ORPHAN NUCLEAR RECEPTOR 4A1 AUGMENTED EXPRESSION TRIGGERS INSULIN SECRETORY DEFECTS BY TARGETING CALCIUM CHANNELS IN CHRONIC PANCREATITIS: THERAPEUTIC IMPLICATIONS FOR PANCREATOGENIC DIABETES IN HUMANS

Background: The incidence of hypertriglyceridemia-associated acute pancreatitis (HTG-AP) has been increasing recently. Plasmapheresis can effectively remove triglyceride(TG) from plasma, but its clinical value in HTG-AP is unclear due to the lack of solid evidence. This study aimed to assess the impact of plasmapheresis on the evolution of organ failure in HTG-AP patients.
Methods: This study is a prospective, multicenter cohort study. HTG-AP patients with clinical worrisome features and abdominal pain onset within 72 hours were included. Patients who underwent plasmapheresis were compared with those receiving conventional medical treatment. The primary outcome was organ failure-free days (OFFD) to 14 days of enrollment. Secondary outcomes included other measures for the evolution of organ failure, need for intensive care unit (ICU) admission, length of ICU and hospital stay, 60-day mortality, and the incidence of infected pancreatic necrosis. Propensity score matching (PSM) and inverse probability of treatment weighting (IPTW) analysis were used to control potential confounders.
Results: During the study period, 267 HTG-AP patients were enrolled from 28 sites, among whom 211 underwent conventional medical treatment and 56 underwent plasmapheresis. The PSM created 47 pairs of patients with balanced baseline characteristics. In the matched cohort, no difference was detected concerning OFFD between patients undergoing plasmapheresis or not (median (IQR), 12(8,14) vs. 13 (8,14); P = 0.94) (Table 1). Kaplan-Meier curves and Cox proportional hazards model also showed similar probability of organ failure resolution in both groups (HR:1.05, 95%CI:0.69-1.60, P=0.82) (Figure 1). Moreover, patients in the plasmapheresis group had higher rates of ICU admission (44(93.6%) vs. 24(51.1%), P<0.01). No difference was found in other secondary outcomes. IPTW results conform to the results from the PSM analysis.
Conclusion: In conclusion, early plasmapheresis may not be effective in improving organ failure and may be associated with more ICU admissions.

Background: While transient bacteremia is common during dental and endoscopic procedures [PMID: 18541739, 6354885], infections in sterile diseases like AP can be fatal. Fat necrosis occurs in human AP [PMID: 3950033, 3094241] and contains abundant NEFA from fat lipolysis [PMID: 25500204]. Here we study how NEFAs released in clinical AP may impair bacterial clearance, causing this sterile to infected transition.
Methods: All studies were approved by the IRB, IACUC. Mayo Clinic AZ emergency room patients with ≥ 2/3 AP criteria were included. Those with active malignancy, immuno-suppression, chemotherapy, age >90 years were excluded. Patients who had microorganisms isolated, developed sepsis, or required antibiotics for infection were grouped as infected. Antibiotic prophylaxis was not considered as infection. Admission serums were analyzed for NEFA, albumin-unbound NEFA, bacterial DNA amounts, microbiome, and immune cell injury (annexin V staining). Mice after IL12,18 pancreatitis [PMID: 18515422] +/- the triglyceride of linoleic acid [LA; PMID: 33514548] were analyzed similarly. Multiple groups were compared by ANOVA. A P value <0.05 was considered significant.
Results: There were 53 normal controls, 178 non-infected, 21 infected AP patients. Age, sex, BMI in all three groups were similar. Serum lipase, etiology of AP were similar in both AP groups. Infected AP had higher (p<0.001) serum unsaturated NEFA (737±526µM) vs. non-infected AP (475±293µM), controls (170±99µM). Unbound-NEFA were similarly higher in infected AP (5.6±3.0µM, 2.8±1.9µM, 0.9±0.4µM, P<0.001). Specifically unbound linoleic acid (LA; 3.4±3.1µM vs. 1.4±0.8µM in non-infected, 0.8±0.7µM control), and unbound-oleic acid (OA) were higher in infected AP. Bacterial 16S DNA copies (670±1201, 170±340, 2.9±6.3) were increased in infected AP with altered beta-diversity (Jaccard-Emperor, PERMANOVA <0.0001), and enrichment in Pseudomonadales on level 4 analysis. Annexin V positive myeloid (CD14) and CD3 cells increased only in infected AP to 15±20%, 11±11% respectively vs <3% in controls. In vitro 2µM Unbound-LA, and -OA depolarized mitochondria of macrophages unlike other -medium and -long chain NEFA. The patterns of cell injury, NEFA elevation, microbiome changes, enrichment of Pseudomonadales were replicated only in mice with AP and lipolysis of LA’s triglyceride.
Conclusions: Release of LA, OA during fat necrosis in AP can increase their circulating unbound levels, which injure immune cells. This injury may explain sterile to infected transition during AP and is consistent with bacteremia being common but transient during dental, endoscopic procedures in normal individuals with healthy immune cells. In a separate submission [Control ID:3866559] we mechanistically detail this amphipathic immune cell injury, and the consequent impairment of phagocytosis and bacterial clearance.

Background: Exocrine Pancreatic insufficiency (EPI) can occur following acute pancreatitis (AP), with impairment in pancreatic enzyme secretion and damage to the pancreatic acinar cells thought to be key contributors. The majority with AP experience rapid resolution of their symptoms of abdominal pain, however the timeline of exocrine function recovery is unknown. The aim of this study is to establish the incidence and predictors of EPI at 3 months after AP in a prospective cohort.

Methods: Adult participants (18 years or older) admitted to the hospital with an AP attack, were enrolled in the Post-Acute Pancreatitis Pancreatic Exocrine Insufficiency (PAPPEI) Study at the time of attempt to resume oral or enteral diet at three centers. AP was defined according to the Revised Atlanta Classification. Patients with a pre-existing history of pancreatic cancer, chronic pancreatitis, EPI, or diseases of malabsorption were excluded. Symptoms of EPI, along with blood and stool samples were collected at baseline and at 3-months after enrollment. EPI was evaluated using FE-1 levels from stool samples (FE-1 <200 µg/g indicating EPI).

Results: A total of 113 participants provided stool samples at enrollment, and 90 completed the 3 month assessment. EPI was seen in 36 (40%) at 3 months. At enrollment, 32/90 (36%) of the subjects had confirmed EPI and 25/32 (78%) had persistent EPI at 3 months. Of the 58 subjects without EPI at baseline, 4 developed new EPI at 3 months. Significant differences were seen in the short term rates of EPI between AP participants with different etiologies; gallstone AP made up only 17% of subjects with EPI at 3 months, whereas it comprised almost 55% of those without EPI at 3 months. Idiopathic etiology contributed the largest group of subjects with EPI at 3 months (39%). Severity of AP and presence of pancreatitis necrosis were both associated with EPI at 3 months after AP. (Table 1)

Conclusions: EPI is present in approximately 40% of prospectively assessed patients at 3-months post-AP, most frequently seen in subjects with idiopathic etiology, severe AP and pancreatic necrosis. Longer term data are needed to further understand the persistence of EPI following AP and the contributing mechanisms.

Introduction:
Advancing current knowledge of insulin secretory defects during progression of pancreatitis from acute injury to chronicity would aid in developing newer strategies to manage diabetes associated with chronic pancreatitis (CP). We had reported enhanced Nuclear receptor4A1 (NR4A1) expression in human CP and mice models of CP. In this study, we aim to delineate the molecular mechanism of NR4A1 enhanced expression and insulin secretory defects, evaluate it in mice with L-arginine induced CP and in human CP.
Methods: MIN6 cells were transfected with pLenti NR4A1 overexpression (OE) plasmid employing PEI linear and the resultant transcriptome was studied (NovaSeq6000). DESeq2, gene ontology and KEGG pathway analysis were employed to identify differentially expressed genes and pathways. Transcription factors of insulin gene were evaluated by qPCR and western blotting. Cytokine exposure and inBio Discover were employed for identifying upstream signal for NR4A1 OE. Intracellular Ca²⁺ levels were measured by fluorimetric assay. Ca²⁺ imaging of OE/cytokine treated MIN6 cells, islets from CP patients and L-Arginine induced CP mice was performed using Fura2AM (EVOS M7000). CP mice were treated with IFN-γ neutralizing antibodies to assess the modulation of NR4A1 and effect on insulin secretion.
Results: NR4A1 OE in MIN6 cells (13.2±0.1 fold) resulted in decreased insulin secretion (18.1 ± 13.6 ng/mg protein). Higher expression of NR4A1 in CP patients and CP mice (19.8±0.1, 4.11±0.1 respectively) correlated with decreased insulin secretion by isolated islets (humans 3.7±0.2, r = -0.89, mice 2.1±0.1, r = -1.0). Transcriptomic analysis of OE MIN6 cells revealed decreased Ca²⁺ channel expression (L type -3.66/T type -4.29; p<0.001) and RFX6 expression (insulin gene transcription factor -1.6 fold). Transcriptome of CP patients and CP mice confirmed Ca²⁺ channel downregulation (human-1.4; mice-1.24 folds). KEGG pathway analysis of OE MIN6 identified downregulation of calcium and cAMP signalling. Fura2 AM based Ca²⁺ imaging of islets from CP patients, CP mice and OE MIN6 showed a decrease in Ca²⁺ Fluorescence intensity units (FIU) by 2.64, 2.4, 1.3 folds (p=0.0007,0.0004, <0.0001) compared to respective controls. IFN-γ was identified as the upstream signal for NR4A1 OE (2.31±0.03) and decreased insulin secretion (-4.21±0.56), Fig.1. Administration of IFN-γ neutralizing antibodies to CP mice resulted in decreased NR4A1 expression by 3.4 ± 0.11fold (p=0.03), improved insulin secretion by 4.4±0.2 fold, p=0.01) associated with increased Ca²⁺ levels (2.39 ± 0.06 fold, p=0.009) Fig.2.
Conclusion: Modulation of NR4A1 expression to enhance Ca²⁺ channel activity can be a therapeutic strategy to improve insulin secretory functions of β cells in chronic pancreatitis to prevent progression to pancreatogenic diabetes.