Protocol for assessing the effects of exogenous hormone administration on human postprandial glucose metabolism, appetite sensations, and food intake.

Here, we present a protocol for a randomized, double-blind, placebo-controlled, crossover trial to evaluate the effects of a continuous intravenous infusion of a native liver-derived hormone, liver-expressed antimicrobial peptide 2 (LEAP2), on postprandial glucose metabolism, appetite and satiety sensations, and ad libitum food intake in humans. We describe the preparation of the exogenous hormone administration and participants. We then detail the liquid mixed meal, ad libitum meal test, and blood sampling procedures for assessing postprandial glucose metabolism and food intake. For complete details on the use and execution of this protocol, please refer to Hagemann et al. (2022).1.

A liver secretome gene signature-based approach for determining circulating biomarkers of NAFLD severity.

Non-invasive biomarkers of non-alcoholic fatty liver disease (NAFLD) supporting diagnosis and monitoring disease progression are urgently needed. The present study aimed to establish a bioinformatics pipeline capable of defining and validating NAFLD biomarker candidates based on paired hepatic global gene expression and plasma bioanalysis from individuals representing different stages of histologically confirmed NAFLD (no/mild, moderate, more advanced NAFLD). Liver secretome gene signatures were generated in a patient cohort of 26 severely obese individuals with the majority having no or mild fibrosis. To this end, global gene expression changes were compared between individuals with no/mild NAFLD and moderate/advanced NAFLD with subsequent filtering for candidate gene products with liver-selective expression and secretion. Four candidate genes, including LPA (lipoprotein A), IGFBP-1 (insulin-like growth factor-binding protein 1), SERPINF2 (serpin family F member 2) and MAT1A (methionine adenosyltransferase 1A), were differentially expressed in moderate/advanced NAFLD, which was confirmed in three independent RNA sequencing datasets from large, publicly available NAFLD studies. The corresponding gene products were quantified in plasma samples but could not discriminate among different grades of NAFLD based on NAFLD activity score. Conclusion: We demonstrate a novel approach based on the liver transcriptome allowing for identification of secreted hepatic gene products as potential circulating diagnostic biomarkers of NAFLD. Using this approach in larger NAFLD patient cohorts may yield potential circulating biomarkers for NAFLD severity.

LEAP2 reduces postprandial glucose excursions and ad libitum food intake in healthy men.

The gastric hormone ghrelin stimulates food intake and increases plasma glucose through activation of the growth hormone secretagogue receptor (GHSR). Liver-expressed antimicrobial peptide 2 (LEAP2) has been proposed to inhibit actions of ghrelin through inverse effects on GHSR activity. Here, we investigate the effects of exogenous LEAP2 on postprandial glucose metabolism and ad libitum food intake in a randomized, double-blind, placebo-controlled, crossover trial of 20 healthy men. We report that LEAP2 infusion lowers postprandial plasma glucose and growth hormone concentrations and decreases food intake during an ad libitum meal test. In wild-type mice, plasma glucose and food intake are reduced by LEAP2 dosing, but not in GHSR-null mice, pointing to GHSR as a potential mediator of LEAP2's glucoregulatory and appetite-suppressing effects in mice.

Beta-Hydroxybutyrate Suppresses Hepatic Production of the Ghrelin Receptor Antagonist LEAP2.

INTRODUCTION: Liver-expressed antimicrobial peptide-2 (LEAP2) is an endogenous ghrelin receptor antagonist, which is upregulated in the fed state and downregulated during fasting. We hypothesized that the ketone body beta-hydroxybutyrate (BHB) is involved in the downregulation of LEAP2 during conditions with high circulating levels of BHB. METHODS: Hepatic and intestinal Leap2 expression were determined in 3 groups of mice with increasing circulating levels of BHB: prolonged fasting, prolonged ketogenic diet, and oral BHB treatment. LEAP2 levels were measured in lean and obese individuals, in human individuals following endurance exercise, and in mice after BHB treatment. Lastly, we investigated Leap2 expression in isolated murine hepatocytes challenged with BHB. RESULTS: We confirmed increased circulating LEAP2 levels in individuals with obesity compared to lean individuals. The recovery period after endurance exercise was associated with increased plasma levels of BHB levels and decreased LEAP2 levels in humans. Leap2 expression was selectively decreased in the liver after fasting and after exposure to a ketogenic diet for 3 weeks. Importantly, we found that oral administration of BHB increased circulating levels of BHB in mice and decreased Leap2 expression levels and circulating LEAP2 plasma levels, as did Leap2 expression after direct exposure to BHB in isolated murine hepatocytes. CONCLUSION: From our data, we suggest that LEAP2 is downregulated during different states of energy deprivation in both humans and rodents. Furthermore, we here provide evidence that the ketone body, BHB, which is highly upregulated during fasting metabolism, directly downregulates LEAP2 levels. This may be relevant in ghrelin receptor-induced hunger signaling during energy deprivation.

Layer-specific and whole wall global longitudinal strain predict major adverse cardiovascular events in patients with stable angina pectoris.

Global longitudinal strain (GLS) has proven to be a powerful prognostic marker in various patient populations, but the prognostic value of layer-specific GLS has not yet been investigated in patients with suspected stable angina pectoris (SAP). We sought to investigate the prognostic value of layer-specific and whole wall GLS in patients with suspected SAP. From September 2008 to March 2011, 296 consecutive patients with clinically suspected SAP, normal ejection fraction, and no previous cardiac history were enrolled in a prospective cohort study. Patients underwent echocardiography including two-dimensional speckle tracking at rest, exercise stress test, and coronary angiography. The end-point was a composite of incident heart failure, acute myocardial infarction, and cardiovascular death (MACE). Out of the 285 included patients (mean age 61 years, 50% male), 24 (8%) developed MACE during a median follow-up of 3.5 years. Both endocardial [hazard ratio (HR) 1.21, 95% CI 1.08-1.35, p = 0.001], epicardial (HR 1.29, 95% CI 1.12-1.50, p = 0.001) and whole wall GLS (HR 1.25, 1.10-1.42, p = 0.001) were significantly associated with an increased risk of developing MACE during follow-up in univariable Cox regression analysis. In multivariable analysis, only epicardial (HR 1.23, 95% CI 1.00-1.51, p = 0.046) and whole wall GLS (HR 1.20, 95% CI 1.00-1.43, p = 0.049) remained significantly associated with an increased risk of MACE independent of various baseline clinical variables, left ventricular ejection fraction (LVEF), E/e' and Duke Score. Layer-specific and whole wall GLS were significant predictors of MACE in this cohort of patients with suspected SAP independent of various baseline clinical variables, LVEF, E/e' and Duke Score.

Identification and Metabolic Profiling of a Novel Human Gut-derived LEAP2 Fragment.

CONTEXT: The mechanisms underlying Roux-en-Y gastric bypass (RYGB) surgery-induced weight loss and the immediate postoperative beneficial metabolic effects associated with the operation remain uncertain. Enteroendocrine cell (EEC) secretory function has been proposed as a key factor in the marked metabolic benefits from RYGB surgery. OBJECTIVE: To identify novel gut-derived peptides with therapeutic potential in obesity and/or diabetes by profiling EEC-specific molecular changes in obese patients following RYGB-induced weight loss. SUBJECTS AND METHODS: Genome-wide expression analysis was performed in isolated human small intestinal EECs obtained from 20 gut-biopsied obese subjects before and after RYGB. Targets of interest were profiled for preclinical and clinical metabolic effects. RESULTS: Roux-en-Y gastric bypass consistently increased expression levels of the inverse ghrelin receptor agonist, liver-expressed antimicrobial peptide 2 (LEAP2). A secreted endogenous LEAP2 fragment (LEAP238-47) demonstrated robust insulinotropic properties, stimulating insulin release in human pancreatic islets comparable to the gut hormone glucagon-like peptide-1. LEAP238-47 showed reciprocal effects on growth hormone secretagogue receptor (GHSR) activity, suggesting that the insulinotropic action of the peptide may be directly linked to attenuation of tonic GHSR activity. The fragment was infused in healthy human individuals (n = 10), but no glucoregulatory effect was observed in the chosen dose as compared to placebo. CONCLUSIONS: Small intestinal LEAP2 expression was upregulated after RYGB. The corresponding circulating LEAP238-47 fragment demonstrated strong insulinotropic action in vitro but failed to elicit glucoregulatory effects in healthy human subjects.

Early diastolic strain rate by two-dimensional speckle tracking echocardiography is a predictor of coronary artery disease and cardiovascular events in stable angina pectoris.

This study aimed to clarify the diagnostic and prognostic potential of strain rate in patients with suspected stable angina pectoris (SAP). Strain rate by 2-dimensional speckle tracking echocardiography (2DSTE) has been suggested to be able to diagnose coronary artery disease (CAD) and predict cardiovascular events in various patient groups. Prospectively enrolled patients (n = 296) with suspected SAP, no previous cardiac disease, and normal left ventricular ejection fraction were examined by 2DSTE, exercise ECG, and coronary angiography. Obstructive CAD was defined as stenosis ≥ 70% in ≥ 1 coronary artery on coronary angiography (n = 107). Major adverse cardiac events (MACE) included myocardial infarction, heart failure, atrial fibrillation, and stroke. In multivariable analysis adjusted for baseline data, conventional echocardiography, and Duke score, early diastolic strain rate (SRe) was independently associated with significant CAD with a 1.35 increased risk of having CAD per 0.1 decrease in SRe (OR = 1.35, 95% CI 1.03-1.76, P = 0.027). Peak velocity of early diastolic filling (E)/SRe was not associated with significant CAD (OR = 1.14, 95% CI 0.81-1.62, P = 0.445). MACE occurred in 34 patients (12%) during follow-up (median 3.5 years) and both SRe (HR 1.26, 95% CI (1.07-1.49), P = 0.006) and E/SRe (HR 1.24, 95% CI (1.04-1.47), P = 0.017) were independent predictors after multivariable adjustment. In patients with suspected SAP, SRe by 2DSTE was independently associated with presence of CAD. In addition, SRe and E/SRe provided independent and incremental prognostic value for predicting future MACE.

Usefulness of layer-specific strain in diagnosis of coronary artery disease in patients with stable angina pectoris.

Novel software allows for layer-specific evaluation of myocardial strain by speckle tracking echocardiography (2DSTE). However, the potential of layer-specific strain at rest for diagnosing coronary artery disease (CAD) in patients with suspected stable angina pectoris (SAP) remains unknown. Our objective was to evaluate the usefulness of layer-specific 2DSTE at rest for diagnosis of CAD in patients with SAP. In total, 285 patients referred with clinically suspected SAP, normal ejection fraction, and no previous cardiac history were prospectively enrolled. All patients were examined by echocardiography, including 2DSTE, exercise ECG, and coronary angiography (CAG). Layer-specific 2DSTE was performed in three apical views to provide longitudinal peak systolic strains. Stenosis ≥ 70% in ≥ 1 major coronary artery on CAG was considered as significant CAD. Of 285 patients included, 104 had significant CAD (36%). Endocardial, epicardial, and mid-myocardial GLS were all significantly impaired in CAD patients (P < 0.001). Multivariable analysis including baseline clinical parameters, conventional echocardiographic measurements, Duke score, and layer-specific strain measurements revealed epicardial [odds ratio 1.19 (P = 0.048)] and mid-myocardial [odds ratio 1.16 (P = 0.047)] global longitudinal strain (GLS) as the only independent predictors of CAD. In direct comparison, epicardial and mid-myocardial GLS had a significantly higher diagnostic performance compared to endocardial GLS (P = 0.038 and P = 0.031, respectively). In conclusion, layer-specific GLS from 2DSTE at rest was significantly impaired in patients with significant CAD. In addition, epicardial and mid-myocardial GLS were independent predictors of CAD.

Bariatric surgery in patients with non-alcoholic fatty liver disease - from pathophysiology to clinical effects.

Non-alcoholic fatty liver disease (NAFLD) is increasingly recognized as a significant liver disease, and it covers the disease spectrum from simple steatosis with a risk of development of non-alcoholic steatohepatitis (NASH) to fibrosis, subsequent cirrhosis, end-stage liver failure, and liver cancer with a potential need for liver transplantation. NAFLD and NASH are closely related to obesity, metabolic syndrome, and type 2 diabetes (T2D). The role of gut hormones, especially glucagon-like peptide 1 (GLP-1), is important in NAFLD. Bariatric surgery has the potential for inducing great weight loss and may improve the symptoms of metabolic syndrome and T2D. Recent data demonstrated significant effects of bariatric surgery on GLP-1 and other gut hormones and important lipid metabolic and inflammatory abnormalities in the pathophysiology of NAFLD. Therefore, bariatric surgery may reverse the pathological liver changes in NAFLD and NASH patients. In the present review, we describe NAFLD and NASH pathophysiology and the primary effects of bariatric surgery on metabolic pathways. We performed a systematic review of the beneficial and harmful effects and focused on changes in liver disease severity in NAFLD and NASH patients. The specific focus was liver histopathology as assessed by the invasive liver biopsy. Additionally, we reviewed several non-invasive methods used for the assessment of liver disease severity following bariatric surgery.