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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

Scholars have long debated the merits of advocacy-based research versus research considered from the quest for objective truth. Building upon reflections from multiple sources, a set of 11 brief reflections on three posed questions are presented. Tsang concludes our discussion with additional insights on how moving beyond the “interestingness” advocacy will be beneficial to the continued professional development of the management discipline.

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.

Chromosomal instability in cancer consists of dynamic changes to the number and structure of chromosomes1,2. The resulting diversity in somatic copy number alterations (SCNAs) may provide the variation necessary for tumour evolution1,3,4. Here we use multi-sample phasing and SCNA analysis of 1,421 samples from 394 tumours across 22 tumour types to show that continuous chromosomal instability results in pervasive SCNA heterogeneity. Parallel evolutionary events, which cause disruption in the same genes (such as BCL9, MCL1, ARNT (also known as HIF1B), TERT and MYC) within separate subclones, were present in 37% of tumours. Most recurrent losses probably occurred before whole-genome doubling, that was found as a clonal event in 49% of tumours. However, loss of heterozygosity at the human leukocyte antigen (HLA) locus and loss of chromosome 8p to a single haploid copy recurred at substantial subclonal frequencies, even in tumours with whole-genome doubling, indicating ongoing karyotype remodelling. Focal amplifications that affected chromosomes 1q21 (which encompasses BCL9, MCL1 and ARNT), 5p15.33 (TERT), 11q13.3 (CCND1), 19q12 (CCNE1) and 8q24.1 (MYC) were frequently subclonal yet appeared to be clonal within single samples. Analysis of an independent series of 1,024 metastatic samples revealed that 13 focal SCNAs were enriched in metastatic samples, including gains in chromosome 8q24.1 (encompassing MYC) in clear cell renal cell carcinoma and chromosome 11q13.3 (encompassing CCND1) in HER2+ breast cancer. Chromosomal instability may enable the continuous selection of SCNAs, which are established as ordered events that often occur in parallel, throughout tumour evolution.

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 ).

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 FROM AUTHOR]

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.