants identified so far involve exon 11 sequence changes that substantially alter the protein's tail region.

Genome-wide association study identifies inversion in the CTRB1-CTRB2 locus to modify risk for alcoholic and non-alcoholic chronic pancreatitis.

OBJECTIVE: Alcohol-related pancreatitis is associated with a disproportionately large number of hospitalisations among GI disorders. Despite its clinical importance, genetic susceptibility to alcoholic chronic pancreatitis (CP) is poorly characterised. To identify risk genes for alcoholic CP and to evaluate their relevance in non-alcoholic CP, we performed a genome-wide association study and functional characterisation of a new pancreatitis locus. DESIGN: 1959 European alcoholic CP patients and population-based controls from the KORA, LIFE and INCIPE studies (n=4708) as well as chronic alcoholics from the GESGA consortium (n=1332) were screened with Illumina technology. For replication, three European cohorts comprising 1650 patients with non-alcoholic CP and 6695 controls originating from the same countries were used. RESULTS: We replicated previously reported risk loci CLDN2-MORC4, CTRC, PRSS1-PRSS2 and SPINK1 in alcoholic CP patients. We identified CTRB1-CTRB2 (chymotrypsin B1 and B2) as a new risk locus with lead single-nucleotide polymorphism (SNP) rs8055167 (OR 1.35, 95% CI 1.23 to 1.6). We found that a 16.6 kb inversion in the CTRB1-CTRB2 locus was in linkage disequilibrium with the CP-associated SNPs and was best tagged by rs8048956. The association was replicated in three independent European non-alcoholic CP cohorts of 1650 patients and 6695 controls (OR 1.62, 95% CI 1.42 to 1.86). The inversion changes the expression ratio of the CTRB1 and CTRB2 isoforms and thereby affects protective trypsinogen degradation and ultimately pancreatitis risk. CONCLUSION: An inversion in the CTRB1-CTRB2 locus modifies risk for alcoholic and non-alcoholic CP indicating that common pathomechanisms are involved in these inflammatory disorders.

Lipopolysaccharide enhances TGF-ß1 signalling pathway and rat pancreatic fibrosis.

Pancreatic stellate cells (PSCs) play a critical role in fibrogenesis during alcoholic chronic pancreatitis (ACP). Transforming growth factor-beta1 (TGF-ß1) is a key regulator of extracellular matrix production and PSC activation. Endotoxin lipopolysaccharide (LPS) has been recognized as a trigger factor in the pathogenesis of ACP. This study aimed to investigate the mechanisms by which LPS modulates TGF-ß1 signalling and pancreatic fibrosis. Sprague-Dawley rats fed with a Lieber-DeCarli alcohol (ALC) liquid diet for 10 weeks with or without LPS challenge during the last 3 weeks. In vitro studies were performed using rat macrophages (Mφs) and PSCs (RP-2 cell line). The results showed that repeated LPS challenge resulted in significantly more collagen production and PSC activation compared to rats fed with ALC alone. LPS administration caused overexpression of pancreatic TLR4 or TGF-ß1 which was paralleled by an increased number of TLR4-positive or TGF-ß1-positive Mφs or PSCs in ALC-fed rats. In vitro, TLR4 or TGF-ß1 production in Mφs or RP-2 cells was up-regulated by LPS. LPS alone or in combination with TGF-ß1 significantly increased type I collagen and α-SMA production and Smad2 and 3 phosphorylation in serum-starved RP-2 cells. TGF-ß pseudoreceptor BAMBI production was repressed by LPS, which was antagonized by Si-TLR4 RNA or by inhibitors of MyD88/NF-kB. Additionally, knockdown of Bambi with Si-Bambi RNA significantly increased TGF-ß1 signalling in RP-2 cells. These findings indicate that LPS increases TGF-ß1 production through paracrine and autocrine mechanisms and that LPS enhances TGF-ß1 signalling in PSCs by repressing BAMBI via TLR4/MyD88/NF-kB activation.

Genetic Variants in the Manganese Superoxide Dismutase 2 Gene and in the Catalase Gene are not Associated With Alcoholic Chronic Pancreatitis.

AIMS: Oxidative stress may contribute to the development of chronic pancreatitis (CP). The enzymes manganese superoxide dismutase 2 (MnSOD, SOD2) and catalase (CAT) counteract free radical activity within the mitochondria and the cytosol. Moreover, CAT activity contributes to the transformation of ethanol to acetaldehyde, a toxic intermediate product of ethanol metabolism, which has been associated with pancreatic damage. Common functional polymorphisms have been described in the MnSOD gene [rs4880, NM_000636.3:c.47 T > C, alanine (ALA) to valine (Val)] and in the CAT promoter region [rs1001179, NG_013339.1:g.4760 C > T]. We investigated whether these polymorphisms are associated with alcoholic CP. METHODS: We genotyped 470 patients with alcoholic CP for these MnSOD and CAT polymorphisms. We also analysed these variants in 357 healthy control subjects, and in an additional control group of 113 individuals with non-alcoholic CP. We used the age at onset of CP as marker of disease severity and investigated whether different genotypes are associated with different ages at onset. In patients with alcoholic CP, we investigated whether an interaction exists between smoking behaviour and genotypes by comparing genotype distributions in smokers and non-smokers. RESULTS: We did not observe significant differences of genotype frequencies between patient groups and controls. In patient groups, we did not find significant differences in the ages at onset between different genotypes. We did not observe an interaction between these polymorphisms. We did not find an association of these variants with smoking behaviour. CONCLUSIONS: The investigated MnSOD and CAT polymorphisms do not predispose to the development of alcoholic CP. SHORT SUMMARY: Patients with alcoholic pancreatitis and controls were genotyped for polymorphisms in oxidative stress genes. There were no significant differences of genotype frequencies between patients and controls, and no association with the age at onset of disease was observed. The polymorphisms are not associated with the development of alcoholic pancreatitis.

PRSS1 and SPINK1 Mutations in Alcoholic and Idiopathic Chronic Pancreatitis.

Several recent studies have reported associations between gene mutations and chronic pancreatitis (CP); however, little is known about their association with risk of CP in the Chinese Han population. The aim of this study was to describe mutations in the cationic trypsinogen (PRSS1) and serine protease inhibitor Kazal type 1 (SPINK1) genes in patients with alcoholic chronic pancreatitis (ACP) and idiopathic chronic pancreatitis (ICP) and to investigate their influence on the clinical course of the disease. One hundred patients (24 with ACP, 76 with ICP) and 100 healthy volunteers (control group) were enrolled in the study. PRSS1 (R122H) mutations were detected in one (1.3%) patient with ICP and SPINK1 (N34S) mutations were present in one (4.1%) patient with ACP. PRSS1 and SPINK1 mutations were not detected in the control populations. There were no statistically significant differences between the CP patients and the control group. Those preliminary data suggest low prevalence of SPINK1 and PRSS1 mutations in the Chinese population, generally, as well as in CP patients, indicating that these mutations do not contribute to the development of CP.

Polymorphisms at PRSS1-PRSS2 and CLDN2-MORC4 loci associate with alcoholic and non-alcoholic chronic pancreatitis in a European replication study.

OBJECTIVE: Several genetic risk factors have been identified for non-alcoholic chronic pancreatitis (NACP). A genome-wide association study reported an association of chronic pancreatitis (CP) with variants in PRSS1-PRSS2 (rs10273639; near the gene encoding cationic trypsinogen) and CLDN2-MORC4 loci (rs7057398 in RIPPLY1 and rs12688220 in MORC4). We aimed to refine these findings in a large European cohort. DESIGN: We studied 3062 patients with alcohol-related CP (ACP) or NACP and 5107 controls. Also, 1559 German patients with alcohol-associated cirrhosis or alcohol dependence were included for comparison. We performed several meta-analyses to examine genotype-phenotype relationships. RESULTS: Association with ACP was found for rs10273639 (OR, 0.63; 95% CI 0.55 to 0.72). ACP was also associated with variants rs7057398 and rs12688220 in men (OR, 2.26; 95% CI 1.94 to 2.63 and OR, 2.66; 95% CI 2.21 to 3.21, respectively) and in women (OR, 1.57; 95% CI 1.14 to 2.18 and OR 1.71; 95% CI 1.41 to 2.07, respectively). Similar results were obtained when German patients with ACP were compared with those with alcohol-associated cirrhosis or alcohol dependence. In the overall population of patients with NACP, association with rs10273639 was absent (OR, 0.93; 95% CI 0.79 to 1.01), whereas rs7057398 of the X chromosomal single nucleotide polymorphisms was associated with NACP in women only (OR, 1.32; 95% CI 1.15 to 1.51). CONCLUSIONS: The single-nucleotide polymorphisms rs10273639 at the PRSS1-PRSS2 locus and rs7057398 and rs12688220 at the CLDN2-MORC4 locus are associated with CP and strongly associate with ACP, but only rs7057398 with NACP in female patients.

Functional characteristics of L1156F-CFTR associated with alcoholic chronic pancreatitis in Japanese.

Although cystic fibrosis is rare in Japanese, measurement of sweat Cl(-) has suggested mild dysfunction of cystic fibrosis transmembrane conductance regulator (CFTR) in some patients with chronic pancreatitis. In the present study, we have investigated the association of CFTR variants and chronic pancreatitis in Japanese and the functional characteristics of a Japanese- and pancreatitis-specific CFTR variant, L1156F. Seventy patients with alcoholic chronic pancreatitis, 18 patients with idiopathic chronic pancreatitis, and 180 normal subjects participated. All exons and their boundaries and promoter region of the CFTR gene were sequenced. Human embryonic kidney-293 cells were transfected with three CFTR variants (M470V, L1156F, and M470V+L1156F), and the protein expression was examined. Xenopus laevis oocytes were injected with the CFTR variants, and bicarbonate (HCO3 (-)) transport activity was examined. CFPAC-1 cells were transfected with the CFTR variants and Cl(-)/HCO3 (-) exchange activity was examined. Six variants (E217G, I556V, M470V, L1156F, Q1352H, and R1453W) were identified in the coding region of the CFTR gene. Cystic fibrosis-causing mutations were not found. The allele frequencies of L1156F and Q1352H in alcoholic chronic pancreatitis (5.0 and 7.9%) were significantly (P < 0.01) higher than those in normal subjects (0.6 and 1.9%). L1156F was linked with a worldwide CFTR variant, M470V. Combination of M470V and L1156F significantly reduced CFTR expression to ∼60%, impaired CFTR-mediated HCO3 (-)/Cl(-) transport activity to 50-60%, and impaired CFTR-coupled Cl(-)/HCO3 (-) exchange activity to 20-30%. The data suggest that the Japanese-specific CFTR variant L1156F causes mild dysfunction of CFTR and increases the risk of alcoholic chronic pancreatitis in Japanese.

Chronic alcohol exposure affects pancreatic acinar mitochondrial thiamin pyrophosphate uptake: studies with mouse 266-6 cell line and primary cells.

Thiamin is essential for normal metabolic activity of all mammalian cells, including those of the pancreas. Cells obtain thiamin from their surroundings and enzymatically convert it into thiamin pyrophosphate (TPP) in the cytoplasm; TPP is then taken up by mitochondria via a specific carrier the mitochondrial TPP transporter (MTPPT; product of the SLC25A19 gene). Chronic alcohol exposure negatively impacts the health of pancreatic acinar cells (PAC), but its effect on physiological/molecular parameters of MTPPT is not known. We addressed this issue using mouse pancreatic acinar tumor cell line 266-6 and primary PAC of wild-type and transgenic mice carrying the SLC25A19 promoter that were fed alcohol chronically. Chronic alcohol exposure of 266-6 cells (but not to its nonoxidative metabolites ethyl palmitate and ethyl oleate) led to a significant inhibition in mitochondrial TPP uptake, which was associated with a decreased expression of MTPPT protein, mRNA, and activity of the SLC25A19 promoter. Similarly, chronic alcohol feeding of mice led to a significant inhibition in expression of MTPPT protein, mRNA, heterogeneous nuclear RNA, as well as in activity of SLC25A19 promoter in PAC. While chronic alcohol exposure did not affect DNA methylation of the Slc25a19 promoter, a significant decrease in histone H3 euchromatin markers and an increase in H3 heterochromatin marker were observed. These findings show, for the first time, that chronic alcohol exposure negatively impacts pancreatic MTPPT, and that this effect is exerted, at least in part, at the level of Slc25a19 transcription and appears to involve epigenetic mechanism(s).

The effect of renin angiotensin system genetic variants in acute pancreatitis.

OBJECTIVES: We sought association of genetic variants in the renin-angiotensin system (RAS) and vitamin D system with acute pancreatitis (AP) development and severity. BACKGROUND: The endocrine RAS is involved in circulatory homeostasis through the pressor action of angiotensin II at its AT1 receptor. However, local RAS regulate growth and inflammation in diverse cells and tissues, and their activity may be suppressed by vitamin D. Intrapancreatic angiotensin II generation has been implicated in the development of AP. METHODS: Five hundred forty-four white patients with AP from 3 countries (United Kingdom, 22; Germany, 136; and The Netherlands 386) and 8487 control subjects (United Kingdom 7833, The Netherlands 717) were genotyped for 8 polymorphisms of the RAS/vitamin D systems, chosen on the basis of likely functionality. RESULTS: The angiotensin-converting enzyme I (rather than D) allele was significantly associated with alcohol-related AP when all cohorts were combined (P = 0.03). The renin rs5707 G (rather than A) allele was associated with AP (P = 0.002), infected necrosis (P = 0.025) and mortality (P = 0.046). CONCLUSIONS: The association of 2 RAS polymorphisms with AP suggests the need for further detailed analysis of the role of RAS/vitamin D in the genesis or severity of AP, particularly given the ready potential for pharmacological manipulation of this system using existing marketed agents. However, further replication studies will be required before any such association is considered robust, particularly given the significant heterogeneity of AP causation and clinical course.

Genetic susceptibility factors for alcohol-induced chronic pancreatitis.

Chronic pancreatitis is a progressive inflammatory disease of the pancreas and frequently associated with immoderate alcohol consumption. Since only a small proportion of alcoholics eventually develop chronic pancreatitis genetic susceptibility factors have long been suspected to contribute to the pathogenesis of the disease. Smaller studies in ethnically defined populations have found that not only polymorphism in proteins involved in the metabolism of ethanol, such as Alcohol Dehydrogenase and Aldehyde Dehydrogenase, can confer a risk for developing chronic pancreatitis but also mutations that had previously been reported in association with idiopathic pancreatitis, such as SPINK1 mutations. In a much broader approach employing genome wide search strategies the NAPS study found that polymorphisms in the Trypsin locus (PRSS1 rs10273639), and the Claudin 2 locus (CLDN2-RIPPLY1-MORC4 locus rs7057398 and rs12688220) confer an increased risk of developing alcohol-induced pancreatitis. These results from North America have now been confirmed by a European consortium. In another genome wide approach polymorphisms in the genes encoding Fucosyltransferase 2 (FUT2) non-secretor status and blood group B were not only found in association with higher serum lipase levels in healthy volunteers but also to more than double the risk for developing alcohol-associated chronic pancreatitis. These novel genetic associations will allow to investigate the pathophysiological and biochemical basis of alcohol-induced chronic pancreatitis on a cellular level and in much more detail than previously possible.

Targeted next-generation sequencing effectively analyzed the cystic fibrosis transmembrane conductance regulator gene in pancreatitis.

BACKGROUND: The cystic fibrosis transmembrane conductance regulator (CFTR) gene, responsible for the development of cystic fibrosis, is known as a pancreatitis susceptibility gene. Direct DNA sequencing of PCR-amplified CFTR gene segments is a first-line method to detect unknown mutations, but it is a tedious and labor-intensive endeavor given the large size of the gene (27 exons, 1,480 amino acids). Next-generation sequencing (NGS) is becoming standardized, reducing the cost of DNA sequencing, and enabling the generation of millions of reads per run. We here report a comprehensive analysis of CFTR variants in Japanese patients with chronic pancreatitis using NGS coupling with target capture. METHODS: Exon sequences of the CFTR gene from 193 patients with chronic pancreatitis (121 idiopathic, 46 alcoholic, 17 hereditary, and nine familial) were captured by HaloPlex target enrichment technology, followed by NGS. RESULTS: The sequencing data covered 91.6 % of the coding regions of the CFTR gene by ≥ 20 reads with a mean read depth of 449. We could identify 12 non-synonymous variants including three novel ones [c.A1231G (p.K411E), c.1753G>T (p.E585X) and c.2869delC (p.L957fs)] and seven synonymous variants including three novel ones in the exonic regions. The frequencies of the c.4056G>C (p.Q1352H) and the c.3468G>T (p.L1156F) variants were higher in patients with chronic pancreatitis than those in controls. CONCLUSIONS: Target sequence capture combined with NGS is an effective method for the analysis of pancreatitis susceptibility genes.

Genetic polymorphisms in alcohol dehydrogenase, aldehyde dehydrogenase and alcoholic chronic pancreatitis susceptibility: a meta-analysis.

PURPOSE: This study was aimed to determine the relationship of alcohol-metabolizing enzymes ADH2, ADH3, and ALDH2 polymorphisms with the susceptibility to alcoholic chronic pancreatitis (ACP). METHODS: Meta-analyses that evaluated the association of ADH2, ADH3, and ALDH2 variations with ACP were performed. RESULTS: Eight case-control studies were selected for analysis. The overall data revealed a significant association of ADH2 polymorphism (OR=1.56, 95% CI=1.42-1.72, P=0.000 for dominant model; OR=1.63, 95% CI=1.55-1.71, P=0.000 for homozygote comparison model; OR=1.11, 95% CI=1.01-1.22, P=0.030 for allelic contrast model), ADH3 polymorphism (OR=0.95, 95% CI=0.86-1.06, P=0.389 for dominant; OR=0.64, 95% CI=0.44-0.93, P=0.020 for homozygote comparison; and OR=0.87, 95% CI=0.77-0.99, P=0.039 for allelic contrast model) and ALDH2 polymorphism (OR=0.57, 95% CI=0.40-0.81, P=0.002 for dominant; OR=0.50, 95% CI=0.23-1.08, P=0.079 for homozygote comparison; and OR=0.58, 95% CI=0.41-0.84, P=0.003 for allelic contrast model) with ACP risk. The subgroup analyses suggested that the variant ADH2*2/*2+*1/*2, ADH2*2/*2 genotype and ADH2*2 allele significantly increased ACP risk among Asian individuals; the variant ADH3*2/*2 genotype and ADH3*2 allele significantly decreased ACP risk among non-Asian individuals; and the variant ALDH2*2/*2+*1/*2 genotype and ALDH2*2 allele significantly decreased ACP risk among Asians. CONCLUSIONS: ADH2, ADH3 and ALDH2 polymorphisms may be susceptibility facts of ACP, and it may be ethnic and race-dependent.

Regulation of pancreatic inflammation by connective tissue growth factor (CTGF/CCN2).

Pancreatitis is caused by long-term heavy alcohol consumption, which results in injury and death of pancreatic acinar cells (PAC). The PAC play a pivotal role in mediating early inflammatory responses but the underlying mechanisms remain poorly understood. Treatment of C57BL/6 mice with ethanol and cerulein resulted in increased staining for acinar interleukin- 1b (IL-1b), chemokine (C-C motif) ligand 3 (CCL3), or connective tissue growth factor (CTGF/CCN2) by Day 16 and this was associated with increased infiltration of F4/80-positive macrophages and increased expression of pancreatic CTGF/CCN2 mRNA. Compared with wild-type Swiss Webster mice, ethanol treatment of pan-green fluorescent protein (GFP)-CTGF/CCN2 transgenic mice caused enhanced acinar staining for GFP or CTGF/CCN2 and a significant increase in pancreatic infiltration of F4/80-positive macrophages or NIMP-R14-positive neutrophils. Treatment of primary mouse PAC or the rat AR42J PAC line with ethanol or CTGF/CCN2 resulted in enhanced expression of IL-1b or CCL3. Conditioned medium from CTGF/CCN2-treated AR42J cells induced chemotaxis in NR8383 macrophages and this response was abrogated in a dose dependent manner by addition of BX471, an inhibitor of chemokine (C-C motif) receptor 1. These results reveal that acinar CTGF/CCN2 plays a novel role in alcohol-induced inflammatory processes in the pancreas by increasing infiltration of macrophages and neutrophils and increasing acinar production of inflammatory mediators such as IL-1b or CCL3. The early production of CTGF/CCN2 by PAC to drive inflammation is distinct from its previously reported production by pancreatic stellate cells to drive fibrosis at later stages of pancreatic injury.