miR-122-5p is involved in posttranscriptional regulation of the mitochondrial thiamin pyrophosphate transporter (SLC25A19) in pancreatic acinar cells.

Thiamin (vitamin B1) plays a vital role in cellular energy metabolism/ATP production. Pancreatic acinar cells (PACs) obtain thiamin from circulation and convert it to thiamin pyrophosphate (TPP) in the cytoplasm. TPP is then taken up by the mitochondria via a carrier-mediated process that involves the mitochondrial TPP transporter (MTPPT; encoded by the gene SLC25A19). We have previously characterized different aspects of the mitochondrial carrier-mediated TPP uptake process, but nothing is known about its possible regulation at the posttranscriptional level. We address this issue in the current investigations focusing on the role of miRNAs in this regulation. First, we subjected the human (and rat) 3'-untranslated region (3'-UTR) of the SLC25A19 to three in-silico programs, and all have identified putative binding sites for miR-122-5p. Transfecting pmirGLO-hSLC25A19 3'-UTR into rat PAC AR42J resulted in a significant reduction in luciferase activity compared with cells transfected with pmirGLO-empty vector. Mutating as well as truncating the putative miR-122-5p binding sites in the hSLC25A19 3'-UTR led to abrogation of inhibition in luciferase activity in PAC AR42J. Furthermore, transfecting/transducing PAC AR42J and human primary PACs with mimic of miR-122-5p led to a significant inhibition in the level of expression of the MTPPT mRNA and protein as well as in mitochondrial carrier-mediated TPP uptake. Conversely, transfecting PAC AR42J with an inhibitor of miR-122-5p increased MTPPT expression and function. These findings show, for the first time, that expression and function of the MTPPT in PACs are subject to posttranscriptional regulation by miR-122-5p.NEW & NOTEWORTHY This study shows that the expression and function of mitochondrial TPP transporter (MTPPT) are subject to posttranscriptional regulation by miRNA-122-5p in pancreatic acinar cells.

Pancreatic Stellate Cells Prolong Ex Vivo Islet Viability and Function and Improve Engraftment.

Preserving islet health and function is critical during pretransplant culture to improve islet transplantation outcome and for ex vivo modeling of diabetes for pharmaceutical drug discovery. The limited islet engraftment potential is primarily attributable to loss of extracellular matrix (ECM) support and interaction. Multipotent cells with ECM depositing competency improve islet survival during short coculture period. However, role of pancreatic stellate cells (PSCs) and their ECM support in preserving ex vivo islet physiology remains largely unknown. Here, we report novel cytoprotective effects of culture-adapted porcine PSCs and role of their ECM-mediated intercellular communication on pig, mouse and human islets ex vivo. Using direct-contact coculture system, we demonstrate that porcine PSCs preserve and significantly prolong islet viability and function from 7 ± 3 days to more than 28 ± 5 (P < .001) days in vitro. These beneficial effects of PSCs on islet health are not species-specific. Using NSC47924 to specifically inhibit 37/67 kDa laminin receptor (LR), we identified that LR-mediated intercellular communication is essential for PSCs to protect functional viability of islets in vitro. Finally, our results demonstrate that PSC co-transplantation improved function and enhanced capacity of syngeneic islets to reverse hyperglycemia in mice with preexisiting diabetes. Cumulatively, our findings unveil novel effects of culture-adapted PSCs on islet health likely mirroring in vivo niche interaction. Furthermore, islet and PSC coculture may aid in development of ex vivo diabetes modeling and also suggests that a combined islet-PSC tissue engineered implant may significantly improve islet transplantation outcome.

Predictors of Glycemic Outcomes at 1 Year Following Pediatric Total Pancreatectomy With Islet Autotransplantation.

OBJECTIVE: Total pancreatectomy with islet autotransplantation (TPIAT) is indicated to alleviate debilitating pancreas-related pain and mitigate diabetes in patients with acute recurrent and chronic pancreatitis when medical/endoscopic therapies fail. Our aim was to evaluate predictors of insulin requirement at 1 year following TPIAT in a cohort of children. RESEARCH DESIGN AND METHODS: This was a review of 43 pediatric patients followed after TPIAT for 1 year or longer. Primary outcome was insulin use at 1 year, categorized as follows: insulin independent, low insulin requirement (<0.5 units/kg/day), or high insulin requirement (≥0.5 units/kg/day). RESULTS: At 1 year after TPIAT, 12 of 41 (29%) patients were insulin independent and 21 of 41 (51%) had low and 8 of 41 (20%) had high insulin requirement. Insulin-independent patients were younger than those with low and high insulin requirement (median age 8.2 vs. 14.6 vs. 13.1 years, respectively; P = 0.03). Patients with insulin independence had a higher number of transplanted islet equivalents (IEQ) per kilogram body weight (P = 0.03) and smaller body surface area (P = 0.02), compared with those with insulin dependence. Preoperative exocrine insufficiency was associated with high insulin requirement (P = 0.03). Higher peak C-peptide measured by stimulated mixed-meal tolerance testing (MMTT) at 3 and 6 months post-TPIAT was predictive of lower insulin requirement at 1 year (P = 0.006 and 0.03, respectively). CONCLUSIONS: We conclude that insulin independence following pediatric TPIAT is multifactorial and associated with younger age, higher IEQ per kilogram body weight transplanted, and smaller body surface area at time of operation. Higher peak C-peptide measured by MMTT following TPIAT confers a higher likelihood of low insulin requirement.

The future treatment for type 1 diabetes: Pig islet- or stem cell-derived ß cells?

Replacement of ß cells is only a curative approach for type 1 diabetes (T1D) patients to avoid the threat of iatrogenic hypoglycemia. In this pursuit, islet allotransplantation under Edmonton's protocol emerged as a medical miracle to attain hypoglycemia-free insulin independence in T1D. Shortage of allo-islet donors and post-transplantation (post-tx) islet loss are still unmet hurdles for the widespread application of this therapeutic regimen. The long-term survival and effective insulin independence in preclinical studies have strongly suggested pig islets to cure overt hyperglycemia. Importantly, CRISPR-Cas9 technology is pursuing to develop "humanized" pig islets that could overcome the lifelong immunosuppression drug regimen. Lately, induced pluripotent stem cell (iPSC)-derived ß cell approaches are also gaining momentum and may hold promise to yield a significant supply of insulin-producing cells. Theoretically, personalized ß cells derived from a patient's iPSCs is one exciting approach, but ß cell-specific immunity in T1D recipients would still be a challenge. In this context, encapsulation studies on both pig islet as well as iPSC-ß cells were found promising and rendered long-term survival in mice. Oxygen tension and blood vessel growth within the capsules are a few of the hurdles that need to be addressed. In conclusion, challenges associated with both procedures, xenotransplantation (of pig-derived islets) and stem cell transplantation, are required to be cautiously resolved before their clinical application.

Correction to: trisected pancreas model for testing tissue dissociation enzyme combinations: a novel methodology for improving human islet yield for clinical islet transplantation.

[This corrects the article DOI: 10.1007/s40200-020-00519-y.].

Circulating miRNA in Patients Undergoing Total Pancreatectomy and Islet Autotransplantation.

Circulating microRNAs (miRNAs) can be biomarkers for diagnosis and progression of several pathophysiological conditions. In a cohort undergoing total pancreatectomy with islet autotransplantation (TPIAT) from the multicenter Prospective Observational Study of TPIAT (POST), we investigated associations between a panel of circulating miRNAs (hsa-miR-375, hsa-miR-29b-3p, hsa-miR-148a-3p, hsa-miR-216a-5p, hsa-miR-320d, hsa-miR-200c, hsa-miR-125b, hsa-miR-7-5p, hsa-miR-221-3p, hsa-miR-122-5p) and patient, disease and islet-isolation characteristics. Plasma samples (n = 139) were collected before TPIAT and miRNA levels were measured by RTPCR. Disease duration, prior surgery, and pre-surgical diabetes were not associated with circulating miRNAs. Levels of hsa-miR-29b-3p (P = 0.03), hsa-miR-148a-3p (P = 0.04) and hsa-miR-221-3p (P = 0.01) were lower in those with genetic risk factors. Levels of hsa-miR-148a-3p (P = 0.04) and hsa-miR-7-5p (P = 0.04) were elevated in toxic/metabolic disease. Participants with exocrine insufficiency had lower hsa-miR-29b-3p, hsa-miR-148a-3p, hsa-miR-320d, hsa-miR-221-3p (P < 0.01) and hsa-miR-375, hsa-miR-200c-3p, and hsa-miR-125b-5p (P < 0.05). Four miRNAs were associated with fasting C-peptide before TPIAT (hsa-miR-29b-3p, r = 0.18; hsa-miR-148a-3p, r = 0.21; hsa-miR-320d, r = 0.19; and hsa-miR-221-3p, r = 0.21; all P < 0.05), while hsa-miR-29b-3p was inversely associated with post-isolation islet equivalents/kg and islet number/kg (r = -0.20, P = 0.02). Also, hsa-miR-200c (r = 0.18, P = 0.03) and hsa-miR-221-3p (r = 0.19, P = 0.03) were associated with islet graft tissue volume. Further investigation is needed to determine the predictive potential of these miRNAs for assessing islet autotransplant outcomes.

Differential cytotoxicity, ER/oxidative stress, dysregulated AMPKα signaling, and mitochondrial stress by ethanol and its metabolites in human pancreatic acinar cells.

BACKGROUND: Alcoholic chronic pancreatitis (ACP) is a serious inflammatory disorder of the exocrine pancreatic gland. A previous study from this laboratory showed that ethanol (EtOH) causes cytotoxicity, dysregulates AMPKα and ER/oxidative stress signaling, and induces inflammatory responses in primary human pancreatic acinar cells (hPACs). Here we examined the differential cytotoxicity of EtOH and its oxidative (acetaldehyde) and nonoxidative (fatty acid ethyl esters; FAEEs) metabolites in hPACs was examined to understand the metabolic basis and mechanism of ACP. METHODS: We evaluated concentration-dependent cytotoxicity, AMPKα inactivation, ER/oxidative stress, and inflammatory responses in hPACs by incubating them for 6 h with EtOH, acetaldehyde, or FAEEs at clinically relevant concentrations reported in alcoholic subjects using conventional methods. Cellular bioenergetics (mitochondrial stress and a real-time ATP production rate) were determined using Seahorse XFp Extracellular Flux Analyzer in AR42J cells treated with acetaldehyde or FAEEs. RESULTS: We observed concentration-dependent increases in LDH release, inactivation of AMPKα along with upregulation of ACC1 and FAS (key lipogenic proteins), downregulation of p-LKB1 (an oxidative stress-sensitive upstream kinase regulating AMPKα) and CPT1A (involved in ß-oxidation of fatty acids) in hPACs treated with EtOH, acetaldehyde, or FAEEs. Concentration-dependent increases in oxidative stress and ER stress as measured by GRP78, unspliced XBP1, p-eIF2α, and CHOP along with activation of p-JNK1/2, p-ERK1/2, and p-P38MAPK were present in cells treated with EtOH, acetaldehyde, or FAEEs, respectively. Furthermore, a significant decrease was observed in the total ATP production rate with subsequent mitochondrial stress in AR42J cells treated with acetaldehyde and FAEEs. CONCLUSIONS: EtOH and its metabolites, acetaldehyde and FAEEs, caused cytotoxicity, ER/oxidative and mitochondrial stress, and dysregulated AMPKα signaling, suggesting a key role of EtOH metabolism in the etiopathogenesis of ACP. Because oxidative EtOH metabolism is negligible in the exocrine pancreas, the pathogenesis of ACP could be attributable to the formation of FAEEs and related pancreatic acinar cell injury.

Preoperative ERCP has no impact on islet yield following total pancreatectomy and islet autotransplantation (TPIAT): Results from the Prospective Observational Study of TPIAT (POST) cohort.

BACKGROUND AND AIMS: Many patients undergoing total pancreatectomy with islet autotransplant (TPIAT) for severe, refractory chronic pancreatitis or recurrent acute pancreatitis have a history of endoscopic retrograde cholangiopancreatography (ERCP). Using data from the multicenter POST (Prospective Observational Study of TPIAT) cohort, we aimed to determine clinical characteristics associated with ERCP and the effect of ERCP on islet yield. METHODS: Using data from 230 participants (11 centers), demographics, pancreatitis history, and imaging features were tested for association with ERCP procedures. Logistic and linear regression were used to assess association of islet yield measures with having any pre-operative ERCPs and with the number of ERCPs, adjusting for confounders. RESULTS: 175 (76%) underwent ERCPs [median number of ERCPs (IQR) 2 (1-4). ERCP was more common in those with obstructed pancreatic duct (p = 0.0009), pancreas divisum (p = 0.0009), prior pancreatic surgery (p = 0.005), and longer disease duration (p = 0.004). A greater number of ERCPs was associated with disease duration (p < 0.0001), obstructed pancreatic duct (p = 0.006), and prior pancreatic surgery (p = 0.006) and increased risk for positive islet culture (p < 0.0001). Mean total IEQ/kg with vs. without prior ERCP were 4145 (95% CI 3621-4669) vs. 3476 (95% CI 2521-4431) respectively (p = 0.23). Adjusting for confounders, islet yield was not significantly associated with prior ERCP, number of ERCPs, biliary or pancreatic sphincterotomy or stent placement. CONCLUSIONS: ERCP did not appear to adversely impact islet yield. When indicated, ERCP need not be withheld to optimize islet yield but the risk-benefit ratio of ERCP should be considered given its potential harms, including risk for excessive delay in TPIAT.

The demise of islet allotransplantation in the United States: A call for an urgent regulatory update.

Islet allotransplantation in the United States (US) is facing an imminent demise. Despite nearly three decades of progress in the field, an archaic regulatory framework has stymied US clinical practice. Current regulations do not reflect the state-of-the-art in clinical or technical practices. In the US, islets are considered biologic drugs and "more than minimally manipulated" human cell and tissue products (HCT/Ps). In contrast, across the world, human islets are appropriately defined as "minimally manipulated tissue" and not regulated as a drug, which has led to islet allotransplantation (allo-ITx) becoming a standard-of-care procedure for selected patients with type 1 diabetes mellitus. This regulatory distinction impedes patient access to islets for transplantation in the US. As a result only 11 patients underwent allo-ITx in the US between 2016 and 2019, and all as investigational procedures in the settings of a clinical trials. Herein, we describe the current regulations pertaining to islet transplantation in the United States. We explore the progress which has been made in the field and demonstrate why the regulatory framework must be updated to both better reflect our current clinical practice and to deal with upcoming challenges. We propose specific updates to current regulations which are required for the renaissance of ethical, safe, effective, and affordable allo-ITx in the United States.

Progress in individualizing autologous islet isolation techniques for pediatric islet autotransplantation after total pancreatectomy in children for chronic pancreatitis.

Total pancreatectomy with islet autotransplantation is performed to treat chronic pancreatitis in children. Successful islet isolation must address the challenges of severe pancreatic fibrosis and young donor age. We have progressively introduced modifications to optimize enzymatic and mechanical dissociation of the pancreas during islet isolation. We evaluated 2 islet isolation metrics in 138 children-digest islet equivalents per gram pancreas tissue (IEQ/g) and digest IEQ per kilogram body weight (IEQ/kg), using multiple regression to adjust for key disease and patient features. Islet yield at digest had an average 4569 (standard deviation 2949) islet equivalent (IEQ)/g and 4946 (4009) IEQ/kg, with 59.1% embedded in exocrine tissue. Cases with very low yield (<2000 IEQ/g or IEQ/kg) have decreased substantially over time, 6.8% and 9.1%, respectively, in the most recent tertile of time compared to 19.2% and 23.4% in the middle and 34.1% and 36.4% in the oldest tertile. IEQ/g and IEQ/kg adjusted for patient and disease factors improved in consistency and yield in the modern era. Minimal mechanical disruption during digestion, warm enzymatic digestion using enzyme collagenase:NP activity ratio < 10:1, coupled with extended distension and trimming time during islet isolation of younger and fibrotic pediatric pancreases, gave increased islet yield with improved patient outcomes.

Circulating unmethylated CHTOP and INS DNA fragments provide evidence of possible islet cell death in youth with obesity and diabetes.

BACKGROUND: Identification of islet ß cell death prior to the onset of type 1 diabetes (T1D) or type 2 diabetes (T2D) might allow for interventions to protect ß cells and reduce diabetes risk. Circulating unmethylated DNA fragments arising from the human INS gene have been proposed as biomarkers of ß cell death, but this gene alone may not be sufficiently specific to report ß cell death. RESULTS: To identify new candidate genes whose CpG sites may show greater specificity for ß cells, we performed unbiased DNA methylation analysis using the Infinium HumanMethylation 450 array on 64 human islet preparations and 27 non-islet human tissues. For verification of array results, bisulfite DNA sequencing of human ß cells and 11 non-ß cell tissues was performed on 5 of the top 10 CpG sites that were found to be differentially methylated. We identified the CHTOP gene as a candidate whose CpGs show a greater frequency of unmethylation in human islets. A digital PCR strategy was used to determine the methylation pattern of CHTOP and INS CpG sites in primary human tissues. Although both INS and CHTOP contained unmethylated CpG sites in non-islet tissues, they occurred in a non-overlapping pattern. Based on Naïve Bayes classifier analysis, the two genes together report 100% specificity for islet damage. Digital PCR was then performed on cell-free DNA from serum from human subjects. Compared to healthy controls (N = 10), differentially methylated CHTOP and INS levels were higher in youth with new onset T1D (N = 43) and, unexpectedly, in healthy autoantibody-negative youth who have first-degree relatives with T1D (N = 23). When tested in lean (N = 32) and obese (N = 118) youth, increased levels of unmethylated INS and CHTOP were observed in obese individuals. CONCLUSION: Our data suggest that concurrent measurement of circulating unmethylated INS and CHTOP has the potential to detect islet death in youth at risk for both T1D and T2D. Our data also support the use of multiple parameters to increase the confidence of detecting islet damage in individuals at risk for developing diabetes.

Activation of AMP-activated protein kinase attenuates ethanol-induced ER/oxidative stress and lipid phenotype in human pancreatic acinar cells.

Primary toxicity targets of alcohol and its metabolites in the pancreas are cellular energetics and endoplasmic reticulum (ER). Therefore, the role of AMP-Activated Protein Kinase (AMPKα) in amelioration of ethanol (EtOH)-induced pancreatic acinar cell injury including ER/oxidative stress, inflammatory responses, the formation of fatty acid ethyl esters (FAEEs) and mitochondrial bioenergetics were determined in human pancreatic acinar cells (hPACs) and AR42J cells incubated with/without AMPKα activator [5-aminoimidazole-4-carboxamide ribonucleotide (AICAR)]. EtOH treated hPACs showed concentration and time-dependent increases for FAEEs and inactivation of AMPKα, along with the upregulation of ACC1 and FAS (key lipogenic proteins) and downregulation of CPT1A (involved ß-oxidation of fatty acids). These cells also showed significant ER stress as evidenced by the increased expression for GRP78, IRE1α, and PERK/CHOP arm of unfolded protein response promoting apoptosis and activating p-JNK1/2 and p-ERK1/2 with increased secretion of cytokines. AR42J cells treated with EtOH showed increased oxidative stress, impaired mitochondrial biogenesis, and decreased ATP production rate. However, AMPKα activation by AICAR attenuated EtOH-induced ER/oxidative stress, lipogenesis, and inflammatory responses as well as the formation of FAEEs and restored mitochondrial function in hPACs as well as AR42J cells. Therefore, it is likely that EtOH-induced inactivation of AMPKα plays a crucial role in acinar cell injury leading to pancreatitis. Findings from this study also suggest that EtOH-induced inactivation of AMPKα is closely related to ER/oxidative stress and synthesis of FAEEs, as activation of AMPKα by AICAR attenuates formation of FAEEs, ER/oxidative stress and lipogenesis, and improves inflammatory responses and mitochondrial bioenergetics.

Trisected pancreas model for testing tissue dissociation enzyme combinations: a novel methodology for improving human islet yield for clinical islet transplantation.

PURPOSE: Human islet isolation requires a defined collagenase-protease enzyme combination for obtaining a successful islet yield. While different islet laboratories use different enzyme combinations, a systematic methodology to identify optimal enzyme combinations and their concentrations within a single donor pancreas has not been tested. In this study, we designed a trisected pancreas model to test efficacy of three clinical grade enzyme blends (VitaCyte, Roche, SERVA) within a single pancreas. METHODS: Islet isolations were performed using brain-dead donor pancreases (n = 15) applying the enzyme-related design of experiments (DOEs) and the trisected model approach. After trimming, split each pancreas into three individual lobes (head, body, tail). As per the DOEs, the lobes were altered between different experiments, to minimize anatomical bias. Islets isolated from each lobe (27 lobes totally) were subjected to functional assessments. Insulin staining and islet area fraction were determined for tissue sections obtained from each lobe. RESULTS: Utilizing the trisected model, we identified that the collagenase dose from three different vendors did not affect the pancreas digestion and islet yield, but islet morphology after isolation with the neutral protease and BP-protease was better than thermolysin. In addition, the head lobe yielded a lower islet mass and higher tissue volume compared to other two lobes, irrespective of enzyme combination used. CONCLUSIONS: This study demonstrates that the trisected model is a promising methodology in assessing donor and isolation associated parameters. Based on this study, we conclude that the donor characteristics and an optimal enzyme dose play a critical role in achieving higher islet yields.

Islet cell transplantation in children.

This paper aims to provide an overview of islet cell transplantation in children, with specific attention to pediatric total pancreatectomy with islet autotransplantation (TPIAT). We will summarize the definition and causes of chronic pancreatitis in children, the TPIAT procedure and potential complications, the process of islet cell isolation and autotransplantation, and long-term results after TPIAT. Lastly, we will briefly discuss islet cell allotransplantation in the adult population and its potential role in treating children.

Myt Transcription Factors Prevent Stress-Response Gene Overactivation to Enable Postnatal Pancreatic ß Cell Proliferation, Function, and Survival.

Although cellular stress response is important for maintaining function and survival, overactivation of late-stage stress effectors cause dysfunction and death. We show that the myelin transcription factors (TFs) Myt1 (Nzf2), Myt2 (Myt1l, Nztf1, and Png-1), and Myt3 (St18 and Nzf3) prevent such overactivation in islet ß cells. Thus, we found that co-inactivating the Myt TFs in mouse pancreatic progenitors compromised postnatal ß cell function, proliferation, and survival, preceded by upregulation of late-stage stress-response genes activating transcription factors (e.g., Atf4) and heat-shock proteins (Hsps). Myt1 binds putative enhancers of Atf4 and Hsps, whose overexpression largely recapitulated the Myt-mutant phenotypes. Moreover, Myt(MYT)-TF levels were upregulated in mouse and human ß cells during metabolic stress-induced compensation but downregulated in dysfunctional type 2 diabetic (T2D) human ß cells. Lastly, MYT knockdown caused stress-gene overactivation and death in human EndoC-ßH1 cells. These findings suggest that Myt TFs are essential restrictors of stress-response overactivity.

Recent Advances in Understanding the Complexity of Alcohol-Induced Pancreatic Dysfunction and Pancreatitis Development.

Chronic excessive alcohol use is a well-recognized risk factor for pancreatic dysfunction and pancreatitis development. Evidence from in vivo and in vitro studies indicates that the detrimental effects of alcohol on the pancreas are from the direct toxic effects of metabolites and byproducts of ethanol metabolism such as reactive oxygen species. Pancreatic dysfunction and pancreatitis development are now increasingly thought to be multifactorial conditions, where alcohol, genetics, lifestyle, and infectious agents may determine the initiation and course of the disease. In this review, we first highlight the role of nonoxidative ethanol metabolism in the generation and accumulation of fatty acid ethyl esters (FAEEs) that cause multi-organellar dysfunction in the pancreas which ultimately leads to pancreatitis development. Further, we discuss how alcohol-mediated altered autophagy leads to the development of pancreatitis. We also provide insights into how alcohol interactions with other co-morbidities such as smoking or viral infections may negatively affect exocrine and endocrine pancreatic function. Finally, we present potential strategies to ameliorate organellar dysfunction which could attenuate pancreatic dysfunction and pancreatitis severity.

Human pancreatic tissue dissociation enzymes for islet isolation: Advances and clinical perspectives.

BACKGROUND AND AIMS: Successful clinical human allo or auto-islet transplantation requires the recovery of a sufficient number of functional islets from either brain-dead or chronic pancreatitis pancreases respectively. METHODS: In the last two decades (2000-2019), significant progress has been made in improving the human islet isolation procedures and in standardizing the use of different tissue dissociation enzyme (TDE; a mixture of collagenase and protease enzymes) blends to recover higher islet yields. RESULTS AND CONCLUSIONS: This review presents information focusing on properties and role of TDE blends during the islet isolation process, particularly emphasizing on the current developments, associated challenges and future perspectives within the field.