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Cancer of unknown primary (CUP) tumors are biologically very heterogeneous, which complicates stratification of patients for treatment. Consequently, these patients face limited treatment options and a poor prognosis. With this study, we aim to expand on the current knowledge of CUP biology by analyzing two cohorts: a well‐characterized cohort of 44 CUP patients, and 213 metastatic patients with known primary. These cohorts were treated at the same institution and characterized by identical molecular assessments. Through comparative analysis of genomic and transcriptomic data, we found that CUP tumors were characterized by high expression of immune‐related genes and pathways compared to other metastatic tumors. Moreover, CUP tumors uniformly demonstrated high levels of tumor‐infiltrating leukocytes and circulating T cells, indicating a strong immune response. Finally, the genetic landscape of CUP tumors resembled that of other metastatic cancers and demonstrated mutations in established cancer genes. In conclusion, CUP tumors possess a distinct immunophenotype that distinguishes them from other metastatic cancers. These results may suggest an immune response in CUP that facilitates metastatic tumor growth while limiting growth of the primary tumor.

Abstract Background Plasma levels of d-dimer are elevated in patients with thromboembolisms. Here we investigated the existence of interfering antibodies as a potential cause for elevated d-dimer levels. Case presentation A 42-year-old white Caucasian woman with a prior history of pulmonary embolism during her first pregnancy (treated with heparin therapy for 6 weeks postnatally) and hypothyroidism had a persistent elevated d-dimer without any clinical or ultrasound-based signs of thromboembolic conditions during her second pregnancy. We obtained informed consent and plasma was obtained from the patient. d-dimer levels were measured using two different assays. We also tested for the presence of rheumatoid factor, performed dilution series, and finally used an antibody depletion strategy. The two d-dimer assays performed similarly. Using our antibody depletion technique, we observed that ~ 1/3 of the increased plasma levels of d-dimer may be attributed to interfering antibodies. Conclusions Our results identify interfering antibodies as a potential contributor to an increased d-dimer in this patient. Our case highlights the potential of heterophilic interference for increased d-dimer and provides a procedure to determine this analytically.

Abstract Multisystem inflammatory syndrome in children (MIS-C) is a severe disease that emerged during the COVID-19 pandemic. Although recognized as an immune-mediated condition, the pathogenesis remains unresolved. Furthermore, the absence of a diagnostic test can lead to delayed immunotherapy. Using state-of-the-art mass-spectrometry proteomics, assisted by artificial intelligence (AI), we aimed to identify a diagnostic signature for MIS-C and to gain insights into disease mechanisms. We identified a highly specific 4-protein diagnostic signature in children with MIS-C. Furthermore, we identified seven clusters that differed between MIS-C and controls, indicating an interplay between apolipoproteins, immune response proteins, coagulation factors, platelet function, and the complement cascade. These intricate protein patterns indicated MIS-C as an immunometabolic condition with global hypercoagulability. Our findings emphasize the potential of AI-assisted proteomics as a powerful and unbiased tool for assessing disease pathogenesis and suggesting avenues for future interventions and impact on pediatric disease trajectories through early diagnosis.

Abstract Background Glucagon is secreted from pancreatic alpha cells in response to low blood glucose and increases hepatic glucose production. Furthermore, glucagon enhances hepatic protein and lipid metabolism during a mixed meal. Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are secreted from gut endocrine cells during meals and control glucose homeostasis by potentiating insulin secretion and inhibiting food intake. Both glucose homeostasis and food intake have been reported to be affected by circadian rhythms and vice versa. In this study, we investigated whether the secretion of glucagon, GLP-1 and GIP was affected by circadian rhythms. Methods A total of 24 healthy men with regular sleep schedules were examined for 24 h at the hospital ward with 15 h of wakefulness and 9 h of sleep. Food intake was standardized, and blood samples were obtained every third hour. Plasma concentrations of glucagon, GLP-1 and GIP were measured, and data were analyzed by rhythmometric statistical methods. Available data on plasma glucose and plasma C-peptide were also included. Results Plasma concentrations of glucagon, GLP-1, GIP, C-peptide and glucose fluctuated with a diurnal 24-h rhythm, with the highest levels during the day and the lowest levels during the night: glucagon (p

Not available.

Objective: In obesity and type 2 diabetes, hyperglucagonaemia may be caused by elevated levels of glucagonotropic amino acids due to hepatic glucagon resistance at the level of amino acid turnover. Here, we investigated the effect of exogenous glucagon on circulating amino acids in obese and non-obese individuals with and without type 2 diabetes. Design: This was a post hoc analysis in a glucagon infusion study performed in individuals with type 2 diabetes (n = 16) and in age, sex, and body mass index-matched control individuals without diabetes (n = 16). Each group comprised two subgroups of eight individuals with and without obesity, respectively. Methods: All participants received a 1-h glucagon infusion (4 ng/kg/min) in the overnight fasted state. Plasma amino acid concentrations were measured with frequent intervals. Results: Compared to the control subgroup without obesity, baseline total amino acid levels were elevated in the control subgroup with obesity and in the type 2 diabetes subgroup without obesity. In all subgroups, amino acid levels decreased by up to 20% in response to glucagon infusion, which resulted in high physiological steady-state glucagon levels (mean concentration: 74 pmol/L, 95% CI [68;79] pmol/L). Following correction for multiple testing, no intergroup differences in changes in amino acid levels reached significance. Conclusion: Obesity and type 2 diabetes status was associated with elevated fasting levels of total amino acids. The glucagon infusion decreased circulating amino acid levels similarly in all subgroups, without significant differences in the response to exogenous glucagon between individuals with and without obesity and type 2 diabetes. Significance statement The hormone glucagon stimulates glucose production from the liver, which may promote hyperglycaemia if glucagon levels are abnormally elevated, as is often seen in type 2 diabetes and obesity. Glucagon levels are closely linked to, and influenced by, the levels of circulating amino acids. To further investigate this link, we measured amino acid levels in individuals with and without obesity and type 2 diabetes before and during an infusion of glucagon. We found that circulating amino acid levels were higher in type 2 diabetes and obesity, and that glucagon infusion decreased amino acid levels in both individuals with and without type 2 diabetes and obesity. The study adds novel information to the link between circulating levels of glucagon and amino acids.

This paper presents formulations for time-based first-order and second-order high-pass, shelf, and notch filters. These formulations are an extension to the existing literature where low-pass filters are already developed using a multiphase controlled oscillator in conjunction with a phase detector and charge pump. The presented high-pass filter expands the circuit by introducing a current-controlled delay line (CCDL) that provides a direct path from input to output. By combining the high-pass filter with the low-pass filter, we show that shelf and notch filters can be obtained without an increase in circuit complexity compared to the high-pass filter. The results show good matching between the ideal small signal and the simulated time-based large signal frequency response. The simulated of total harmonic distortion for the filters shows an increase in distortion due to the nonlinearities introduced by the CCDL for the high-pass, notch, and shelf filter compared to the existing low-pass filter. The derivation of the new filter types allows the creation of complex high-order time-based filters by combining multiple first- or second-order filters.

Aims: Hyperglucagonaemia contributes to the pathophysiology in type 2 diabetes (T2D), but the mechanisms behind the inappropriate glucagon secretion are not fully understood. Glucagon and amino acids are regulated in a feedback loop referred to as the liver–α cell axis. Individuals with non-alcoholic fatty liver disease (NAFLD) appear to be glucagon resistant, disrupting the liver–α cell axis resulting in hyperglucagonaemia and hyperaminoacidaemia. We investigated the associations between circulating glucagon, amino acids, and liver fat content in a cohort of individuals with T2D. Methods: We included 110 individuals with T2D in this cross-sectional study. Liver fat content was quantified using 1H magnetic resonance spectroscopy (MRS). Associations between liver fat content and plasma glucagon and amino acids, respectively, were estimated in multivariate linear regression analyses. Results: Individuals with NAFLD (n = 52) had higher plasma glucagon concentrations than individuals without NAFLD (n = 58). The positive association between plasma glucagon concentrations and liver fat content was confirmed in the multivariable regression analyses. Plasma concentrations of isoleucine and glutamate were increased, and glycine and serine concentrations were decreased in individuals with NAFLD. Concentrations of other amino acids were similar between individuals with and without NAFLD, and no clear association was seen between liver fat content and amino acids in the regression analyses. Conclusion: MRS-diagnosed NAFLD in T2D is associated with hyperglucagonaemia and elevated plasma concentrations of isoleucine and glutamate and low plasma concentrations of glycine and serine. Whether NAFLD and glucagon resistance per se induce these changes remains to be elucidated.

Introduction: Health systems are confronted with not only the growing worldwide childhood obesity epidemic but also associated comorbidities. These subsequently cause variations in distinct metabolic pathways, leading to metabolic dysfunction-associated steatotic liver disease (MASLD). The aim of this evidence map is to systematically evaluate the evidence and to identify research gaps on glucagon-induced amino acid (AA) turnover and its metabolic interaction with MASLD. Methodology: A systematic literature search was conducted up to April 2023 in three electronic databases. Studies were required to include at least two of the main research areas, glucagon, AA metabolism and MASLD. Two independent reviewers screened titles and abstracts according to prespecified eligibility criteria, as well as full-text articles. Results are summarized in tables stratified by human and animal studies and study population age. Results: Thirty-four references were ultimately included. The publication years dated back to 1965 showed a great increase from 2012 to 2023. In total, there were 19 animal studies and 15 human studies. Among the human studies, except for two studies in adolescents, all the studies were conducted in adults. In human studies, the methods used to evaluate metabolic changes differed among hyperinsulinemic-euglycemic clamp and oral glucose tolerance tests. Thirteen studies focused on the metabolic effects of MASLD, while only two studies explored the interaction between MASLD, glucagon and AA metabolism in humans. The other 19 studies focused on metabolomics, beta cell function or just one topic of a research area and not on interactions between one another. Conclusion: Research on the interaction between MASLD, glucagon and AA metabolism in humans is sparse and complete lacking in pediatrics. Furthermore, longitudinal studies with a focus on hyperglucagonemia independent of diabetes but related to MASLD present an unambiguous research gap.

Digital integrated electronics benefits from its higher abstraction level, allowing optimisation methods and automated workflows. However, analogue integrated circuit design is still predominantly done manually, leading to lengthy design cycles. This paper proposes a new systematic design approach for the sizing of analogue integrated circuits to address this issue. The method utilises a surrogate optimisation technique that approximates a simple monomial function based on few simulation results. These monomials are convex and can be optimised using a simple linear optimisation routine, resulting in a single global optimal solution. We show that monomial functions, in many cases, have an analytic relation to integrated circuits, making them well suited for the application. The method is demonstrated by designing a 14 MHz pseudodifferential voltage-controlled oscillator (VCO) with minimised current consumption and is manufactured in a 180 nm process. The measured total current matches the predicted and is lower than that for other similar state-of-the-art VCOs.

Abstract Background Carbonic anhydrases catalyze CO2/HCO3 – buffer reactions with implications for effective H+ mobility, pH dynamics, and cellular acid–base sensing. Yet, the integrated consequences of carbonic anhydrases for cancer and stromal cell functions, their interactions, and patient prognosis are not yet clear. Methods We combine (a) bioinformatic analyses of human proteomic data and bulk and single-cell transcriptomic data coupled to clinicopathologic and prognostic information; (b) ex vivo experimental studies of gene expression in breast tissue based on quantitative reverse transcription and polymerase chain reactions, intracellular and extracellular pH recordings based on fluorescence confocal microscopy, and immunohistochemical protein identification in human and murine breast cancer biopsies; and (c) in vivo tumor size measurements, pH-sensitive microelectrode recordings, and microdialysis-based metabolite analyses in mice with experimentally induced breast carcinomas. Results Carbonic anhydrases—particularly the extracellular isoforms CA4, CA6, CA9, CA12, and CA14—undergo potent expression changes during human and murine breast carcinogenesis. In patients with basal-like/triple-negative breast cancer, elevated expression of the extracellular carbonic anhydrases negatively predicts survival, whereas, surprisingly, the extracellular carbonic anhydrases positively predict patient survival in HER2/ErbB2-enriched breast cancer. Carbonic anhydrase inhibition attenuates cellular net acid extrusion and extracellular H+ elimination from diffusion-restricted to peripheral and well-perfused regions of human and murine breast cancer tissue. Supplied in vivo, the carbonic anhydrase inhibitor acetazolamide acidifies the microenvironment of ErbB2-induced murine breast carcinomas, limits tumor immune infiltration (CD3+ T cells, CD19+ B cells, F4/80+ macrophages), lowers inflammatory cytokine (Il1a, Il1b, Il6) and transcription factor (Nfkb1) expression, and accelerates tumor growth. Supporting the immunomodulatory influences of carbonic anhydrases, patient survival benefits associated with high extracellular carbonic anhydrase expression in HER2-enriched breast carcinomas depend on the tumor inflammatory profile. Acetazolamide lowers lactate levels in breast tissue and blood without influencing breast tumor perfusion, suggesting that carbonic anhydrase inhibition lowers fermentative glycolysis. Conclusions We conclude that carbonic anhydrases (a) elevate pH in breast carcinomas by accelerating net H+ elimination from cancer cells and across the interstitial space and (b) raise immune infiltration and inflammation in ErbB2/HER2-driven breast carcinomas, restricting tumor growth and improving patient survival.

Desmoplasia is a common feature of aggressive cancers, driven by a complex interplay of protein production and degradation. Basigin is a type 1 integral membrane receptor secreted in exosomes or released by ectodomain shedding from the cell surface. Given that soluble basigin is increased in the circulation of patients with a poor cancer prognosis, we explored the putative role of the ADAM12-generated basigin ectodomain in cancer progression. We show that recombinant basigin ectodomain binds β1 integrin and stimulates gelatin degradation and the migration of cancer cells in a matrix metalloproteinase (MMP)- and β1-integrin-dependent manner. Subsequent in vitro and in vivo experiments demonstrated the altered expression of extracellular matrix proteins, including fibronectin and collagen type 5. Thus, we found increased deposits of collagen type 5 in the stroma of nude mice tumors of the human tumor cell line MCF7 expressing ADAM12—mimicking the desmoplastic response seen in human cancer. Our findings indicate a feedback loop between ADAM12 expression, basigin shedding, TGFβ signaling, and extracellular matrix (ECM) remodeling, which could be a mechanism by which ADAM12-generated basigin ectodomain contributes to the regulation of desmoplasia, a key feature in human cancer progression.

With the advent of robust and high-throughput mass spectrometric technologies and bioinformatics tools to analyze large data sets, proteomics has penetrated broadly into basic and translational life sciences research. More than 95% of FDA-approved drugs currently target proteins, and most diagnostic tests are protein-based. The introduction of proteomics to the clinic, for instance to guide patient stratification and treatment, is already ongoing. Importantly, ethical challenges come with this success, which must also be adequately addressed by the proteomics and medical communities. Consortium members of the H2020 European Union-funded proteomics initiative: European Proteomics Infrastructure Consortium-providing access (EPIC-XS) met at the Core Technologies for Life Sciences (CTLS) conference to discuss the emerging role and implementation of proteomics in the clinic. The discussion, involving leaders in the field, focused on the current status, related challenges, and future efforts required to make proteomics a more mainstream technology for translational and clinical research. Here we report on that discussion and provide an expert update concerning the feasibility of clinical proteomics, the ethical implications of generating and analyzing large-scale proteomics clinical data, and recommendations to ensure both ethical and effective implementation in real-world applications.

Summary: Inhibitors of neprilysin improve glycemia in patients with heart failure and type 2 diabetes (T2D). The effect of weight loss by diet, surgery, or pharmacotherapy on neprilysin activity (NEPa) is unknown. We investigated circulating NEPa and neprilysin protein concentrations in obesity, T2D, metabolic dysfunction-associated steatotic liver disease (MASLD), and following bariatric surgery, or GLP-1-receptor-agonist therapy. NEPa, but not neprilysin protein, was enhanced in obesity, T2D, and MASLD. Notably, MASLD associated with NEPa independently of BMI and HbA1c. NEPa decreased after bariatric surgery with a concurrent increase in OGTT-stimulated GLP-1. Diet-induced weight loss did not affect NEPa, but individuals randomized to 52-week weight maintenance with liraglutide (1.2 mg/day) decreased NEPa, consistent with another study following 6-week liraglutide (3 mg/day). A 90-min GLP-1 infusion did not alter NEPa. Thus, MASLD may drive exaggerated NEPa, and lowered NEPa following bariatric surgery or liraglutide therapy may contribute to the reported improved cardiometabolic effects.

The application of next-generation sequencing (NGS) has transformed cancer research. As costs have decreased, NGS has increasingly been applied to generate multiple layers of molecular data from the same samples, covering genomics, transcriptomics, and methylomics. Integrating these types of multi-omics data in a combined analysis is now becoming a common issue with no obvious solution, often handled on an ad hoc basis, with multi-omics data arriving in a tabular format and analyzed using computationally intensive statistical methods. These methods particularly ignore the spatial orientation of the genome and often apply stringent p-value corrections that likely result in the loss of true positive associations. Here, we present GENIUS (GEnome traNsformatIon and spatial representation of mUltiomicS data), a framework for integrating multi-omics data using deep learning models developed for advanced image analysis. The GENIUS framework is able to transform multi-omics data into images with genes displayed as spatially connected pixels and successfully extract relevant information with respect to the desired output. We demonstrate the utility of GENIUS by applying the framework to multi-omics datasets from the Cancer Genome Atlas. Our results are focused on predicting the development of metastatic cancer from primary tumors, and demonstrate how through model inference, we are able to extract the genes which are driving the model prediction and are likely associated with metastatic disease progression. We anticipate our framework to be a starting point and strong proof of concept for multi-omics data transformation and analysis without the need for statistical correction.

With the advent of robust and high-throughput mass spectrometric technologies and bioinformatics tools to analyze large data sets, proteomics has penetrated broadly into basic and translational life sciences research. More than 95% of FDA-approved drugs currently target proteins, and most diagnostic tests are protein-based. The introduction of proteomics to the clinic, for instance to guide patient stratification and treatment, is already ongoing. Importantly, ethical challenges come with this success, which must also be adequately addressed by the proteomics and medical communities. Consortium members of the H2020 European Union-funded proteomics initiative: European Proteomics Infrastructure Consortium-providing access (EPIC-XS) met at the Core Technologies for Life Sciences (CTLS) conference to discuss the emerging role and implementation of proteomics in the clinic. The discussion, involving leaders in the field, focused on the current status, related challenges, and future efforts required to make proteomics a more mainstream technology for translational and clinical research. Here we report on that discussion and provide an expert update concerning the feasibility of clinical proteomics, the ethical implications of generating and analyzing large-scale proteomics clinical data, and recommendations to ensure both ethical and effective implementation in real-world applications.

Abstract Fatty liver disease has mainly been characterized under fasting conditions. However, as the liver is essential for postprandial homeostasis, identifying postprandial disturbances may be important. Here, we investigated postprandial changes in markers of metabolic dysfunction between healthy individuals, obese individuals with non‐alcoholic fatty liver disease (NAFLD) and patients with cirrhosis. We included individuals with biopsy‐proven NAFLD (n = 9, mean age 50 years, mean BMI 35 kg/m2, no/mild fibrosis), cirrhosis with hepatic steatosis (n = 10, age 62 years, BMI 32 kg/m2, CHILD A/B) and healthy controls (n = 10, age 23, BMI 25 kg/m2), randomized 1:1 to fasting or standardized mixed meal test (postprandial). None of the patients randomized to mixed meal test had type 2 diabetes (T2D). Peripheral blood was collected for 120 min. After 60 min, a transjugular liver biopsy and liver vein blood was taken. Plasma levels of glucose, insulin, C‐peptide, glucagon, and fibroblast growth factor 21 (FGF21) were measured. Postprandial peak glucose and C‐peptide were significantly increased in NAFLD, and cirrhosis compared with healthy. Patients with NAFLD and cirrhosis had hyperglucagonemia as a potential sign of glucagon resistance. FGF21 was increased in NAFLD and cirrhosis independent of sampling from the liver vein versus peripheral blood. Glucagon levels were higher in the liver vein compared with peripheral blood. Patients with NAFLD and cirrhosis without T2D showed impaired glucose tolerance, hyperinsulinemia, and hyperglucagonemia after a meal compared to healthy individual. Postprandial characterization of patients with NAFLD may be important to capture their metabolic health.

Impaired mitochondrial function in liver tissue may contribute to the pathogenesis and disease progression of nonalcoholic fatty liver disease (NAFLD). Here the authors report that patients with obesity have lower mitochondrial capacity in adipose tissues but higher capacity in the liver, without overall associations to NAFLD severity, and that bariatric surgery increases hepatic mitochondrial respiration and mitochondrial biogenesis.

Abstract Deeper understanding of liver pathophysiology would benefit from a comprehensive quantitative proteome resource at cell type resolution to predict outcome and design therapy. Here, we quantify more than 150,000 sequence‐unique peptides aggregated into 10,000 proteins across total liver, the major liver cell types, time course of primary cell cultures, and liver disease states. Bioinformatic analysis reveals that half of hepatocyte protein mass is comprised of enzymes and 23% of mitochondrial proteins, twice the proportion of other liver cell types. Using primary cell cultures, we capture dynamic proteome remodeling from tissue states to cell line states, providing useful information for biological or pharmaceutical research. Our extensive data serve as spectral library to characterize a human cohort of non‐alcoholic steatohepatitis and cirrhosis. Dramatic proteome changes in liver tissue include signatures of hepatic stellate cell activation resembling liver cirrhosis and providing functional insights. We built a web‐based dashboard application for the interactive exploration of our resource ( www.liverproteome.org ).

Insulin and IGF-1 receptors (IR and IGF1R) are highly homologous and share similar signaling systems, but each has a unique physiological role, with IR primarily regulating metabolic homeostasis and IGF1R regulating mitogenic control and growth. Here, we show that replacement of a single amino acid at position 973, just distal to the NPEY motif in the intracellular juxtamembrane region, from leucine, which is highly conserved in IRs, to phenylalanine, the highly conserved homologous residue in IGF1Rs, resulted in decreased IRS-1/PI3K/Akt/mTORC1 signaling and increased Shc/Gab1/MAPK cell cycle signaling. As a result, cells expressing L973F-IR exhibited decreased insulin-induced glucose uptake, increased cell growth, and impaired receptor internalization. Mice with knockin of the L973F-IR showed similar alterations in signaling in vivo, and this led to decreased insulin sensitivity, a modest increase in growth, and decreased weight gain when mice were challenged with a high-fat diet. Thus, leucine-973 in the juxtamembrane region of the IR acts as a crucial residue differentiating IR signaling from IGF1R signaling.

Immunotherapy has revolutionised cancer treatment. However, not all cancer patients benefit, and current stratification strategies based primarily on PD1 status and mutation burden are far from perfect. We hypothesised that high activation of an innate response relative to the adaptive response may prevent proper tumour neoantigen identification and decrease the specific anticancer response, both in the presence and absence of immunotherapy. To investigate this, we obtained transcriptomic data from three large publicly available cancer datasets, the Cancer Genome Atlas (TCGA), the Hartwig Medical Foundation (HMF), and a recently published cohort of metastatic bladder cancer patients treated with immunotherapy. To analyse immune infiltration into bulk tumours, we developed an RNAseq-based model based on previously published definitions to estimate the overall level of infiltrating innate and adaptive immune cells from bulk tumour RNAseq data. From these, the adaptive-to-innate immune ratio (A/I ratio) was defined. A meta-analysis of 32 cancer types from TCGA overall showed improved overall survival in patients with an A/I ratio above median (Hazard ratio (HR) females 0.73, HR males 0.86, P < 0.05). Of particular interest, we found that the association was different for males and females for eight cancer types, demonstrating a gender bias in the relative balance of the infiltration of innate and adaptive immune cells. For patients with metastatic disease, we found that responders to immunotherapy had a significantly higher A/I ratio than non-responders in HMF (P = 0.036) and a significantly higher ratio in complete responders in a separate metastatic bladder cancer dataset (P = 0.022). Overall, the adaptive-to-innate immune ratio seems to define separate states of immune activation, likely linked to fundamental immunological reactions to cancer. This ratio was associated with improved prognosis and improved response to immunotherapy, demonstrating potential relevance to patient stratification. Furthermore, by demonstrating a significant difference between males and females that associates with response, we highlight an important gender bias which likely has direct clinical relevance.

Objective: Treatment with glucagon receptor antagonists (GRAs) reduces blood glucose but causes dyslipidemia and accumulation of fat in the liver. We investigated the acute and chronic effects of glucagon on lipid metabolism in mice. Methods: Chronic effects of glucagon receptor signaling on lipid metabolism were studied using oral lipid tolerance tests (OLTTs) in overnight fasted glucagon receptor knockout (Gcgr−/−) mice, and in C57Bl/6JRj mice treated with a glucagon receptor antibody (GCGR Ab) or a long-acting glucagon analogue (GCGA) for eight weeks. Following treatment, liver tissue was harvested for RNA-sequencing and triglyceride measurements. Acute effects were studied in C57Bl/6JRj mice treated with a GRA or GCGA 1 h or immediately before OLTTs, respectively. Direct effects of glucagon on hepatic lipolysis were studied using isolated perfused mouse liver preparations. To investigate potential effects of GCGA and GRA on gastric emptying, paracetamol was, in separate experiments, administered immediately before OLTTs. Results: Plasma triglyceride concentrations increased 2-fold in Gcgr−/− mice compared to their wild-type littermates during the OLTT (P = 0.001). Chronic treatment with GCGR Ab increased, whereas GCGA treatment decreased, plasma triglyceride concentrations during OLTTs (P

Summary: The pancreatic hormone, glucagon, is known to regulate hepatic glucose production, but recent studies suggest that its regulation of hepatic amino metabolism is equally important. Here, we show that chronic glucagon receptor activation with a long-acting glucagon analog increases amino acid catabolism and ureagenesis and causes alpha cell hypoplasia in female mice. Conversely, chronic glucagon receptor inhibition with a glucagon receptor antibody decreases amino acid catabolism and ureagenesis and causes alpha cell hyperplasia and beta cell loss. These effects were associated with the transcriptional regulation of hepatic genes related to amino acid uptake and catabolism and by the non-transcriptional modulation of the rate-limiting ureagenesis enzyme, carbamoyl phosphate synthetase-1. Our results support the importance of glucagon receptor signaling for amino acid homeostasis and pancreatic islet integrity in mice and provide knowledge regarding the long-term consequences of chronic glucagon receptor agonism and antagonism.

Multiple molecular profiling methods are required to study urothelial non-muscle-invasive bladder cancer (NMIBC) due to its heterogeneity. Here the authors integrate multi-omics data of 834 NMIBC patients, identifying a molecular subgroup associated with multiple alterations and worse outcomes.

Objective: Glucagon and glucagon-like peptide-1 (GLP-1) originate from the common precursor, proglucagon, and their plasma concentrations have been reported to be increased during inflammatory conditions. Increased blood glucose levels are frequently observed in septic patients, and therefore we hypothesized that glucagon, but not GLP-1, is increased in individuals with inflammation. Design: Prospective longitudinal cohort study. Materials and methods: We measured glucagon and GLP-1 in plasma sampled consecutively in three cohorts consisting of patients with infective endocarditis (n = 16), urosepsis (n = 28) and post-operative inflammation following percutaneous aortic valve implantation or thoracic endovascular aortic repair (n = 5). Correlations between C-reactive protein (CRP), a marker of systemic inflammat ion, and glucagon and GLP-1 concentrations were investigated. Additionally, glucagon and GLP-1 concentrations were measured after a bolus infusion of lipopolysaccharide (LPS, 1 ng/kg) in nine healthy young males. Results: Glucagon and CRP were positively and significantly correlated ( r = 0.27; P = 0.0003), whereas no significant association between GLP-1 and CRP was found (r = 0.08, P = 0.30). LPS infusion resulted in acute systemic inflammation reflected by increased temperature, pulse, tumor necrosis factor-α (TNFα), interleukin-6 (IL-6) and concomitantly increased concentrations of glucagon (P < 0.05) but not GLP-1. Conclusions: Systemic inflammation caused by bacterial infections or develop ed as a non-infected condition is associated with increased plasma concentration of glucagon, but not GLP-1. Hyperglucagonemia may contribute to the impaired glucose control in patients with systemic inflammatory diseases.

Breast cancer heterogeneity in histology and molecular subtype influences metabolic and proliferative activity and hence the acid load on cancer cells. We hypothesized that acid-base transporters and intracellular pH (pHi) dynamics contribute inter-individual variability in breast cancer aggressiveness and prognosis. We show that Na+,HCO3– cotransport and Na+/H+ exchange dominate cellular net acid extrusion in human breast carcinomas. Na+/H+ exchange elevates pHi preferentially in estrogen receptor-negative breast carcinomas, whereas Na+,HCO3– cotransport raises pHi more in invasive lobular than ductal breast carcinomas and in higher malignancy grade breast cancer. HER2-positive breast carcinomas have elevated protein expression of Na+/H+ exchanger NHE1/SLC9A1 and Na+,HCO3– cotransporter NBCn1/SLC4A7. Increased dependency on Na+,HCO3– cotransport associates with severe breast cancer: enlarged CO2/HCO3–-dependent rises in pHi predict accelerated cell proliferation, whereas enhanced CO2/HCO3–-dependent net acid extrusion, elevated NBCn1 protein expression, and reduced NHE1 protein expression predict lymph node metastasis. Accordingly, we observe reduced survival for patients suffering from luminal A or basal-like/triple-negative breast cancer with high SLC4A7 and/or low SLC9A1 mRNA expression. We conclude that the molecular mechanisms of acid-base regulation depend on clinicopathological characteristics of breast cancer patients. NBCn1 expression and dependency on Na+,HCO3– cotransport for pHi regulation, measured in biopsies of human primary breast carcinomas, independently predict proliferative activity, lymph node metastasis, and patient survival.

Insulin signaling represses Forkhead transcription factor FoxO activity, which contributes to organismal metabolism. Here, the authors use proteomics to identify positively regulated insulin signaling targets FoxK1/K2 and demonstrate their role in lipid metabolism and mitochondrial regulation.

Abstract Non‐alcoholic fatty liver disease (NAFLD) affects 25% of the population and can progress to cirrhosis with limited treatment options. As the liver secretes most of the blood plasma proteins, liver disease may affect the plasma proteome. Plasma proteome profiling of 48 patients with and without cirrhosis or NAFLD revealed six statistically significantly changing proteins (ALDOB, APOM, LGALS3BP, PIGR, VTN, and AFM), two of which are already linked to liver disease. Polymeric immunoglobulin receptor (PIGR) was significantly elevated in both cohorts by 170% in NAFLD and 298% in cirrhosis and was further validated in mouse models. Furthermore, a global correlation map of clinical and proteomic data strongly associated DPP4, ANPEP, TGFBI, PIGR, and APOE with NAFLD and cirrhosis. The prominent diabetic drug target DPP4 is an aminopeptidase like ANPEP, ENPEP, and LAP3, all of which are up‐regulated in the human or mouse data. Furthermore, ANPEP and TGFBI have potential roles in extracellular matrix remodeling in fibrosis. Thus, plasma proteome profiling can identify potential biomarkers and drug targets in liver disease.

Abstract Background Attentional biases are a relatively robust phenomenon among clinical populations but less pronounced in healthy participants. However, regarding the components of attentional biases and the directions of attention allocation, there are several inconsistencies in the literature. The present study examined whether these inconsistencies can be traced back to previous experiences of relational peer victimization in clinical populations. Methods Participants were subjects with a diagnosed psychiatric disorder (n = 30) and healthy controls (n = 31). Additionally, the sample was divided into two subgroups according to the participants’ reports of previous relational peer victimization (high peer victimization: n = 28; low peer victimization: n = 33). Attentional biases were measured by the Emotional Stroop task and a dot-probe task. Results In both samples, peer victimized participants showed delayed response times when color-naming negative and positive compared to neutral adjectives in the Emotional Stroop task. Likewise, the dot-probe task indicated attentional avoidance of both negative and positive words in peer victimized participants with and without a psychiatric disorder. Interestingly, presence of a psychiatric disorder did not have a significant effect on attentional biases. Conclusion Both tasks could detect that attentional processes were linked to the experience of peer victimization rather than to the current diagnostic status of the participants. Attentional avoidance of emotional stimuli may prevent victimized individuals from responding adequately to environmental stimuli, which may increase the risk for the development of psychopathology.

BackgroundAltered bile acid (BA) turnover has been suggested to be involved in the improved glucose regulation after Roux-en-Y gastric bypass (RYGB), possibly via stimulation of GLP-1 secretion. We investigated the role of exogenous as well as endogenous BAs for GLP-1 secretion after RYGB by administering chenodeoxycholic acid (CDCA) and the BA sequestrant colesevelam (COL) both in the presence and the absence of a meal stimulus.MethodsTwo single-blinded randomized cross-over studies were performed. In study 1, eight RYGB operated participants ingested 200 ml water with 1) CDCA 1.25 g or 2) CDCA 1.25 g + colesevelam 3.75 g on separate days. In study 2, twelve RYGB participants ingested on separate days a mixed meal with addition of 1) CDCA 1.25 g, 2) COL 3.75 g or 3) COL 3.75 g × 2, or 4) no additions.ResultsIn study 1, oral intake of CDCA increased circulating BAs, GLP-1, C-peptide, glucagon, and neurotensin. Addition of colesevelam reduced all responses. In study 2, addition of CDCA enhanced meal-induced increases in plasma GLP-1, glucagon and FGF-19 and lowered plasma glucose and C-peptide concentrations, while adding colesevelam lowered circulating BAs but did not affect meal-induced changes in plasma glucose or measured gastrointestinal hormones.ConclusionIn RYGB-operated persons, exogenous CDCA enhanced meal-stimulated GLP-1 and glucagon secretion but not insulin secretion, while the BA sequestrant colesevelam decreased CDCA-stimulated GLP-1 secretion but did not affect meal-stimulated GLP-1, C-peptide or glucagon secretion, or glucose tolerance. These findings suggest a limited role for endogenous bile acids in the acute regulation of postprandial gut hormone secretion or glucose metabolism after RYGB.

Somatostatin (SS) inhibits glucagon-like peptide-1 (GLP-1) secretion in a paracrine manner. We hypothesized that blocking somatostatin subtype receptor 2 (SSTR2) and 5 (SSTR5) would improve glycemia by enhancing GLP-1 secretion. In the perfused mouse small intestine, the selective SSTR5 antagonist (SSTR5a) stimulated glucose-induced GLP-1 secretion to a larger degree than the SSTR2 antagonist (SSTR2a). In parallel, mice lacking the SSTR5R showed increased glucose-induced GLP-1 secretion. Both antagonists improved glycemia in vivo in a GLP-1 receptor–dependent (GLP-1R–dependent) manner, as the glycemic improvements were absent in mice with impaired GLP-1R signaling and in mice treated with a GLP-1R–specific antagonist. SSTR5a had no direct effect on insulin secretion in the perfused pancreas, whereas SSTR2a increased insulin secretion in a GLP-1R–independent manner. Adding a dipeptidyl peptidase 4 inhibitor (DPP-4i) in vivo resulted in additive effects on glycemia. However, when glucose was administered intraperitoneally, the antagonist was incapable of lowering blood glucose. Oral administration of SSTR5a, but not SSTR2a, lowered blood glucose in diet-induced obese mice. In summary, we demonstrate that selective SSTR antagonists can improve glucose control primarily through the intestinal GLP-1 system in mice.

Objective: Glucagon is well known to regulate blood glucose but may be equally important for amino acid metabolism. Plasma levels of amino acids are regulated by glucagon-dependent mechanism(s), while amino acids stimulate glucagon secretion from alpha cells, completing the recently described liver-alpha cell axis. The mechanisms underlying the cycle and the possible impact of hepatic steatosis are unclear. Methods: We assessed amino acid clearance in vivo in mice treated with a glucagon receptor antagonist (GRA), transgenic mice with 95% reduction in alpha cells, and mice with hepatic steatosis. In addition, we evaluated urea formation in primary hepatocytes from ob/ob mice and humans, and we studied acute metabolic effects of glucagon in perfused rat livers. We also performed RNA sequencing on livers from glucagon receptor knock-out mice and mice with hepatic steatosis. Finally, we measured individual plasma amino acids and glucagon in healthy controls and in two independent cohorts of patients with biopsy-verified non-alcoholic fatty liver disease (NAFLD). Results: Amino acid clearance was reduced in mice treated with GRA and mice lacking endogenous glucagon (loss of alpha cells) concomitantly with reduced production of urea. Glucagon administration markedly changed the secretion of rat liver metabolites and within minutes increased urea formation in mice, in perfused rat liver, and in primary human hepatocytes. Transcriptomic analyses revealed that three genes responsible for amino acid catabolism (Cps1, Slc7a2, and Slc38a2) were downregulated both in mice with hepatic steatosis and in mice with deletion of the glucagon receptor. Cultured ob/ob hepatocytes produced less urea upon stimulation with mixed amino acids, and amino acid clearance was lower in mice with hepatic steatosis. Glucagon-induced ureagenesis was impaired in perfused rat livers with hepatic steatosis. Patients with NAFLD had hyperglucagonemia and increased levels of glucagonotropic amino acids, including alanine in particular. Both glucagon and alanine levels were reduced after diet-induced reduction in Homeostatic Model Assessment for Insulin Resistance (HOMA-IR, a marker of hepatic steatosis). Conclusions: Glucagon regulates amino acid metabolism both non-transcriptionally and transcriptionally. Hepatic steatosis may impair glucagon-dependent enhancement of amino acid catabolism.

Obesity and obesity-related diseases are major public health concerns that have been exponentially growing in the last decades. Bariatric surgery is an effective long-term treatment to achieve weight loss and obesity comorbidity remission. Post-bariatric hypoglycemia (PBH) is a late complication of bariatric surgery most commonly reported after Roux-en-Y gastric bypass (RYGB). PBH is the end result of postprandial hyperinsulinemia but additional endocrine mechanisms involved are still under debate. Our aim was to characterize entero-pancreatic hormone dynamics associated with postprandial hypoglycemia after RYGB. Individuals previously submitted to RYGB (N=23) in a single tertiary hospital presenting PBH symptoms (Sym, n=14) and asymptomatic weight-matched controls (Asy, n=9) were enrolled. Participants underwent a mixed-meal tolerance test (MMTT) to assess glucose, total amino acids (total AA), insulin, C-peptide, glucagon, glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), and neurotensin (NT). We found that hypoglycemia during the MMTT was equally frequent in Sym and Asy groups (p=1.000). Re-grouped according to glucose nadir during the MMTT (Hypo n=11 vs NoHypo n=12; nadir 0.05), despite distinct late glycemic outcomes (t=60 min and t=90 min: p0.05). In sum, after RYGB, postprandial hyperinsulinemia is key in triggering PBH, but a parallel and earlier rise in endogenous glucagon might sustain the inter-individual variability in glycemic outcome beyond the effect of hyperinsulinism, advocating a potential pivotal role for glucagon in preventing hyperinsulinemic hypoglycemia.

Introduction The aim of this study is to investigate the effects of time-restricted eating (TRE) on change in body weight and describe changes in behaviour and metabolism in individuals at high risk of type 2 diabetes.Methods and analysis The REStricted Eating Time (RESET) study is a randomised controlled parallel-group open-label trial. 100 women and men with (1) overweight (body mass index (BMI)≥25 kg/m2) and prediabetes (glycated haemoglobin 39–47 mmol/mol); or (2) obesity (BMI≥30 kg/m2) will be randomised to a control group (habitual living) or TRE (self-selected 10-hours eating window within the period from 06:00 to 20:00 in a 1:1 ratio. Testing is scheduled at baseline and after 6 weeks (mid-intervention), 3 months (post-intervention) and 6 months (follow-up). The primary outcome is change in body weight after 3 months of intervention. Secondary outcomes include changes in body composition; measures of glucose metabolism including glycaemic variability, hormones and metabolites; subjective and metabolic markers of appetite, food preferences and reward; dietary intake; physical activity, sleep, chronotype; gastric emptying, gastrointestinal transit time and motility; respiratory and glycolytic capacities; the plasma proteome and metabolome; blood pressure, resting heart rate and heart rate variability; and resting energy expenditure and substrate oxidation. Motivation and feasibility will be examined based on interviews at baseline and after 3 months. After the 3-month intervention, a 3-month follow-up period and subsequent testing are scheduled to assess maintenance and longer-term effects.Ethics and dissemination The study has been approved by the Ethics Committee of the Capital Region of Denmark (H-18059188) and the Danish Data Protection Agency. The study will be conducted in accordance with the Declaration of Helsinki. Results from the study will address whether TRE is effective and feasible in improving health outcomes in individuals at risk of lifestyle-related diseases and can potentially inform the design of feasible health recommendations.Trial registration number NCT03854656.

Summary: Although thousands of solid tumors have been sequenced to date, a fundamental under-sampling bias is inherent in current methodologies. This is caused by a tissue sample input of fixed dimensions (e.g., 6 mm biopsy), which becomes grossly under-powered as tumor volume scales. Here, we demonstrate representative sequencing (Rep-Seq) as a new method to achieve unbiased tumor tissue sampling. Rep-Seq uses fixed residual tumor material, which is homogenized and subjected to next-generation sequencing. Analysis of intratumor tumor mutation burden (TMB) variability shows a high level of misclassification using current single-biopsy methods, with 20% of lung and 52% of bladder tumors having at least one biopsy with high TMB but low clonal TMB overall. Misclassification rates by contrast are reduced to 2% (lung) and 4% (bladder) when a more representative sampling methodology is used. Rep-Seq offers an improved sampling protocol for tumor profiling, with significant potential for improved clinical utility and more accurate deconvolution of clonal structure.

Using data from the European Value Survey, covering more than 300,000 respondents in 32 countries between 2002 and 2012, we offer new insight into the consequences for subjective well-being of self-employment. We hypothesize that the positive link between entrepreneurship and well-being is influenced by the extent to which the decision to engage in entrepreneurship reflects voluntary choice and by the ability of the entrepreneur to match entrepreneurial preferences for autonomy, task variety, and challenging tasks to task environments. While the hypotheses are confirmed by our empirical analysis, we also find-rather surprisingly-no evidence that the effects are mediated by autonomy. To handle the endogeneity and simultaneity problems that arise from the fact that the choice to become an entrepreneur is not random and which potentially threaten the validity of our findings, we rely on a novel econometric method which allows us to sidestep the selection problem and establish that the well-being increase associated with entering into entrepreneurial activity is at least approximately equivalent to a one-decile increase in household income.

Cell-in-cell (CIC) structures are frequently observed in cancers; however, it is unclear how their formation contributes to tumorigenesis. Here, the authors show that the high frequency of CIC is linked to mutant p53 status and CIC containing p53 mutant cancer cells are pro-tumorigenic due to enhanced genomic instability.

Objective: Bile acids (BAs) facilitate fat absorption and may play a role in glucose and metabolism regulation, stimulating the secretion of gut hormones. The relative importance and mechanisms involved in BA-stimulated secretion of appetite and metabolism regulating hormones from the gut and pancreas is not well described and was the purpose of this study. Methods: The effects of bile acids on the secretion of gut and pancreatic hormones was studied in rats and compared to the most well described nutritional secretagogue: glucose. The molecular mechanisms that underlie the secretion was studied by isolated perfused rat and mouse small intestine and pancreas preparations and supported by immunohistochemistry, expression analysis, and pharmacological studies. Results: Bile acids robustly stimulate secretion of not only the incretin hormones, glucose-dependent insulinotropic peptide (GIP), and glucagon-like peptide-1 (GLP-1), but also glucagon and insulin in vivo, to levels comparable to those resulting from glucose stimulation. The mechanisms of GLP-1, neurotensin, and peptide YY (PYY) secretion was secondary to intestinal absorption and depended on activation of basolateral membrane Takeda G-protein receptor 5 (TGR5) receptors on the L-cells in the following order of potency: Lithocholic acid (LCA) >Deoxycholicacid (DCA)>Chenodeoxycholicacid (CDCA)> Cholic acid (CA). Thus BAs did not stimulate secretion of GLP-1 and PYY from perfused small intestine in TGR5 KO mice but stimulated robust responses in wild type littermates. TGR5 is not expressed on α-cells or β-cells, and BAs had no direct effects on glucagon or insulin secretion from the perfused pancreas. Conclusion: BAs should be considered not only as fat emulsifiers but also as important regulators of appetite- and metabolism-regulating hormones by activation of basolateral intestinal TGR5. Keywords: Bile-acids, GLP-1, Neurotensin, Insulin, PYY, TGR5

Roux-en-Y Gastric bypass surgery (RYGB) is emerging as a powerful tool for treatment of obesity and may also cause remission of type 2 diabetes. However, the molecular mechanism of RYGB leading to diabetes remission independent of weight loss remains elusive. In this study, we profiled plasma metabolites and proteins of 10 normal glucose-tolerant obese (NO) and 9 diabetic obese (DO) patients before and 1-week, 3-months, 1-year after RYGB. 146 proteins and 128 metabolites from both NO and DO groups at all four stages were selected for further analysis. By analyzing a set of bi-molecular associations among the corresponding network of the subjects with our newly developed computational method, we defined the represented physiological states (called the edge-states that reflect the interactions among the bio-molecules), and the related molecular networks of NO and DO patients, respectively. The principal component analyses (PCA) revealed that the edge states of the post-RYGB NO subjects were significantly different from those of the post-RYGB DO patients. Particularly, the time-dependent changes of the molecular hub-networks differed between DO and NO groups after RYGB. In conclusion, by developing molecular network-based systems signatures, we for the first time reveal that RYGB generates a unique path for diabetes remission independent of weight loss.

Activated PI3K causes cancer, but the role of active PI3K mutations in early stages of malignancy are unclear. Here, the authors show in a mouse model that active PI3K induces centrosome amplification via AKT, ROCK, CDK2/Cyclin E and nucleophosmin, and increased tolerance of genome doubling.

Summary: Glucagon is secreted from pancreatic α cells, and hypersecretion (hyperglucagonemia) contributes to diabetic hyperglycemia. Molecular heterogeneity in hyperglucagonemia is poorly investigated. By screening human plasma using high-resolution-proteomics, we identified several glucagon variants, among which proglucagon 1-61 (PG 1-61) appears to be the most abundant form. PG 1-61 is secreted in subjects with obesity, both before and after gastric bypass surgery, with protein and fat as the main drivers for secretion before surgery, but glucose after. Studies in hepatocytes and in β cells demonstrated that PG 1-61 dose-dependently increases levels of cAMP, through the glucagon receptor, and increases insulin secretion and protein levels of enzymes regulating glycogenolysis and gluconeogenesis. In rats, PG 1-61 increases blood glucose and plasma insulin and decreases plasma levels of amino acids in vivo. We conclude that glucagon variants, such as PG 1-61, may contribute to glucose regulation by stimulating hepatic glucose production and insulin secretion. : Wewer Albrechtsen et al. identify a glucagon-like molecule, PG 1-61, in humans using mass-spectrometry-based proteomics. PG 1-61 activates the glucagon receptor, stimulates insulin secretion, and activates key gluconeogenic enzymes. In dysmetabolic conditions, PG 1-61 is upregulated and may therefore serve as a marker of alpha cell stress. Keywords: glucagon, GLP-1, alpha cells, diabetes, proglucagon, L-cells, proteomics

Abstract Intra-tumour genetic heterogeneity (ITH) fosters drug resistance and is a critical hurdle to clinical treatment. ITH can be well-measured using multi-region sampling but this is costly and challenging to implement. There is therefore a need for tools to estimate ITH in individual samples, using standard genomic data such as SNP-arrays, that could be implemented routinely. We designed two novel scores S and R, respectively based on the Shannon diversity index and Ripley’s L statistic of spatial homogeneity, to quantify ITH in single SNP-array samples. We created in-silico and in-vitro mixtures of tumour clones, in which diversity was known for benchmarking purposes. We found significant but highly-variable associations of our scores with diversity in-silico (p

Abstract Background Medical education is a cornerstone in the global combat against diseases such as diabetes and obesity which together affect more than 500 million humans. Massive Open Online Courses (MOOCs) are educational tools for institutions to teach and share their research worldwide. Currently, millions of people have participated in evidence-based MOOCs, however educational and professional benefit(s) for course participants of such initiatives have not been addressed sufficiently. We therefore investigated if participation in a 6 week open online course in the prevention and treatment of diabetes and obesity had any impact on the knowledge, skills, and career of health care professionals contrasting participants from developing countries versus developed countries. Methods 52.006 participants signed up and 29.469 participants were active in one of the three sessions (2014–2015) of Diabetes - a Global Challenge. Using an online based questionnaire (nine sections) software (Survey Monkey), email invitations were send out using a Coursera based database to the 29.469 course participants. Responses were analyzed and stratified, according to the United Nations stratification method, by developing and developed countries. Results 1.303 (4.4%) of the 29.469 completed the questionnaire. 845 of the 1303 were defined as health care professionals, including medical doctors (34%), researchers (15%), nurses (11%) and medical students (8%). Over 80% of the health care participants report educational benefits, improved knowledge about the prevention and treatment therapies of diabetes and furthermore improved professional life and practice. Over 40% reported that their professional network expanded after course participation. Study participants who did not complete all modules of the course reported similar impact as the ones that completed the entire course(P = 0.9). Participants from developing countries gained more impact on their clinical practice (94%) compared to health care professionals from developed regions (88%) (Mean of differences = 6%, P = 0.03. Conclusions Based on self-reports from course participants, MOOC based medical education seems promising with respect to providing accessible and free research-based education to health professionals in both developing and developed countries. Course participants from developing countries report more benefits from course participation than their counterparts in the developed world.

Abstract Sustained weight loss is a preferred intervention in a wide range of metabolic conditions, but the effects on an individual's health state remain ill‐defined. Here, we investigate the plasma proteomes of a cohort of 43 obese individuals that had undergone 8 weeks of 12% body weight loss followed by a year of weight maintenance. Using mass spectrometry‐based plasma proteome profiling, we measured 1,294 plasma proteomes. Longitudinal monitoring of the cohort revealed individual‐specific protein levels with wide‐ranging effects of losing weight on the plasma proteome reflected in 93 significantly affected proteins. The adipocyte‐secreted SERPINF1 and apolipoprotein APOF1 were most significantly regulated with fold changes of −16% and +37%, respectively (P

Glucagon is secreted from the pancreatic alpha cells upon hypoglycemia and stimulates hepatic glucose production. Type 2 diabetes is associated with dysregulated glucagon secretion, and increased glucagon concentrations contribute to the diabetic hyperglycemia. Antagonists of the glucagon receptor have been considered as glucose-lowering therapy in type 2 diabetes patients, but their clinical applicability has been questioned because of reports of therapy-induced increments in liver fat content and increased plasma concentrations of low-density lipoprotein. Conversely, in animal models, increased glucagon receptor signaling has been linked to improved lipid metabolism. Glucagon acts primarily on the liver and by regulating hepatic lipid metabolism glucagon may reduce hepatic lipid accumulation and decrease hepatic lipid secretion. Regarding whole-body lipid metabolism, it is controversial to what extent glucagon influences lipolysis in adipose tissue, particularly in humans. Glucagon receptor agonists combined with glucagon-like peptide 1 receptor agonists (dual agonists) improve dyslipidemia and reduce hepatic steatosis. Collectively, emerging data support an essential role of glucagon for lipid metabolism.

Glucagon stimulates hepatic glucose production by activating specific glucagon receptors in the liver, which in turn increase hepatic glycogenolysis as well as gluconeogenesis and ureagenesis from amino acids. Conversely, glucagon secretion is regulated by concentrations of glucose and amino acids. Disruption of glucagon signaling in rodents results in grossly elevated circulating glucagon levels but no hypoglycemia. Here, we describe a patient carrying a homozygous G to A substitution in the invariant AG dinucleotide found in a 3′ mRNA splice junction of the glucagon receptor gene. Loss of the splice site acceptor consensus sequence results in the deletion of 70 nucleotides encoded by exon 9, which introduces a frame shift and an early termination signal in the receptor mRNA sequence. The mutated receptor neither bound 125I-labeled glucagon nor induced cAMP production upon stimulation with up to 1 μM glucagon. Despite the mutation, the only obvious pathophysiological trait was hyperglucagonemia, hyperaminoacidemia and massive hyperplasia of the pancreatic α-cells assessed by histology. Our case supports the notion of a hepato–pancreatic feedback system, which upon disruption leads to hyperglucagonemia and α-cell hyperplasia, as well as elevated plasma amino acid levels. Together with the glucagon-induced hypoaminoacidemia in glucagonoma patients, our case supports recent suggestions that amino acids may provide the feedback link between the liver and the pancreatic α-cells.

Low-abundance regulatory peptides, including metabolically important gut hormones, have shown promising therapeutic potential. Here, we present a streamlined mass spectrometry-based platform for identifying and characterizing low-abundance regulatory peptides in humans. We demonstrate the clinical applicability of this platform by studying a hitherto neglected glucose- and appetite-regulating gut hormone, namely, oxyntomodulin. Our results show that the secretion of oxyntomodulin in patients with type 2 diabetes is significantly impaired, and that its level is increased by more than 10-fold after gastric bypass surgery. Furthermore, we report that oxyntomodulin is co-distributed and co-secreted with the insulin-stimulating and appetite-regulating gut hormone glucagon-like peptide-1 (GLP-1), is inactivated by the same protease (dipeptidyl peptidase-4) as GLP-1 and acts through its receptor. Thus, oxyntomodulin may participate with GLP-1 in the regulation of glucose metabolism and appetite in humans. In conclusion, this mass spectrometry-based platform is a powerful resource for identifying and characterizing metabolically active low-abundance peptides.

ABSTRACT I briefly take stock on the microfoundations project which has become influential in macro-management research over the past decade or so. While the project has now moved into distinct theory-building, it still need to engage in serious empirical research. I discuss a number of challenges and solutions associated with the empirical side of microfoundations.

Background The mechanism underlying the beneficial cardiovascular effects of the incretin GLP‐1 (glucagon‐like peptide 1) and its analogues in humans is elusive. We hypothesized that activating receptors located on vascular smooth muscle cells to induce either peripheral or coronary vasodilatation mediates the cardiovascular effect of GLP‐1. Methods and Results Ten stable patients with angina awaiting left anterior descending artery stenting underwent forearm blood flow measurement using forearm plethysmography and post–percutaneous coronary intervention coronary blood flow measurement using a pressure‐flow wire before and after peripheral GLP‐1 administration. Coronary sinus and artery bloods were sampled for GLP‐1 levels. A further 11 control patients received saline rather than GLP‐1 in the coronary blood flow protocol. GLP‐1 receptor (GLP‐1R) expression was assessed by immunohistochemistry using a specific GLP‐1R monoclonal antibody in human tissue to inform the physiological studies. There was no effect of GLP‐1 on absolute forearm blood flow or forearm blood flow ratio after GLP‐1, systemic hemodynamics were not affected, and no binding of GLP‐1R monoclonal antibody was detected in vascular tissue. GLP‐1 reduced resting coronary transit time (mean [SD], 0.87 [0.39] versus 0.63 [0.27] seconds; P=0.02) and basal microcirculatory resistance (mean [SD], 76.3 [37.9] versus 55.4 [30.4] mm Hg/s; P=0.02), whereas in controls, there was an increase in transit time (mean [SD], 0.48 [0.24] versus 0.83 [0.41] seconds; P