Single nucleus RNA-sequencing integrated into risk variant colocalization discovers 17 cell-type-specific abdominal obesity genes for metabolic dysfunction-associated steatotic liver disease.

BACKGROUND: Abdominal obesity increases the risk for non-alcoholic fatty liver disease (NAFLD), now known as metabolic dysfunction-associated steatotic liver disease (MASLD). METHODS: To elucidate the directional cell-type level biological mechanisms underlying the association between abdominal obesity and MASLD, we integrated adipose and liver single nucleus RNA-sequencing and bulk cis-expression quantitative trait locus (eQTL) data with the UK Biobank genome-wide association study (GWAS) data using colocalization. Then we used colocalized cis-eQTL variants as instrumental variables in Mendelian randomization (MR) analyses, followed by functional validation experiments on the target genes of the cis-eQTL variants. FINDINGS: We identified 17 colocalized abdominal obesity GWAS variants, regulating 17 adipose cell-type marker genes. Incorporating these 17 variants into MR discovers a putative tissue-of-origin, cell-type-aware causal effect of abdominal obesity on MASLD consistently with multiple MR methods without significant evidence for pleiotropy or heterogeneity. Single cell data confirm the adipocyte-enriched mean expression of the 17 genes. Our cellular experiments across human adipogenesis identify risk variant -specific epigenetic and transcriptional mechanisms. Knocking down two of the 17 genes, PPP2R5A and SH3PXD2B, shows a marked decrease in adipocyte lipidation and significantly alters adipocyte function and adipogenesis regulator genes, including DGAT2, LPL, ADIPOQ, PPARG, and SREBF1. Furthermore, the 17 genes capture a characteristic MASLD expression signature in subcutaneous adipose tissue. INTERPRETATION: Overall, we discover a significant cell-type level effect of abdominal obesity on MASLD and trace its biological effect to adipogenesis. FUNDING: NIH grants R01HG010505, R01DK132775, and R01HL170604; the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (Grant No. 802825), Academy of Finland (Grants Nos. 333021), the Finnish Foundation for Cardiovascular Research the Sigrid Jusélius Foundation and the Jane and Aatos Erkko Foundation; American Association for the Study of Liver Diseases (AASLD) Advanced Transplant Hepatology award and NIH/NIDDK (P30DK41301) Pilot and Feasibility award; NIH/NIEHS F32 award (F32ES034668); Finnish Diabetes Research Foundation, Kuopio University Hospital Project grant (EVO/VTR grants 2005-2021), the Academy of Finland grant (Contract no. 138006); Academy of Finland (Grant Nos 335443, 314383, 272376 and 266286), Sigrid Jusélius Foundation, Finnish Medical Foundation, Finnish Diabetes Research Foundation, Novo Nordisk Foundation (#NNF20OC0060547, NNF17OC0027232, NNF10OC1013354) and Government Research Funds to Helsinki University Hospital; Orion Research Foundation, Maud Kuistila Foundation, Finish Medical Foundation, and University of Helsinki.

Validation of a Machine Learning Algorithm, EVendo, for Predicting Esophageal Varices in Hepatocellular Carcinoma.

BACKGROUND: Treatment with atezolizumab and bevacizumab has become standard of care for advanced unresectable hepatocellular carcinoma (HCC) but carries an increased gastrointestinal bleeding risk. Therefore, patients are often required to undergo esophagogastroduodenoscopy (EGD) to rule out esophageal varices (EV) prior to initiating therapy, which can delay care and lead to unnecessary procedural risks and health care costs. In 2019, the EVendo score was created and validated as a noninvasive tool to accurately screen out patients who were at low risk for having EV that required treatment. We sought to validate whether the EVendo score could be used to accurately predict the presence of EV and varices needing treatment (VNT) in patients with HCC. METHODS: This was a retrospective multicenter cohort study of patients with HCC from 9/2004 to 12/2021. We included patients who underwent EGDs within 1 year after their HCC diagnosis. We collected clinical parameters needed to calculate an EVendo score at the time of EGD and compared the EVendo model prediction to the gold standard endoscopic report in predicting presence of VNT. RESULTS: 112 with HCC were recruited to this study, with 117 qualifying EGDs. VNT occurred in 39 (33.3%) patients. The EVendo score had a sensitivity of 97.4% and a negative predictive value of 96.9%, supporting the validity in applying EVendo in predicting VNT in HCC. CONCLUSION: In this study, we validated the use of the EVendo score in ruling out VNT in patients with HCC. The application of the EVendo score could safely defer about 30% of EGDs for EV screening in HCC patients. Although additional validation cohorts are needed, this suggests that EVendo score can potentially be applied in patients with HCC to avoid unnecessary EGDs, which can ultimately mitigate healthcare costs and delays in initiating HCC treatment with atezolizumab and bevacizumab.

Sensory neurons regulate stimulus-dependent humoral immunity.

Sensory neurons sense pathogenic infiltration, serving to inform immune coordination of host defense. However, sensory neuron-immune interactions have been predominantly shown to drive innate immune responses. Humoral memory, whether protective or destructive, is acquired early in life - as demonstrated by both early exposure to streptococci and allergic disease onset. Our study further defines the role of sensory neuron influence on humoral immunity in the lung. Using a murine model of Streptococcus pneumonia pre-exposure and infection and a model of allergic asthma, we show that sensory neurons are required for B-cell and plasma cell recruitment and antibody production. In response to S. pneumoniae , sensory neuron depletion resulted in a larger bacterial burden, reduced B-cell populations, IgG release and neutrophil stimulation. Conversely, sensory neuron depletion reduced B-cell populations, IgE and asthmatic characteristics during allergen-induced airway inflammation. The sensory neuron neuropeptide released within each model differed. With bacterial infection, vasoactive intestinal polypeptide (VIP) was preferentially released, whereas substance P was released in response to asthma. Administration of VIP into sensory neuron-depleted mice suppressed bacterial burden and increased IgG levels, while VIP1R deficiency increased susceptibility to bacterial infection. Sensory neuron-depleted mice treated with substance P increased IgE and asthma, while substance P genetic ablation resulted in blunted IgE, similar to sensory neuron-depleted asthmatic mice. These data demonstrate that the immunogen differentially stimulates sensory neurons to release specific neuropeptides which specifically target B-cells. Targeting sensory neurons may provide an alternate treatment pathway for diseases involved with insufficient and/or aggravated humoral immunity.

Female patients delay seeking medical care with alcohol-associated acute pancreatitis.

BACKGROUND/OBJECTIVE: Alcohol consumption is increasing in women, who more frequently report abdominal symptoms compared to men. We aimed to examine differences in presentation of acute pancreatitis [AP] in male and female patients hospitalized with alcohol-associated AP. METHODS: We analyzed 138 patients enrolled in an ongoing case-crossover study of alcohol-associated AP conducted across 5 medical centers in the U.S. Patients meeting the Revised Atlanta Classification of AP and who scored 3 or higher on the AUDIT-C instrument were invited to participate in the study and were interviewed while hospitalized with AP. Sex differences in the timing and type of pancreas-associated pain, alcohol consumption, clinical presentation, and quality of life were examined by Chi-squared tests, Wilcoxon rank sum tests and t-tests. RESULTS: Female patients reported significantly longer interval from onset of pain to deciding to seek medical attention (median 40 h, interquartile range [IQR] 14, 74) as compared to males (14 h, IQR 4, 50; p = 0.005). While male patients were more likely to have been admitted to the intensive care unit [ICU] (21%) as compared to female patients (7%; p = 0.04), the incidence of SIRS or severe AP did not differ by sex. Quality of life measures as reported through the PROMIS-29 instrument were equally suboptimal in both sexes. Anxiety disorders were diagnosed more frequently among females (61%) than in males (41%, p = 0.009). CONCLUSION: In a large case series of alcohol-associated AP, we found that female patients delayed seeking medical care compared to males. However, there were no differences in the type, location and intensity of abdominal pain.

PACAP and VIP Neuropeptides' and Receptors' Effects on Appetite, Satiety and Metabolism.

The overwhelming increase in the prevalence of obesity and related disorders in recent years is one of the greatest threats to the global healthcare system since it generates immense healthcare costs. As the prevalence of obesity approaches epidemic proportions, the importance of elucidating the mechanisms regulating appetite, satiety, body metabolism, energy balance and adiposity has garnered significant attention. Currently, gastrointestinal (GI) bariatric surgery remains the only approach capable of achieving successful weight loss. Appetite, satiety, feeding behavior, energy intake and expenditure are regulated by central and peripheral neurohormonal mechanisms that have not been fully elucidated yet. Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and Vasoactive Intestinal Polypeptide (VIP) are members of a family of regulatory peptides that are widely distributed in parallel with their specific receptors, VPAC1R, VPAC2R and PAC1R, in the central nervous system (CNS) and in the periphery, such as in the gastrointestinal tract and its associated organs and immune cells. PACAP and VIP have been reported to play an important role in the regulation of body phenotype, metabolism and homeostatic functions. The purpose of this review is to present recent data on the effects of PACAP, VIP, VPAC1R, VPAC2R and PAC1R on the modulation of appetite, satiety, metabolism, calorie intake and fat accumulation, to evaluate their potential use as therapeutic targets for the treatment of obesity and metabolic syndrome.

Lipophilic statins inhibit YAP coactivator transcriptional activity in HCC cells through Rho-mediated modulation of actin cytoskeleton.

Hepatocellular carcinoma (HCC) is the third leading cause of liver-related death. Lipophilic statins have been associated with a decrease in HCC incidence, raising the possibility of their use as chemoprevention agents. The Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) have emerged as an important pro-oncogenic mechanism in HCC. Statins modulate YAP/TAZ in other solid tumors, but few studies have assessed their mechanisms in HCC. We aimed to delineate how lipophilic statins regulate YAP protein localization by interrogating the mevalonate pathway in a stepwise manner using pharmacological and genetical approaches in HCC cells. Huh7 and Hep3B HCC cells were treated with the lipophilic statins cerivastatin and atorvastatin. YAP protein localization was determined using quantitative immunofluorescence (IF) imaging. The gene expression of CTGF and CYR61, known YAP/TEA-domain DNA-binding factor (TEAD)-regulated genes, was measured using quantitative real-time PCR. Rescue experiments were conducted using metabolites of the mevalonate pathway including mevalonic acid and geranylgeranyl pyrophosphate (GG-PP). The cellular cytoskeleton was assessed using F-actin IF staining. YAP protein was extruded from the nucleus to the cytoplasm with statin treatment. Consistently, CTGF and CYR61 mRNA expression significantly decreased with statins. Cytoskeletal structure was also compromised with statins. Gene expression, YAP protein localization, and cytoskeletal structure were all restored to baseline with exogenous GG-PP but not with other metabolites of the mevalonate pathway. Direct Rho GTPase inhibitor treatment mirrored the statin effects on YAP. YAP protein localization is regulated by lipophilic statins via Rho GTPases, causing cytoskeletal structural changes and is independent of cholesterol metabolites.NEW & NOTEWORTHY Statins are widely used for the treatment of cardiovascular diseases. Recently, their use has been associated with a decrease in the incidence of hepatocellular carcinoma (HCC); however, their mechanism(s) has remained elusive. In this study, we delineate the mechanism by which statins affect the Yes-associated protein (YAP), which has emerged as a key oncogenic pathway in HCC. We investigate each step of the mevalonate pathway and demonstrate that statins regulate YAP via Rho GTPases.

Machine learning versus regression for prediction of sporadic pancreatic cancer.

BACKGROUND/OBJECTIVES: There is currently no widely accepted approach to identify patients at increased risk for sporadic pancreatic cancer (PC). We aimed to compare the performance of two machine-learning models with a regression-based model in predicting pancreatic ductal adenocarcinoma (PDAC), the most common form of PC. METHODS: This retrospective cohort study consisted of patients 50-84 years of age enrolled in either Kaiser Permanente Southern California (KPSC, model training, internal validation) or the Veterans Affairs (VA, external testing) between 2008 and 2017. The performance of random survival forests (RSF) and eXtreme gradient boosting (XGB) models were compared to that of COX proportional hazards regression (COX). Heterogeneity of the three models were assessed. RESULTS: The KPSC and the VA cohorts consisted of 1.8 and 2.7 million patients with 1792 and 4582 incident PDAC cases within 18 months, respectively. Predictors selected into all three models included age, abdominal pain, weight change, and glycated hemoglobin (A1c). Additionally, RSF selected change in alanine transaminase (ALT), whereas the XGB and COX selected the rate of change in ALT. The COX model appeared to have lower AUC (KPSC: 0.737, 95% CI 0.710-0.764; VA: 0.706, 0.699-0.714), compared to those of RSF (KPSC: 0.767, 0.744-0.791; VA: 0.731, 0.724-0.739) and XGB (KPSC: 0.779, 0.755-0.802; VA: 0.742, 0.735-0.750). Among patients with top 5% predicted risk from all three models (N = 29,663), 117 developed PDAC, of which RSF, XGB and COX captured 84 (9 unique), 87 (4 unique), 87 (19 unique) cases, respectively. CONCLUSIONS: The three models complement each other, but each has unique contributions.

Specific Targeting and Labeling of Colonic Polyps in CPC-APC Mice with Mucin 5AC Fluorescent Antibodies: A Model for Detection of Early Colon Cancer.

Poor visualization of polyps can limit colorectal cancer screening. Fluorescent antibodies to mucin5AC (MUC5AC), a glycoprotein upregulated in adenomas and colorectal cancer, could improve screening colonoscopy polyp detection rate. Adenomatous polyposis coli flox mice with a Cdx2-Cre transgene (CPC-APC) develop colonic polyps that contain both dysplastic and malignant tissue. Mice received MUC5AC-IR800 or IRdye800 as a control IV and were sacrificed after 48 h for near-infrared imaging of their colons. A polyp-to-background ratio (PBR) was calculated for each polyp by dividing the mean fluorescence intensity of the polyp by the mean fluorescence intensity of the background tissue. The mean 25 µg PBR was 1.70 (±0.56); the mean 50 µg PBR was 2.64 (±0.97); the mean 100 µg PBR was 3.32 (±1.33); and the mean 150 µg PBR was 3.38 (±0.87). The mean PBR of the dye-only control was 2.22 (±1.02), significantly less than the 150 µg arm (p-value 0.008). The present study demonstrates the ability of fluorescent anti-MUC5AC antibodies to specifically target and label colonic polyps containing high-grade dysplasia and intramucosal adenocarcinoma in CPC-APC mice. This technology can potentially improve the detection rate and decrease the miss rate of advanced colonic neoplasia and early cancer at colonoscopy.

Cross-tissue omics analysis discovers ten adipose genes encoding secreted proteins in obesity-related non-alcoholic fatty liver disease.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a fast-growing, underdiagnosed, epidemic. We hypothesise that obesity-related inflammation compromises adipose tissue functions, preventing efficient fat storage, and thus driving ectopic fat accumulation into the liver. METHODS: To identify adipose-based mechanisms and potential serum biomarker candidates (SBCs) for NAFLD, we utilise dual-tissue RNA-sequencing (RNA-seq) data in adipose tissue and liver, paired with histology-based NAFLD diagnosis, from the same individuals in a cohort of obese individuals. We first scan for genes that are differentially expressed (DE) for NAFLD in obese individuals' subcutaneous adipose tissue but not in their liver; encode proteins secreted to serum; and show preferential adipose expression. Then the identified genes are filtered to key adipose-origin NAFLD genes by best subset analysis, knockdown experiments during human preadipocyte differentiation, recombinant protein treatment experiments in human liver HepG2 cells, and genetic analysis. FINDINGS: We discover a set of genes, including 10 SBCs, that may modulate NAFLD pathogenesis by impacting adipose tissue function. Based on best subset analysis, we further follow-up on two SBCs CCDC80 and SOD3 by knockdown in human preadipocytes and subsequent differentiation experiments, which show that they modulate crucial adipogenesis genes, LPL, SREBPF1, and LEP. We also show that treatment of the liver HepG2 cells with the CCDC80 and SOD3 recombinant proteins impacts genes related to steatosis and lipid processing, including PPARA, NFE2L2, and RNF128. Finally, utilizing the adipose NAFLD DE gene cis-regulatory variants associated with serum triglycerides (TGs) in extensive genome-wide association studies (GWASs), we demonstrate a unidirectional effect of serum TGs on NAFLD with Mendelian Randomization (MR) analysis. We also demonstrate that a single SNP regulating one of the SBC genes, rs2845885, produces a significant MR result by itself. This supports the conclusion that genetically regulated adipose expression of the NAFLD DE genes may contribute to NAFLD through changes in serum TG levels. INTERPRETATION: Our results from the dual-tissue transcriptomics screening improve the understanding of obesity-related NAFLD by providing a targeted set of 10 adipose tissue-active genes as new serum biomarker candidates for the currently grossly underdiagnosed fatty liver disease. FUNDING: The work was supported by NIH grants R01HG010505 and R01DK132775. The Genotype-Tissue Expression (GTEx) Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health, and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS. The KOBS study (J. P.) was supported by the Finnish Diabetes Research Foundation, Kuopio University Hospital Project grant (EVO/VTR grants 2005-2019), and the Academy of Finland grant (Contract no. 138006). This study was funded by the European Research Council under the European Union's Horizon 2020 research and innovation program (Grant No. 802825 to M. U. K.). K. H. P. was funded by the Academy of Finland (grant numbers 272376, 266286, 314383, and 335443), the Finnish Medical Foundation, Gyllenberg Foundation, Novo Nordisk Foundation (grant numbers NNF10OC1013354, NNF17OC0027232, and NNF20OC0060547), Finnish Diabetes Research Foundation, Finnish Foundation for Cardiovascular Research, University of Helsinki, and Helsinki University Hospital and Government Research Funds. I. S. was funded by the Instrumentarium Science Foundation. Personal grants to U. T. A. were received from the Matti and Vappu Maukonen Foundation, Ella och Georg Ehrnrooths Stiftelse and the Finnish Foundation for Cardiovascular Research.

Isolation of Nuclei from Human Snap-frozen Liver Tissue for Single-nucleus RNA Sequencing.

Single-nucleus RNA sequencing (snRNA-seq) provides a powerful tool for studying cell type composition in heterogenous tissues. The liver is a vital organ composed of a diverse set of cell types; thus, single-cell technologies could greatly facilitate the deconvolution of liver tissue composition and various downstream omics analyses at the cell-type level. Applying single-cell technologies to fresh liver biopsies can, however, be very challenging, and snRNA-seq of snap-frozen liver biopsies requires some optimization given the high nucleic acid content of the solid liver tissue. Therefore, an optimized protocol for snRNA-seq specifically targeted for the use of frozen liver samples is needed to improve our understanding of human liver gene expression at the cell-type resolution. We present a protocol for performing nuclei isolation from snap-frozen liver tissues, as well as guidance on the application of snRNA-seq. We also provide guidance on optimizing the protocol to different tissue and sample types.

Microbial changes from bariatric surgery alters glucose-dependent insulinotropic polypeptide and prevents fatty liver disease.

Bariatric surgery remains a potent therapy for nonalcoholic fatty liver disease (NAFLD), but its inherent risk and eligibility requirement limit its adoption. Therefore, understanding how bariatric surgery improves NAFLD is paramount to developing novel therapeutics. Here, we show that the microbiome changes induced by sleeve gastrectomy (SG) reduce glucose-dependent insulinotropic polypeptide (GIP) signaling and confer resistance against diet-induced obesity (DIO) and NAFLD. We examined a cohort of NALFD patients undergoing SG and evaluated their microbiome, serum metabolites, and GI hormones. We observed significant changes in Bacteroides, lipid-related metabolites, and reduction in GIP. To examine if the changes in the microbiome were causally related to NAFLD, we performed fecal microbial transplants in antibiotic-treated mice from patients before and after their surgery who had significant weight loss and improvement of their NAFLD. Mice transplanted with the microbiome of patients after bariatric surgery were more resistant to DIO and NAFLD development compared to mice transplanted with the microbiome of patients before surgery. This resistance to DIO and NAFLD was also associated with a reduction in GIP levels in mice with post-bariatric microbiome. We further show that the reduction in GIP was related to higher levels of Akkermansia and differing levels of indolepropionate, bacteria-derived tryptophan-related metabolite. Overall, this is one of the few studies showing that GIP signaling is altered by the gut microbiome, and it supports that the positive effect of bariatric surgery on NAFLD is in part due to microbiome changes.

Derivation and External Validation of Machine Learning-Based Model for Detection of Pancreatic Cancer.

INTRODUCTION: There is currently no widely accepted approach to screening for pancreatic cancer (PC). We aimed to develop and validate a risk prediction model for pancreatic ductal adenocarcinoma (PDAC), the most common form of PC, across 2 health systems using electronic health records. METHODS: This retrospective cohort study consisted of patients aged 50-84 years having at least 1 clinic-based visit over a 10-year study period at Kaiser Permanente Southern California (model training, internal validation) and the Veterans Affairs (VA, external testing). Random survival forests models were built to identify the most relevant predictors from >500 variables and to predict risk of PDAC within 18 months of cohort entry. RESULTS: The Kaiser Permanente Southern California cohort consisted of 1.8 million patients (mean age 61.6) with 1,792 PDAC cases. The 18-month incidence rate of PDAC was 0.77 (95% confidence interval 0.73-0.80)/1,000 person-years. The final main model contained age, abdominal pain, weight change, HbA1c, and alanine transaminase change (c-index: mean = 0.77, SD = 0.02; calibration test: P value 0.4, SD 0.3). The final early detection model comprised the same features as those selected by the main model except for abdominal pain (c-index: 0.77 and SD 0.4; calibration test: P value 0.3 and SD 0.3). The VA testing cohort consisted of 2.7 million patients (mean age 66.1) with an 18-month incidence rate of 1.27 (1.23-1.30)/1,000 person-years. The recalibrated main and early detection models based on VA testing data sets achieved a mean c-index of 0.71 (SD 0.002) and 0.68 (SD 0.003), respectively. DISCUSSION: Using widely available parameters in electronic health records, we developed and externally validated parsimonious machine learning-based models for detection of PC. These models may be suitable for real-time clinical application.

Human liver single nucleus and single cell RNA sequencing identify a hepatocellular carcinoma-associated cell-type affecting survival.

BACKGROUND: Hepatocellular carcinoma (HCC) is a common primary liver cancer with poor overall survival. We hypothesized that there are HCC-associated cell-types that impact patient survival. METHODS: We combined liver single nucleus (snRNA-seq), single cell (scRNA-seq), and bulk RNA-sequencing (RNA-seq) data to search for cell-type differences in HCC. To first identify cell-types in HCC, adjacent non-tumor tissue, and normal liver, we integrated single-cell level data from a healthy liver cohort (n = 9 non-HCC samples) collected in the Strasbourg University Hospital; an HCC cohort (n = 1 non-HCC, n = 14 HCC-tumor, and n = 14 adjacent non-tumor samples) collected in the Singapore General Hospital and National University; and another HCC cohort (n = 3 HCC-tumor and n = 3 adjacent non-tumor samples) collected in the Dumont-UCLA Liver Cancer Center. We then leveraged these single cell level data to decompose the cell-types in liver bulk RNA-seq data from HCC patients' tumor (n = 361) and adjacent non-tumor tissue (n = 49) from the Cancer Genome Atlas (TCGA) multi-center cohort. For replication, we decomposed 221 HCC and 209 adjacent non-tumor liver microarray samples from the Liver Cancer Institute (LCI) cohort collected by the Liver Cancer Institute and Zhongshan Hospital of Fudan University. RESULTS: We discovered a tumor-associated proliferative cell-type, Prol (80.4% tumor cells), enriched for cell cycle and mitosis genes. In the liver bulk tissue from the TCGA cohort, the proportion of the Prol cell-type is significantly increased in HCC and associates with a worse overall survival. Independently from our decomposition analysis, we reciprocally show that Prol nuclei/cells significantly over-express both tumor-elevated and survival-decreasing genes obtained from the bulk tissue. Our replication analysis in the LCI cohort confirmed that an increased estimated proportion of the Prol cell-type in HCC is a significant marker for a shorter overall survival. Finally, we show that somatic mutations in the tumor suppressor genes TP53 and RB1 are linked to an increase of the Prol cell-type in HCC. CONCLUSIONS: By integrating liver single cell, single nucleus, and bulk expression data from multiple cohorts we identified a proliferating cell-type (Prol) enriched in HCC tumors, associated with a decreased overall survival, and linked to TP53 and RB1 somatic mutations.

The VIP/VPAC1R Pathway Regulates Energy and Glucose Homeostasis by Modulating GLP-1, Glucagon, Leptin and PYY Levels in Mice.

Vasoactive Intestinal Peptide binds with high affinity to VPAC1R and VPAC2R, thus regulating key physiologic functions. Previously, we documented in VIP-/- mice a leaner body phenotype and altered metabolic hormones. Past reports described in VPAC2-/- mice impaired circadian rhythm, reduced food intake, and altered metabolism. To better define the effects of VPAC1R on body phenotype, energy/glucose homeostasis, and metabolism, we conducted a 12-week study in a VPAC1R null model. Our results reveal that VPAC1-/- mice experienced significant metabolic alterations during the dark cycle with greater numbers of feeding bouts (p = 0.009), lower Total Energy Expenditure (p = 0.025), VO2 (p = 0.029), and VCO2 (p = 0.016); as well as during the light cycle with lower Total Energy Expenditure (p = 0.04), VO2 (p = 0.044), and VCO2 (p = 0.029). Furthermore, VPAC1-/- mice had significantly higher levels of GLP-1 and PYY during fasting, and higher levels of GLP-1, glucagon leptin and PYY during postprandial conditions. In addition, VPAC1-/- mice had lower levels of glucose at 60' and 120', as assessed by insulin tolerance test. In conclusion, this study supports a key role for VPAC1R in the regulation of body glucose/energy homeostasis and metabolism.

Prediction of Pancreatic Cancer in Diabetes Patients with Worsening Glycemic Control.

BACKGROUND: Worsening glycemic control indicates elevated risk of pancreatic ductal adenocarcinoma (PDAC). We developed prediction models for PDAC among those with worsening glycemic control after diabetes diagnosis. METHODS: In 2000-2016 records within the Veterans Affairs Health System (VA), we identified three cohorts with progression of diabetes: (i) insulin initiation (n = 449,685), (ii) initiation of combination oral hypoglycemic medication (n = 414,460), and (iii) hemoglobin A1c (HbA1c) ≥8% with ≥Δ1% within 15 months (n = 593,401). We computed 12-, 36-, and 60-month incidence of PDAC and developed prediction models separately for males and females, with consideration of >30 demographic, behavioral, clinical, and laboratory variables. Models were selected to optimize Akaike's Information Criterion, and performance for predicting 12-, 36-, and 60-month incident PDAC was evaluated by bootstrap. RESULTS: Incidence of PDAC was highest for insulin initiators and greater in males than in females. Optimism-corrected c-indices of the models for predicting 36-month incidence of PDAC in the male population were: (i) 0.72, (ii) 0.70, and (iii) 0.71, respectively. Models performed better for predicting 12-month incident PDAC [c-index (i) 0.78, (ii) 0.73, (iii) 0.76 for males], and worse for predicting 60-month incident PDAC [c-index (i) 0.69, (ii) 0.67, (iii) 0.68 for males]. Model performance was lower among females. For subjects whose model-predicted 36-month PDAC risks were ≥1%, the observed incidences were (i) 1.9%, (ii) 2.2%, and (iii) 1.8%. CONCLUSIONS: Sex-specific models for PDAC can estimate risk of PDAC at the time of progression of diabetes. IMPACT: Our models can identify diabetes patients who would benefit from PDAC screening.

Duodenal Microbiome and Serum Metabolites Predict Hepatocellular Carcinoma in a Multicenter Cohort of Patients with Cirrhosis.

BACKGROUND: Hepatocellular carcinoma (HCC) is rapidly increasing in the U.S. and is a leading cause of mortality for patients with cirrhosis. Discovering novel biomarkers for risk stratification of HCC is paramount. We examined biomarkers of the gut-liver axis in a prospective multicenter cohort. METHODS: Patients with cirrhosis without a history of HCC were recruited between May 2015 and March 2020 and prospectively followed at 3 tertiary care hospitals in Los Angeles. Microbiome analysis was performed on duodenal biopsies and metabolomic analysis was performed on serum samples, collected at the time of enrollment. Optimal microbiome-based survival analysis and Cox proportional hazards regression analysis were used to determine microbiota and metabolite associations with HCC development, respectively. RESULTS: A total of 227 participants with liver cirrhosis contributed a total of 459.58 person-years of follow-up, with 14 incident HCC diagnoses. Male sex (HR = 7.06, 95% CI = 1.02-54.86) and baseline hepatic encephalopathy (HE, HR = 4.65, 95% CI = 1.60-13.52) were associated with developing HCC over follow-up. Adjusting for age, sex, baseline HE, and alkaline phosphatase, an increased risk of HCC were observed for participants with the highest versus lowest three quartiles for duodenal Alloprevotella (HR = 3.22, 95% CI = 1.06-9.73) and serum taurocholic acid (HR = 6.87, 95% CI = 2.32-20.27), methionine (HR = 9.97, 95% CI = 3.02-32.94), and methioninesulfoxide (HR = 5.60, 95% CI = 1.84-17.10). Being in the highest quartile for Alloprevotella or methionine had a sensitivity and specificity for developing HCC of 85.71% and 60.56%, respectively, with an odds ratio of 10.92 (95% CI = 2.23-53.48). CONCLUSION: Alloprevotella and methionine, methioninesulfoxide, and taurocholic acid predicted future HCC development in a high-risk population of participants with liver cirrhosis.

Familial Duodenal Somatostatinomatosis Not Associated With a Known Genetic Syndrome.

ABSTRACT: We report a father and his daughter who both had multiple somatostatinomas in the duodenal bulb without a known syndrome. The father, at age 68 years, was incidentally found to harbor 4 approximately 1.5-cm well-differentiated neuroendocrine tumors in the duodenal bulb. His preoperative somatostatin level was elevated. He underwent partial duodenectomy and regional lymph node dissection; one lymph node was positive for metastasis. One year postoperatively, a recurrence was found in the surgical bed; he was treated with octreotide for 2 years, which stabilized the recurrent tumor. Ten years postoperatively, the mucosa of his remaining duodenum was normal. His daughter, at age 53 years, was found to harbor multiple small neuroendocrine tumors in the duodenal bulb. Immunostaining of available specimens showed that the neuroendocrine tumors from the father and daughter both were strongly positive for somatostatin. Micronodules of somatostatin-expressing neuroendocrine cells were found in the parts of the specimens uninvolved with the tumors. Both patients exhibited no evidence of known syndromes associated with somatostatinoma. The daughter did not harbor mutations in 93 genes commonly found in genetic tumor syndromes. The 2 cases thus suggest a novel, autosomal dominant, genetic syndrome of familial duodenal somatostatinomatosis.

ß2-spectrin (SPTBN1) as a therapeutic target for diet-induced liver disease and preventing cancer development.

The prevalence of nonalcoholic steatohepatitis (NASH) and liver cancer is increasing. De novo lipogenesis and fibrosis contribute to disease progression and cancerous transformation. Here, we found that ß2-spectrin (SPTBN1) promotes sterol regulatory element (SRE)­binding protein (SREBP)­stimulated lipogenesis and development of liver cancer in mice fed a high-fat diet (HFD) or a western diet (WD). Either hepatocyte-specific knockout of SPTBN1 or siRNA-mediated therapy protected mice from HFD/WD-induced obesity and fibrosis, lipid accumulation, and tissue damage in the liver. Biochemical analysis suggested that HFD/WD induces SPTBN1 and SREBP1 cleavage by CASPASE-3 and that the cleaved products interact to promote expression of genes with sterol response elements. Analysis of human NASH tissue revealed increased SPTBN1 and CASPASE-3 expression. Thus, our data indicate that SPTBN1 represents a potential target for therapeutic intervention in NASH and liver cancer.

Longitudinal Changes in Liver Enzyme Levels Among Transgender People Receiving Gender Affirming Hormone Therapy.

BACKGROUND: The effect of gender affirming hormone therapy (GAHT) on clinical laboratory parameters, including levels of liver enzymes alanine aminotransferase (ALT) and aspartate transaminase (AST), is an area of uncertainty in transgender health. AIM: We sought to estimate the distribution parameters of liver enzyme levels among transmasculine (TM) and transfeminine (TF) persons receiving GAHT relative to the corresponding measures in cisgender reference groups, and to evaluate longitudinal changes in these laboratory measures following GAHT initiation. METHODS: The data for this longitudinal study included 624 TF and 438 transmasculine (TM) people as well as 4,090 cisgender males and 4,797 cisgender females enrolled in 3 integrated health systems. Time under observation was divided into 2 intervals: from the first blood test to the date of the first filled GAHT prescription and from GAHT initiation to the most recent ALT or AST measurement. Linear mixed models were used to compare changes in log-transformed ALT and AST values among transgender cohort members before and after GAHT initiation, and relative to the reference groups. The results were expressed as relative differences (in %) and the ratios of these differences (ratios-of-ratios) along with the 95% confidence intervals (CIs). OUTCOMES: Changes in ALT and AST levels among transgender people over time and relative to the corresponding changes in cisgender referents. RESULTS: Among TM study participants, the post GAHT ratios-of-ratios for AST were 1.61 (95% CI: 1.13, 2.31) and 1.57 (95% CI: 1.06, 2.31) relative to cisgender males and females respectively. For ALT, the corresponding comparisons yielded the ratios-of-ratios (95% CIs) of 2.06 (1.67, 2.54) and 1.90 (1.50, 2.40). No statistically significant changes were observed among TF participants. Other factors associated with higher liver enzyme levels included alcohol use/abuse and obesity. CLINICAL IMPLICATIONS: TM persons may experience modest increases in ALT and AST concentrations following testosterone initiation; however, clinical significance of the observed association remains unclear and requires further investigation. By contrast, feminizing GAHT is unlikely to induce appreciable changes in liver enzyme levels. STRENGTH AND LIMITATIONS: The strengths of this study are the longitudinal design and the ability to assemble an unselected cohort nested within large health systems. The main limitations include the lack of information on hormone levels and the inability to take into account GAHT doses and routes of administration. CONCLUSION: The influence of long-term GAHT on ALT and AST levels appears modest and not likely to reflect clinically meaningful changes in liver function. Hashemi L, Zhang Q, Getahun D, et al. Longitudinal Changes in Liver Enzyme Levels Among Transgender People Receiving Gender Affirming Hormone Therapy. J Sex Med 2021;18:1662-1675.