Dietary Green Tea Extract and Antioxidants Improve Insulin Secretory Functions of Pancreatic β-Cells in Mild and Severe Experimental Rodent Model of Chronic Pancreatitis

Chronic pancreatitis (CP) is a progressive inflammatory disorder of the pancreas. It is predominantly idiopathic (with an unknown cause) in India and mostly due to alcohol in the West. Diabetes that occur secondary to chronic pancreatitis (T3c Diabetes) is often brittle, and is difficult to attain normoglycemia with conventional treatment requiring multiple doses of insulin. Mild and severe model of CP was induced in mice by repeated intraperitoneal injections of cerulein and L-arginine respectively with an intent to study islet dysfunction and develop therapeutic strategy in animal models of CP. Dietary intervention of epigallocatechin-3-gallate (EGCG) was tested in both the models of CP for its beneficial effects on insulin secretory functions. Pancreata collected upon euthanasia were used to study alterations in the morphology of pancreatic parenchyma and inflammation by staining with H&E and fibrotic changes by Masson’s trichrome and picrosirius staining. Insulin secretory functions of islets were evaluated to test the efficacy of the dietary intervention on β-cell functions. Intraperitoneal glucose tolerance test was performed to monitor the glucose homeostasis before and after the dietary intervention. Both the models resulted in CP with dispersed acini, inflammation and fibrosis. The loss of acini and extent of fibrosis was more in L-arginine model. 2-fold improvement in glucose-stimulated insulin secretory functions of islets was observed with 0.5% EGCG dietary intervention in cerulein model of CP and 1.6-fold in L-arginine model of CP. A further improvement in insulin secretion by 3.2-fold was observed with additional dietary supplements like N-acetyl cysteine, curcumin in combination with EGCG. Our results thus demonstrate and highlight the therapeutic potential of dietary green tea (EGCG) supplementation in reversing islet dysfunction and improving glucose homeostasis in experimental chronic pancreatitis in mice.

role of the normal gut microbiota

Relation between the gut microbiota and human health is being increasingly recognised. It is now well established that a healthy gut flora is largely responsible for overall health of the host. The normal human gut microbiota comprises of two major phyla, namely Bacteroidetes and Firmicutes. Though the gut microbiota in an infant appears haphazard, it starts resembling the adult flora by the age of 3 years. Nevertheless, there exist temporal and spatial variations in the microbial distribution from esophagus to the rectum all along the individual's life span. Developments in genome sequencing technologies and bioinformatics have now enabled scientists to study these microorganisms and their function and microbehost interactions in an elaborate manner both in health and disease. The normal gut microbiota imparts specific function in host nutrient metabolism, xenobiotic and drug metabolism, maintenance of structural integrity of the gut mucosal barrier, immunomodulation, and protection against pathogens. Several factors play a role in shaping the normal gut microbiota. They include(1) the mode of delivery(vaginal or caesarean);(2) diet during infancy(breast milk or formula feeds) and adulthood(vegan based or meat based); and(3) use of antibiotics or antibiotic like molecules that are derived from the environment or the gut commensal community. A major concern of antibiotic use is the long-term alteration of the normal healthy gut microbiota and horizontal transfer of resistance genes that could result in reservoir of organisms with a multidrug resistant gene pool.

Pancreatic stellate cell: Pandora's box for pancreatic disease biology

Pancreatic stellate cells(PSCs) were identified in the early 1980 s, but received much attention after 1998 when the methods to isolate and culture them from murine and human sources were developed. PSCs contribute to a small proportion of all pancreatic cells under physiological condition, but are essential for maintaining the normal pancreatic architecture. Quiescent PSCs are characterized by the presence of vitamin A laden lipid droplets. Upon PSC activation, these perinuclear lipid droplets disappear from the cytosol, attain a myofibroblast like phenotype and expresses the activation marker, alpha smooth muscle actin. PSCs maintain their activated phenotype via an autocrine loop involving different cytokines and contribute to progressive fibrosis in chronic pancreatitis(CP) and pancreatic ductal adenocarcinoma(PDAC). Several pathways(e.g., JAK-STAT, Smad, Wnt signaling, Hedgehog etc.), transcription factors and mi RNAs have been implicated in the inflammatory and profibrogenic function of PSCs. The role of PSCs goes much beyond fibrosis/desmoplasia in PDAC. It is now shown that PSCs are involved in significant crosstalk between the pancreatic cancer cells and the cancer stroma. These interactions result in tumour progression, metastasis, tumour hypoxia, immune evasion and drug resistance. This is the rationale for therapeutic preclinical and clinical trials that have targeted PSCs and the cancer stroma.

Autologous mobilized peripheral blood CD34^+ cell infusion in non-viral decompensated liver cirrhosis

AIM: To study the effect of mobilized peripheral blood autologous CD34 positive(CD34+) cell infusion in patients with non-viral decompensated cirrhosis.METHODS: Cirrhotic patients of non-viral etiology were divided into 2 groups based on their willingness to be listed for deceased donor liver transplant(DDLT)(control, n = 23) or to receive autologous CD34+ cell infusion through the hepatic artery(study group, n= 22). Patients in the study group were admitted to hospital and received granulocyte colony stimulating factor injections 520 μg/d for 3 consecutive days to mobilize CD34+ cells from the bone marrow. On day 4,leukapheresis was done and CD34+ cells were isolated using CliniMAC magnetic cell sorter. The isolated CD34+ cells were infused into the hepatic artery under radiological guidance. The patients were discharged within 48 h. The control group received standard of care treatment for liver cirrhosis and were worked up for DDLT as per protocol of the institute. Both groups were followed up every week for 4 wk and then every month for 3 mo.RESULTS: In the control and the study group, the cause of cirrhosis was cryptogenic in 18(78.2%) and16(72.72%) and alcohol related in 5(21.7%) and6(27.27%), respectively. The mean day 3 cell count(cells/μL) was 27.00 ± 20.43 with a viability of 81.84± 11.99%. and purity of 80%-90%. Primary end point analysis revealed that at 4 wk, the mean serum albumin in the study group increased significantly(2.83± 0.36 vs 2.43 ± 0.42, P = 0.001) when compared with controls. This improvement in albumin was,however, not sustained at 3 mo. However, at the end of3 mo there was a statistically significant improvement in serum creatinine in the study group(0.96 ± 0.33 vs 1.42 ± 0.70, P = 0.01) which translated into a significant improvement in the Model for End-Stage Liver Disease score(15.75 ± 5.13 vs 19.94 ± 6.68,P = 0.04). On statistical analysis of secondary end points, the transplant free survival at the end of 1 mo and 3 mo did not show any significant difference(P =0.60) when compared to the control group. There was no improvement in aspartate transaminase, alanine transaminase, and bilirubin at any point in the study population. There was no mortality benefit in the study group. The procedure was safe with no procedural or treatment related complications.CONCLUSION: Autologous CD 34+ cell infusion is safe and effectively improves liver function in the short term and may serve as a bridge to liver transplantation.

Thinking outside the liver: Induced pluripotent stem cells for hepatic applications

The discovery of induced pluripotent stem cells (iPSCs) unraveled a mystery in stem cell research, after identification of four re-programming factors for generating pluripotent stem cells without the need of embryos. This breakthrough in generating iPSCs from somatic cells has overcome the ethical issues and immune rejection involved in the use of human embryonic stem cells. Hence, iPSCs form a great potential source for developing disease models, drug toxicity screening and cell-based therapies. These cells have the potential to differentiate into desired cell types, including hepatocytes, under in vitro as well as under in vivo conditions given the proper microenvironment. iPSC-derived hepatocytes could be useful as an unlimited source, which can be utilized in disease modeling, drug toxicity testing and producing autologous cell therapies that would avoid immune rejection and enable correction of gene defects prior to cell transplantation. In this review, we discuss the induction methods, role of reprogramming factors, and characterization of iPSCs, along with hepatocyte differentiation from iPSCs and potential applications. Further, we discuss the location and detection of liver stem cells and their role in liver regeneration. Although tumor formation and genetic mutations are a cause of concern, iPSCs still form a promising source for clinical applications.

Genetics of acute and chronic pancreatitis: An update

Progress made in identifying the genetic susceptibility underlying acute and chronic pancreatitis has benefitted the clinicians in understanding the pathogenesis of the disease in a better way. The identification of mutations in cationic trypsinogen gene(PRSS1 gene; functional gain mutations) and serine protease inhibitor kazal type 1(SPINK1 gene; functional loss mutations) and other potential susceptibility factors in genes that play an important role in the pancreatic secretory functions or response to inflammation during pancreatic injury has changed the current concepts and understanding of a complex multifactorial disease like pancreatitis. An indi-vidual's susceptibility to the disease is governed by ge-netic factors in combination with environmental factors. Candidate gene and genetic linkage studies have iden-tified polymorphisms in cationic trypsinogen(PRSS1), SPINK1, cystic fibrosis trans-membrane conductance regulator(CFTR), Chymotrypsinogen C(CTRC), Ca-thepsin B(CTSB) and calcium sensing receptor(CASR). Individuals with polymorphisms in the mentioned genes and other as yet identified genes are at an enhanced risk for the disease. Recently, polymorphisms in genes other than those involved in "intra-pancreatic trypsin regulatory mechanism" namely Claudin-2(CLDN2) andCarboxypeptidase A1(CPA1) gene have also been iden-tified for their association with pancreatitis. With ever growing number of studies trying to identify the genetic susceptibility in the form of single nucleotide polymor-phisms, this review is an attempt to compile the avail-able information on the topic.

Pooled genetic analysis in ultrasound measured non-alcoholic fatty liver disease in Indian subjects:A pilot study

AIM: To investigate genetic susceptibility in Indian subjects with non-alcoholic fatty liver disease(NAFLD) by performing a pooled genetic study.METHODS: Study subjects(n = 306) were recruited and categorized into NAFLD and control groups based on ultrasound findings of fatty infiltration. Of the 306 individuals, 156 individuals had fatty infiltration and thus comprised the NAFLD group. One hundred and fifty(n = 150) individuals were normal, without fatty infiltration of the liver, comprising the control group. Blood samples, demographic and anthropometric data from the individuals were collected after obtaining informed consent. Anthropometric data, blood glucose, lipids and liver function tests were estimated using standard methods. Genome wide association stud-ies done to date on NAFLD were identified, 19 single nucleotide polymorphisms(SNPs) were selected from these studies that were reported to be significantly associated with NAFLD and genotyping was performed on the Sequenom platform. Student's t test for continuous variables and χ2 test was applied to variant carriers from both groups. Required corrections were applied as multiple testing was done.RESULTS The mean age of the control group was 39.78 ± 10.83 and the NAFLD group was 36.63 ± 8.20 years. The waist circumference of males and females in the control and NAFLD groups were 80.13 ± 10.35; 81.77 ± 13.65 and 94.09 ± 10.53; 92.53 ± 8.27 respectively. The mean triglyceride and alanine transaminase(ALT) levels in the control and NAFLD groups were 135.18 ± 7.77; 25.39 ± 14.73 and 184.40 ± 84.31; 110.20 ± 67.05 respectively. When χ2 test was applied to the number of individuals carrying the variant risk alleles between the control and NAFLD group, a significant association was seen between rs738409 of the patatin-like phospholipase domain containing 3(PNPLA3) gene(P = 0.001), rs2073080 of the PARVB gene(P = 0.02), rs2143571 of SAMM50 gene(P = 0.05) and rs6487679 of the pregnancy zone protein(PZP) gene(P = 0.01) with the disease. Variant single nucleotide polymorphisms(SNPs) in NCAN and PNPLA3 gene were associated with higher levels of ALT, whereas variant SNPs in APOC3, PNPLA3, EFCAB4 B and COL13A1 were associated with high triglyceride levels. Apart from the above associations, rs2073080, rs343062 and rs6591182 were significantly associated with high BMI; rs2854117 and rs738409 with high triglyceride levels; and rs2073080, rs2143571, rs2228603, rs6487679 and rs738409 with high ALT levels.CONCLUSION: Pooled genetic analysis revealed an association of SNPs in PNPLA3, PARVB, SAMM50 and PZP genes with NAFLD. SNPs in NCAN and PNPLA3gene were associated with higher levels of ALT,whereas variant SNPs in APOC3, PNPLA3, EFCAB4 B and COL13A1 were associated with high triglyceride levels.

Genetics of non-alcoholic fatty liver disease: From susceptibility and nutrient interactions to management

Genetics plays an important role in determining the susceptibility of an individual to develop a disease. Complex, multi factorial diseases of modern day(diabetes, cardiovascular disease, hypertension and obesity) are a result of disparity between the type of food consumed and genes, suggesting that food which does not match the host genes is probably one of the major reasons for developing life style diseases. Non-alcoholic fatty liver is becoming a global epidemic leading to substantial morbidity. While various genotyping approaches such as whole exome sequencing using next generation sequencers and genome wide association studies have identified susceptibility loci for non-alcoholic fatty liver disease(NAFLD) including variants in patatin-like phospholipase domain containing 3 and transmembrane 6 superfamily member 2 genes apart from others; nutrient based studies emphasized on a combination of vitamin D, E and omega-3 fatty acids to manage fatty liver disease. However majority of the studies were conducted independent of each other and very few studies explored the interactions between the genetic susceptibility and nutrient interactions. Identifying such interactions will aid in optimizing the nutrition tailor made to an individual's genetic makeup, thereby aiding in delaying the onset of the disease and its progression. The present topic focuses on studies that identified the genetic susceptibility for NAFLD, nutritional recommendations, and their interactions for better management of NAFLD.

Pain in chronic pancreatitis: Managing beyond the pancreatic duct

Chronic pancreatitis(CP)continues to be a clinical challenge.Persistent or recurrent abdominal pain is the most compelling symptom that drives patients to seek medical care.Unfortunately,in spite of using several treatment approaches in the clinical setting,there is no single specific treatment modality that can be earmarked as a cure for this disease.Traditionally,ductal hypertension has been associated with causation of pain in CP;and patients are often subjected to endotherapy and surgery with a goal to decompress the pancreatic duct.Recent studies on humans(clinical and laboratory based)and experimental models have put forward several mechanisms,including neuroimmune alterations,which could be responsible for pain.This might explain the partial or no response to single modality treatment in a significant proportion of patients.The current review discusses the recent concepts of pain generation in CP and evidence based therapeutic approaches(other than ductal decompression)to handle persistent or recurrent pain.We focus primarily on parenchymal and neural components;and discuss the role of antioxidants and the existing controversies,drugs that interfere with neural transmission,pancreatic enzyme supplementation,celiac neurolysis,and pancreatic resection procedures.The review concludes with the treatment approach that we follow at our institute.

Regional differences in genetic susceptibility to nonalcoholic liver disease in two distinct Indian ethnicities

AIM To validate the association of variants in PNPLA3(rs2281135) and TM6SF2(rs58542926) genes with ultrasound detected non-alcoholic fatty liver disease(NAFLD).METHODS A total of 503 individuals with and without fatty infiltration were recruited. Fatty infiltration was confirmed based on ultrasound findings. Anthropometric data and blood samples were collected from the study group. DNA was isolated from peripheral blood, quality and quantity was assessed by gel electrophoresis and spectrophotometer respectively. Genotyping of the variants in PNPLA3 and TM6SF2 genes was carried out by employing taqman probes(C_15875080_10 for PNPLA3 and C_8946351_10 for TM6SF2 SNP) on real time PCR(Stepone-Lifetechnologies). Genotype data was tested for deviations from Hardy-Weinbergequilibrium. χ~2 test was used to analyze the statistical significance of the difference in genotype distribution of the studied variants in patients and controls and the strength of association was expressed as odds ratio(95%CI). A two-tailed P value of ≤ 0.05 was considered statistically significant. RESULTS The study group comprised of 503 individuals of which 256 had fatty infiltration and 247 without fatty infiltration and thus formed the patient and control groups respectively. As the patient group could be divided in to two distinct ethnicities(ancestral South Indians-ASI and North-East Indians-NEI), further recruitment of control cohort and association analyses was carried out based on ethnicities. Of the 256 with fatty infiltration 93 were ASI and 163 were NEI and of the 247 controls 138 were ASI and 109 were NEI. As expected, there were significant differences in the anthropometric and other clinical data between the control and the patient groups. However significant differences within the ethnicities were also noted. While rs2281135 in PNPLA3 gene was significantly associated(P = 0.03) with higher risk(odds 1.9, 95%CI: 1.5-3.14, P = 0.03) of NAFLD in NEI ethnicity, rs58542926 in TM6SF2 gene was significantly associated with NAFLD with a 2.7 fold higher risk(odds 2.7, 95%CI: 1.37-5.3, P = 0.0004) of the disease. There were significantly higher proportions of individuals with variants in both the genes in the patient group in both ASI(patients-14/93 and controls-7/138; P = 0.009) and NEI ethnicities(patients-17/163 and controls-7/109; P = 0.01). CONCLUSION Although the study identified distinct genetic susceptibility in the two ethnicities, transheterozygosity of the variants suggests higher risk of NAFLD in individuals with both the variants.

Transcriptome Profiles and Gene Expression of Min6 Cells Are Altered by Pancreatic Stellate Cells

Aim: To identify the influence of pancreatic stellate cell (PSCs) secretions on gene expression profiles of Min6 cells by whole transcriptome sequencing. Methods: Pancreatic stellate cells (PSCs) were isolated from C57BL6J mice and propagated in vitro to acquire the activated phenotype. Total RNA was isolated from monocultured (MC) and PSC cocultured (CC) Min6 cells to prepare cDNA libraries, which were subjected to whole transcriptome sequencing for identifying differential expression of β-cell transcription factors (Pdx-1, Rfx6 and NeuroD1) related to insulin gene transcription and GSIS related genes such as Glut2, Gck, Abcc8, Kcnj11 and L-type Ca2+ channels (Cacnb2, Cacna1c). qRT-PCR was used to validate the gene expression. GSIS of Min6 cells was examined by estimating insulin levels in response to high glucose challenge. Results: Transcriptome analysis of discovery set revealed that coculture of Min6 cells with PSCs caused increased expression of β-cell specific genes (Ins1, Rfx6 and NeuroD1) concomitant with decreased expression of Pdx-1, MafA and Nkx2-2. Expression of GSIS associated genes (Glut2, Gck, Abcc8, Kcnj11 and Cacnb2) was decreased in such conditions. Validation by qRT-PCR in Min6 cells cocultured with PSCs revealed increased significant expression of Ins1 (2.1 ± 0.22 folds;p ≤ 0.001), Rfx6 (1.68 ± 0.23 folds;p ≤ 0.002) and NeuroD1 (0.96 ± 0.11 folds;p ≤ 0.01), accompanied by downregulation of Cacnb2 (-0.93 ± 0.57 folds;p ≤ 0.05). PSC secretions did not restore the GSIS from glucose unresponsive higher passage Min6 cells (MC: 1.33 ± 0.42;CC: 1.55 ± 0.72 pmol/mg protein;p = ns) upon high glucose stimulation. However, glucose responsive higher passage Min6 cells cocultured with PSCs presented increased insulin secretion (MC: 7.025 ± 0.64;CC: 14.84 ± 1.01 pmol/mg protein;p ≤ 0.04) concomitant with marginal increase of insulin contents. Conclusion: PSC secretions increase Ins1, Rfx6 and NeuroD1 gene expression, GSIS from glucose responsive Min6 cells, but do not restore the GSIS from glucose unresponsive Min6 cells.

Characterization of Islets from Chronic Calcific Pancreatitis Patients of Tropical Region with Distinct Phenotype

Background and Objective: Islet autotransplantation is performed to preserve endocrine function in patients undergoing pancreatic resections for painful chronic pancreatitis. We characterized islets isolated from chronic pancreatitis patients (CP) of tropical region. Patients and Methods: Pancreatic tissues were obtained from CP patients with and without diabetes undergoing pancreatic resections (n = 35) and brain-dead multi organ donors (n = 6;considered as controls). Islets isolated were assessed for yield, purity, viability and in vitro islet function (Glucose stimulated insulin release, GSIR) as per standard protocols. Results: Islets from CP patients without diabetes were similar to controls in yield (control 4120 - 6100 IE/g, CP 3550 - 5660 IE/g), purity (control 78% ± 12%, CP 70% ± 8.2%) and viability (control 85% ± 8%, CP 81% ± 10%) and islets from CP patients with diabetes showed decreases in yield (3002 - 2300 IE/g), purity (61% ± 16%) and viability (62% ± 21%). Islets measuring 50 - 200 μ were similar in abundance in controls (94.74% ± 3.2%) and CP patients with and without diabetes, 86.31% ± 4.9%, 91.03% ± 3.8%. GSIR of islets from CP patients and controls were similar at 5.5 mM glucose (2.8 - 3.1 μU/ml). However, GSIR at 16.5 mM glucose was decreased in CP patients (control 18.5 ± 0.6, CP without diabetes 11.8 ± 0.3, CP with diabetes 4.3 ± 0.3 μU/ml). Conclusion: Our results demonstrate suitability of islets isolated from CP patients of tropical region for autotransplantation.

Dysregulated miRNA Associated with Transcription Factors of Insulin Gene Expression in Chronic Pancreatitis

Background/Aim: MicroRNAs with regulatory functions in gene expression are implicated in different diseases. The present study investigated differentially expressed miRNAs that possibly influence transcription factors involved in insulin gene expression in Chronic Pancreatitis (CP) employing bioinformatics approaches. Methods: Pancreatic tissues were collected from CP patients undergoing partial pancreatectomy (n = 16) and controls (n = 15) undergoing resections for non-pancreatic malignancies. MiRNA profiles obtained using microarrays were validated by qRT-PCR. Target search involving miRWalk and TarBase as well as functional annotation employing KEGG (Kyoto encyclopedia of genes and genomes) and DAVID (Database for Annotation) databases were performed. Ingenuity pathway analysis (IPA) was used to construct networks relating miRNAs to their target genes. mRNA and proteins related to insulin gene transcription factors and hormones were evaluated by qRT-PCR and western blotting followed by confirmation upon immunofluorescent staining. Results: Microarray data revealed 10 up-regulated and 15 down-regulated miRNAs in CP as compared to controls (Log2 FC > 2). Bioinformatic analysis showed 8399 target genes and KEGG pathway analysis suggested a role for the dysregulated miRNAs in modulating cytokine signaling, fibrosis, JAK-STAT signaling and insulin synthesis. IPA analysis suggested a simplified network attributing dysregulated miRNAs to NFκB-dependent cytokine signaling. Further, associations could be noted between miRNA 200b with Maf A, 138-1 with Neuro D and 27b with FoxO1. Decreases in mRNA levels of Pdx1, Neuro D and increases of Maf A and FoxO1 transcription factors could be noted (P Conclusion: Our results identified dysregulation of miRNAs 138-1, 27b and 200b which were found to be associated with insulin gene transcription factors Neuro D, FoxO1 and Maf A respectively.