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Abstract Background First-degree relatives of type 2 diabetics (FDR) exhibit a high risk of developing type 2 diabetes (T2D) and feature subcutaneous adipocyte hypertrophy, independent of obesity. In FDR, adipose cell abnormalities contribute to early insulin-resistance and are determined by adipocyte precursor cells (APCs) early senescence and impaired recruitment into the adipogenic pathway. Epigenetic mechanisms signal adipocyte differentiation, leading us to hypothesize that abnormal epigenetic modifications cause adipocyte dysfunction and enhance T2D risk. To test this hypothesis, we examined the genome-wide histone profile in APCs from the subcutaneous adipose tissue of healthy FDR. Results Sequencing-data analysis revealed 2644 regions differentially enriched in lysine 4 tri-methylated H3-histone (H3K4me3) in FDR compared to controls (CTRL) with significant enrichment in mitochondrial-related genes. These included TFAM, which regulates mitochondrial DNA (mtDNA) content and stability. In FDR APCs, a significant reduction in H3K4me3 abundance at the TFAM promoter was accompanied by a reduction in TFAM mRNA and protein levels. FDR APCs also exhibited reduced mtDNA content and mitochondrial-genome transcription. In parallel, FDR APCs exhibited impaired differentiation and TFAM induction during adipogenesis. In CTRL APCs, TFAM-siRNA reduced mtDNA content, mitochondrial transcription and adipocyte differentiation in parallel with upregulation of the CDKN1A and ZMAT3 senescence genes. Furthermore, TFAM-siRNA significantly expanded hydrogen peroxide (H2O2)-induced senescence, while H2O2 did not affect TFAM expression. Conclusions Histone modifications regulate APCs ability to differentiate in mature cells, at least in part by modulating TFAM expression and affecting mitochondrial function. Reduced H3K4me3 enrichment at the TFAM promoter renders human APCs senescent and dysfunctional, increasing T2D risk. Graphical abstract

Abstract The association between type 2 diabetes (T2D) and the development of cardiac arrhythmias and conduction disturbances has not been extensively studied. Arrhythmia was defined as atrial fibrillation and flutter (AF/AFl), ventricular tachycardia (VT) and ventricular fibrillation (VF), and conduction abnormality as sinus node disease (SND), atrioventricular (AV) block or pacemaker implantation, and intraventricular conduction blocks (IVCB). Incidence rates and Cox regression were used to compare outcomes, and to assess optimal levels for cardiometabolic risk factors and risk associated with multifactorial risk factor control (i.e., HbA1c, LDL-C, systolic blood pressure (SBP), BMI and eGFR), between patients with versus without T2D. The analyses included data from 617,000 patients with T2D and 2,303,391 matched controls. Patients with diabetes and the general population demonstrated a gradual increase in rates for cardiac conduction abnormalities and virtually all age-groups for AF/AFI showed increased incidence during follow-up. For patients with versus without T2D, risks for cardiac arrhythmias were higher, including for AF/AFl (HR 1.17, 95% CI 1.16–1.18), the composite of SND, AV-block or pacemaker implantation (HR 1.40, 95% CI 1.37–1.43), IVCB (HR 1.23, 95% CI 1.18–1.28) and VT/VF (HR 1.08, 95% CI 1.04–1.13). For patients with T2D who had selected cardiometabolic risk factors within target ranges, compared with controls, risk of arrythmia and conduction abnormalities for T2D vs not were: AF/AFl (HR 1.09, 95% CI 1.05–1.14), the composite of SND, AV-block or pacemaker implantation (HR 1.06, 95% CI 0.94–1.18), IVCB (HR 0.80, 95% CI 0.60–0.98), and for VT/VF (HR 0.97, 95% CI 0.80–1.17). Cox models showed a linear risk increase for SBP and BMI, while eGFR showed a U-shaped association. Individuals with T2D had a higher risk of arrhythmias and conduction abnormalities than controls, but excess risk associated with T2D was virtually not evident among patients with T2D with all risk factors within target range. BMI, SBP and eGFR displayed significant associations with outcomes among patients with T2D.

Cell senescence (CS) is at the nexus between aging and associated chronic disorders, and aging increases the burden of CS in all major metabolic tissues. However, CS is also increased in adult obesity, type 2 diabetes (T2D), and nonalcoholic fatty liver disease independent of aging. Senescent tissues are characterized by dysfunctional cells and increased inflammation, and both progenitor cells and mature, fully differentiated and nonproliferating cells are afflicted. Recent studies have shown that hyperinsulinemia and associated insulin resistance (IR) promote CS in both human adipose and liver cells. Similarly, increased CS promotes cellular IR, showing their interdependence. Furthermore, the increased adipose CS in T2D is independent of age, BMI, and degree of hyperinsulinemia, suggesting premature aging. These results suggest that senomorphic/senolytic therapy may become important for treating these common metabolic disorders.

Objective: Cellular senescence, an irreversible proliferative cell arrest, is caused by excessive intracellular or extracellular stress/damage. Increased senescent cells have been identified in multiple tissues in different metabolic and other aging-related diseases. Recently, several human and mouse studies emphasized the involvement of senescence in development and progression of NAFLD. Hyperinsulinemia, seen in obesity, metabolic syndrome, and other conditions of insulin resistance, has been linked to senescence in adipocytes and neurons. Here, we investigate the possible direct role of chronic hyperinsulinemia in the development of senescence in human hepatocytes. Methods: Using fluorescence microscopy, immunoblotting, and gene expression, we tested senescence markers in human hepatocytes subjected to chronic hyperinsulinemia in vitro and validated the data in vivo by using liver-specific insulin receptor knockout (LIRKO) mice. The consequences of hyperinsulinemia were also studied in senescent hepatocytes following doxorubicin as a model of stress-induced senescence. Furthermore, the effects of senolytic agents in insulin- and doxorubicin-treated cells were analyzed. Results: Results showed that exposing the hepatocytes to prolonged hyperinsulinemia promotes the onset of senescence by increasing the expression of p53 and p21. It also further enhanced the senescent phenotype in already senescent hepatocytes. Addition of insulin signaling pathway inhibitors prevented the increase in cell senescence, supporting the direct contribution of insulin. Furthermore, LIRKO mice, in which insulin signaling in the liver is abolished due to deletion of the insulin receptor gene, showed no differences in senescence compared to their wild-type counterparts despite having marked hyperinsulinemia indicating these are receptor-mediated effects. In contrast, the persistent hyperinsulinemia in LIRKO mice enhanced senescence in white adipose tissue. In vitro, senolytic agents dasatinib and quercetin reduced the prosenescent effects of hyperinsulinemia in hepatocytes. Conclusion: Our findings demonstrate a direct link between chronic hyperinsulinemia and hepatocyte senescence. This effect can be blocked by reducing the levels of insulin receptors or administration of senolytic drugs, such as dasatinib and quercetin.

Abstract The study of metabolomics has improved our knowledge of the biology behind type 2 diabetes and its related metabolic physiology. We aimed to investigate markers of adipose tissue morphology, as well as insulin and glucose metabolism in 53 non-obese male individuals. The participants underwent extensive clinical, biochemical and magnetic resonance imaging phenotyping, and we also investigated non-targeted serum metabolites. We used a multi-modal machine learning approach to evaluate which serum metabolomic compounds predicted markers of glucose and insulin metabolism, adipose tissue morphology and distribution. Fasting glucose was associated with metabolites of intracellular insulin action and beta-cell dysfunction, namely cysteine-s-sulphate and n-acetylgarginine, whereas fasting insulin was predicted by myristoleoylcarnitine, propionylcarnitine and other metabolites of beta-oxidation of fatty acids. OGTT-glucose levels at 30 min were predicted by 7-Hoca, a microbiota derived metabolite, as well as eugenol, a fatty acid. Both insulin clamp and HOMA-IR were predicted by metabolites involved in beta-oxidation of fatty acids and biodegradation of triacylglycerol, namely tartrate and 3-phosphoglycerate, as well as pyruvate, xanthine and liver fat. OGTT glucose area under curve (AUC) and OGTT insulin AUC, was associated with bile acid metabolites, subcutaneous adipocyte cell size, liver fat and fatty chain acids and derivates, such as isovalerylcarnitine. Finally, subcutaneous adipocyte size was associated with long chain fatty acids, markers of sphingolipid metabolism, increasing liver fat and dopamine-sulfate 1. Ectopic liver fat was predicted by methylmalonate, adipocyte cell size, glutathione derived metabolites and fatty chain acids. Ectopic heart fat was predicted visceral fat, gamma-glutamyl tyrosine and 2-acetamidophenol sulfate. Adipocyte cell size, age, alpha-tocopherol and blood pressure were associated with visceral fat. We identified several biomarkers associated with adipose tissue pathophysiology and insulin and glucose metabolism using a multi-modal machine learning approach. Our approach demonstrated the relative importance of serum metabolites and they outperformed traditional clinical and biochemical variables for most endpoints.

Cellular senescence is a state of irreversible cell cycle arrest and has been shown to play a key role in many diseases, including metabolic diseases. To investigate the potential contribution of hepatocyte cellular senescence to the metabolic derangements associated with non-alcoholic steatohepatitis (NASH), we treated human hepatocyte cell lines HepG2 and IHH with the senescence-inducing drugs nutlin-3a, doxorubicin and etoposide. The senescence-associated markers p16, p21, p53 and beta galactosidase were induced upon drug treatment, and this was associated with increased lipid storage, increased expression of lipid transporters and the development of hepatic steatosis. Drug-induced senescence also led to increased glycogen content, and increased VLDL secretion from hepatocytes. Senescence was also associated with an increase in glucose and fatty acid oxidation capacity, while de novo lipogenesis was decreased. Surprisingly, cellular senescence caused an overall increase in insulin signaling in hepatocytes, with increased insulin-stimulated phosphorylation of IR, Akt, and MAPK. Together, these data indicate that hepatic senescence plays a causal role in the development of NASH pathogenesis, by modulating glucose and lipid metabolism, favoring steatosis. Our findings contribute to a better understanding of the mechanisms linking cellular senescence and fatty liver disease and support the development of new therapies targeting senescent cells for the treatment of NASH.

Objective: Bone morphogenetic protein 4 (BMP4) adeno-associated viral vectors of serotype 8 (AAV8) gene therapy targeting the liver prevents the development of obesity in initially lean mice by browning the large subcutaneous white adipose tissue (WAT) and enhancing energy expenditure. Here, we examine whether this approach could also reduce established obesity. Methods: Dietary-induced obese C57BL6/N mice received AAV8 BMP4 gene therapy at 17–18 weeks of age. They were kept on a high-fat diet and phenotypically characterized for an additional 10–12 weeks. Following termination, the mice underwent additional characterization in vitro. Results: Surprisingly, we observed no effect on body weight, browning of WAT, or energy expenditure in these obese mice, but whole-body insulin sensitivity and glucose tolerance were robustly improved. Insulin signaling and insulin-stimulated glucose uptake were increased in both adipose cells and skeletal muscle. BMP4 also decreased hepatic glucose production and reduced gluconeogenic enzymes in the liver, but not in the kidney, in addition to enhancing insulin action in the liver. Conclusions: Our findings show that BMP4 prevents, but does not reverse, established obesity in adult mice, while it improves insulin sensitivity independent of weight reduction. The BMP antagonist Noggin was increased in WAT in obesity, which may account for the lack of browning. Keywords: Obesity, Gene therapy, Insulin sensitivity, Adipose tissue, Skeletal muscle, Liver

Adipose tissue hypertrophy in obesity is associated with insulin resistance and other metabolic complications. Here, the authors analyze subcutaneous adipose tissue from patients with hypertrophic obesity and insulin resistance and find that adipocyte progenitor cells show features of senescence and have poor differentiation capacity.

ObjectiveGremlin 1 (GREM1) is a secreted BMP2/4 inhibitor which regulates commitment and differentiation of human adipose precursor cells and prevents the browning effect of BMP4. GREM1 is an insulin antagonist and serum levels are high in type 2 diabetes (T2D). We here examined in vivo effects of AAV8 (Adeno-Associated Viral vectors of serotype eight) GREM 1 targeting the liver in mature mice to increase its systemic secretion and also, in a separate study, injected recombinant GREM 1 intraperitoneally. The objective was to characterize systemic effects of GREM 1 on insulin sensitivity, glucose tolerance, body weight, adipose cell browning and other local tissue effects.MethodsAdult mice were injected with AAV8 vectors expressing GREM1 in the liver or receiving regular intra-peritoneal injections of recombinant GREM1 protein. The mice were fed with a low fat or high fat diet (HFD) and followed over time.ResultsLiver-targeted AAV8-GREM1 did not alter body weight, whole-body glucose and insulin tolerance, or adipose tissue gene expression. Although GREM1 protein accumulated in liver cells, GREM1 serum levels were not increased suggesting that it may not have been normally processed for secretion. Hepatic lipid accumulation, inflammation and fibrosis were also not changed. Repeated intraperitoneal rec-GREM1 injections for 5 weeks were also without effects on body weight and insulin sensitivity. UCP1 was slightly but significantly reduced in both white and brown adipose tissue but this was not of sufficient magnitude to alter body weight. We validated that recombinant GREM1 inhibited BMP4-induced pSMAD1/5/9 in murine cells in vitro, but saw no direct inhibitory effect on insulin signalling and pAkt (ser 473 and thr 308) activation.ConclusionGREM1 accumulates intracellularly when overexpressed in the liver cells of mature mice and is apparently not normally processed/secreted. However, also repeated intraperitoneal injections were without effects on body weight and insulin sensitivity and adipose tissue UCP1 levels were only marginally reduced. These results suggest that mature mice do not readily respond to GREMLIN 1 but treatment of murine cells with GREMLIN 1 protein in vitro validated its inhibitory effect on BMP4 signalling while insulin signalling was not altered.

ABSTRACT Multiple myeloma is characterized by accumulation of malignant plasma cells in the bone marrow. Most patients suffer from an osteolytic bone disease, caused by increased bone degradation and reduced bone formation. Bone morphogenetic protein 4 (BMP4) is important for both pre‐ and postnatal bone formation and induces growth arrest and apoptosis of myeloma cells. BMP4‐treatment of myeloma patients could have the potential to reduce tumor growth and restore bone formation. We therefore explored BMP4 gene therapy in a human‐mouse model of multiple myeloma where humanized bone scaffolds were implanted subcutaneously in RAG2−/− γC−/−mice. Mice were treated with adeno‐associated virus serotype 8 BMP4 vectors (AAV8‐BMP4) to express BMP4 in the liver. When mature BMP4 was detectable in the circulation, myeloma cells were injected into the scaffolds and tumor growth was examined by weekly imaging. Strikingly, the tumor burden was reduced in AAV8‐BMP4 mice compared with the AAV8‐CTRL mice, suggesting that increased circulating BMP4 reduced tumor growth. BMP4‐treatment also prevented bone loss in the scaffolds, most likely due to reduced tumor load. To delineate the effects of BMP4 overexpression on bone per se, without direct influence from cancer cells, we examined the unaffected, non‐myeloma femurs by μCT. Surprisingly, the AAV8‐BMP4 mice had significantly reduced trabecular bone volume, trabecular numbers, as well as significantly increased trabecular separation compared with the AAV8‐CTRL mice. There was no difference in cortical bone parameters between the two groups. Taken together, BMP4 gene therapy inhibited myeloma tumor growth, but also reduced the amount of trabecular bone in mice. Our data suggest that care should be taken when considering using BMP4 as a therapeutic agent. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Along with insulin resistance and increased risk of type 2 diabetes (T2D), lean first-degree relatives of T2D subjects (FDR) feature impaired adipogenesis in subcutaneous adipose tissue (SAT) and subcutaneous adipocyte hypertrophy well before diabetes onset. The molecular mechanisms linking these events have only partially been clarified. In the present report, we show that silencing of the transcription factor Homeobox A5 (HOXA5) in human preadipocytes impaired differentiation in mature adipose cells in vitro. The reduced adipogenesis was accompanied by inappropriate WNT-signaling activation. Importantly, in preadipocytes from FDR individuals, HOXA5 expression was attenuated, with hypermethylation of the HOXA5 promoter region found responsible for its downregulation, as revealed by luciferase assay. Both HOXA5 gene expression and DNA methylation were significantly correlated with SAT adipose cell hypertrophy in FDR, whose increased adipocyte size marks impaired adipogenesis. In preadipocytes from FDR, the low HOXA5 expression negatively correlated with enhanced transcription of the WNT signaling downstream genes NFATC1 and WNT2B. In silico evidence indicated that NFATC1 and WNT2B were directly controlled by HOXA5. The HOXA5 promoter region also was hypermethylated in peripheral blood leukocytes from these same FDR individuals, which was further revealed in peripheral blood leukocytes from an independent group of obese subjects. Thus, HOXA5 controlled adipogenesis in humans by suppressing WNT signaling. Altered DNA methylation of the HOXA5 promoter contributed to restricted adipogenesis in the SAT of lean subjects who were FDR of type 2 diabetics and in obese individuals.

Branched-chain amino acids (BCAAs) metabolite, 3-Hydroxyisobutyric acid (3-HIB) has been identified as a secreted mediator of endothelial cell fatty acid transport and insulin resistance (IR) using animal models. To identify if 3-HIB is a marker of human IR and future risk of developing Type 2 diabetes (T2D), we measured plasma levels of 3-HIB and associated metabolites in around 10,000 extensively phenotyped individuals. The levels of 3-HIB were increased in obesity but not robustly associated with degree of IR after adjusting for BMI. Nevertheless, also after adjusting for obesity and plasma BCAA, 3-HIB levels were associated with future risk of incident T2D. We also examined the effect of 3-HIB on fatty acid uptake in human cells and found that both HUVEC and human cardiac endothelial cells respond to 3-HIB whereas human adipose tissue-derived endothelial cells do not respond to 3-HIB. In conclusion, we found that increased plasma level of 3-HIB is a marker of future risk of T2D and 3-HIB may be important for the regulation of metabolic flexibility in heart and muscles.

Abstract We performed integrative network analyses to identify targets that can be used for effectively treating liver diseases with minimal side effects. We first generated co‐expression networks (CNs) for 46 human tissues and liver cancer to explore the functional relationships between genes and examined the overlap between functional and physical interactions. Since increased de novo lipogenesis is a characteristic of nonalcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC), we investigated the liver‐specific genes co‐expressed with fatty acid synthase (FASN). CN analyses predicted that inhibition of these liver‐specific genes decreases FASN expression. Experiments in human cancer cell lines, mouse liver samples, and primary human hepatocytes validated our predictions by demonstrating functional relationships between these liver genes, and showing that their inhibition decreases cell growth and liver fat content. In conclusion, we identified liver‐specific genes linked to NAFLD pathogenesis, such as pyruvate kinase liver and red blood cell (PKLR), or to HCC pathogenesis, such as PKLR, patatin‐like phospholipase domain containing 3 (PNPLA3), and proprotein convertase subtilisin/kexin type 9 (PCSK9), all of which are potential targets for drug development.

Summary: We examined the effect of Bone Morphogenetic Protein 4 (BMP4) on energy expenditure in adult mature mice by targeting the liver with adeno-associated viral (AAV) BMP4 vectors to increase circulating levels. We verified the direct effect of BMP4 in inducing a brown oxidative phenotype in differentiating preadipocytes in vitro. AAV-BMP4-treated mice display marked browning of subcutaneous adipocytes, with increased mitochondria and Uncoupling Protein 1 (UCP1). These mice are protected from obesity on a high-fat diet and have increased whole-body energy expenditure, improved insulin sensitivity, reduced liver fat, and reduced adipose tissue inflammation. On a control diet, they show unchanged body weight but improved insulin sensitivity. In contrast, AAV-BMP4-treated mice showed beiging of BAT with reduced UCP1, increased lipids, and reduced hormone-sensitive lipase (HSL). Thus, BMP4 exerts different effects on WAT and BAT, but the overall effect is to enhance insulin sensitivity and whole-body energy expenditure by browning subcutaneous adipose tissue. : Hoffmann et al. show that increased circulating BMP4 in mature mice targets subcutaneous WAT, promoting its browning with increased UCP1 and mitochondria and increased energy expenditure. Increased BMP4 also targets BAT, resulting in increased lipids and reduced UCP1. Together, these findings underscore the potential of browning WAT. Keywords: BMP4, white adipose tissue, brown adipose tissue, obesity, insulin resistance, glucose tolerance, energy expenditure

Abstract Background Insulin resistance (IR) has previously been associated with an increased risk of developing Alzheimer’s disease (AD), although the relationship between IR and AD is not yet clear. Here, we examined the influence of IR on AD using plasma and cerebrospinal fluid (CSF) biomarkers related to IR and AD in cognitively healthy men. We also aimed to characterise the shared protein signatures between IR and AD. Methods Fifty-eight cognitively healthy men, 28 IR and 30 non-IR (age and APOE ε4 matched), were drawn from the Metabolic Syndrome in Men study in Kuopio, Finland. CSF AD biomarkers (amyloid β-peptide (Aβ), total tau and tau phosphorylated at the Thr181 epitope) were examined with respect to IR. Targeted proteomics using ELISA and Luminex xMAP assays were performed to assess the influence of IR on previously identified CSF and plasma protein biomarker candidates of AD pathology. Furthermore, CSF and plasma SOMAscan was performed to discover proteins that associate with IR and CSF AD biomarkers. Results CSF AD biomarkers did not differ between IR and non-IR groups, although plasma insulin correlated with CSF Aβ/tau across the whole cohort. In total, 200 CSF and 487 plasma proteins were differentially expressed between IR and non-IR subjects, and significantly enriched pathways, many of which have been previously implicated in AD, were identified. CSF and plasma proteins significantly associated with CSF AD biomarkers were also discovered, and those sensitive to both IR and AD were identified. Conclusions These data indicate that IR is not directly related to the level of CSF AD pathology in cognitively healthy men. Proteins that associated with both AD and IR are potential markers indicative of shared pathology.

Abstract To elucidate the molecular mechanisms underlying non‐alcoholic fatty liver disease (NAFLD), we recruited 86 subjects with varying degrees of hepatic steatosis (HS). We obtained experimental data on lipoprotein fluxes and used these individual measurements as personalized constraints of a hepatocyte genome‐scale metabolic model to investigate metabolic differences in liver, taking into account its interactions with other tissues. Our systems level analysis predicted an altered demand for NAD+ and glutathione (GSH) in subjects with high HS. Our analysis and metabolomic measurements showed that plasma levels of glycine, serine, and associated metabolites are negatively correlated with HS, suggesting that these GSH metabolism precursors might be limiting. Quantification of the hepatic expression levels of the associated enzymes further pointed to altered de novo GSH synthesis. To assess the effect of GSH and NAD+ repletion on the development of NAFLD, we added precursors for GSH and NAD+ biosynthesis to the Western diet and demonstrated that supplementation prevents HS in mice. In a proof‐of‐concept human study, we found improved liver function and decreased HS after supplementation with serine (a precursor to glycine) and hereby propose a strategy for NAFLD treatment.

Adiposity is strongly associated with cardiovascular (CV) morbidity. Uncoupling protein 1 (UCP1) increases energy expenditure in adipocytes and may counteract adiposity. Our objective was to investigate a connection between UCP1 expression and cardiovascular health in patients with rheumatoid arthritis (RA) in a longitudinal observational study. Transcription of UCP1 was measured by qPCR in the subcutaneous adipose tissue of 125 female RA patients and analyzed with respect to clinical parameters and the estimated CV risk. Development of new CV events and diabetes mellitus was followed for five years. Transcription of UCP1 was identified in 89 (71%) patients. UCP1 positive patients had often active RA disease (p = 0.017), high serum levels of IL6 (p = 0.0025) and were frequently overweight (p = 0.015). IL-6hiBMIhi patients and patients treated with IL6 receptor inhibitor tocilizumab had significantly higher levels of UCP1 compared to other RA patients (p < 0.0001, p = 0.032, respectively). Both UCP1hi groups displayed unfavorable metabolic profiles with high plasma glucose levels and high triglyceride-to-HDL ratios, which indicated insulin resistance. Prospective follow-up revealed no significant difference in the incidence of new CV and metabolic events in the UCP1hi groups and remaining RA patients. The study shows that high transcription of UCP1 in adipose tissue is related to IL6-driven processes and reflects primarily metabolic CV risk in female RA patients.

The pre-absorptive cephalic phase of insulin secretion is elicited during the first ten min of a meal and before glucose levels rise. Its importance for insulin release during the post-absorptive phase has been well documented in animals but its presence or importance in man has become increasingly controversial. We here examined the presence of an early cephalic phase of insulin release in 31 well matched individuals without (n = 15) or with (n = 16) a known family history of type 2 diabetes (first-degree relatives; FDR). We also examined the potential differences in individuals with or without impaired fasting (IFG) and impaired glucose tolerance (IGT). We here demonstrate that a cephalic phase of insulin secretion was present in all individuals examined and without any differences between control persons and FDR or IFG/IGT. However, the overall importance of the cephalic phase is conjectural since it was unrelated to the subsequent post-absorptive insulin release or glucose tolerance. One of the best predictors of the incremental cephalic phase of insulin release was fasting insulin level and, thus, a relation to degree of insulin sensitivity is likely. In conclusion, an early pre-absorptive and cephalic phase of insulin release is robustly present in man. However, we could not document any relation to family history of Type 2 diabetes nor to the post-absorptive phase and, thus, confirm its importance for subsequent degree of insulin release or glucose tolerance.

No clinical trials have assessed the effects or cost-effectiveness of health check strategies to detect and manage vascular disease. We used a mathematical model to estimate the cost-effectiveness of several health check strategies in six European countries.We used country-specific data from Denmark, France, Germany, Italy, Poland, and the United Kingdom to generate simulated populations of individuals aged 40-75 eligible for health checks in those countries (e.g. individuals without a previous diagnosis of diabetes, myocardial infarction, stroke, or serious chronic kidney disease). For each country, we used the Archimedes model to compare seven health check strategies consisting of assessments for diabetes, hypertension, lipids, and smoking. For patients diagnosed with vascular disease, treatment was simulated in a standard manner. We calculated the effects of each strategy on the incidence of type 2 diabetes, major adverse cardiovascular events (MACE), and microvascular complications in addition to quality of life, costs, and cost per quality-adjusted life-year (QALY).Compared with current care, health checks reduced the incidence of MACE (6-17 events prevented per 1000 people screened) and diabetes related microvasular complications (5-11 events prevented per 1000 people screened), and increased QALYs (31-59 discounted QALYs) over 30 years, in all countries. The cost per QALY of offering a health check to all individuals in the study cohort ranged from €14903 (France) to cost saving (Poland). Pre-screening the population and offering health checks only to higher risk individuals lowered the cost per QALY. Pre-screening on the basis of obesity had a cost per QALY of €10200 (France) or less, and pre-screening with a non-invasive risk score was similar.A vascular disease health check would likely be cost effective at 30 years in Denmark, France, Germany, Italy, Poland, and the United Kingdom.

We investigated the levels of non-cholesterol sterols as predictors for the development of hyperglycemia (an increase in the glucose area under the curve in an oral glucose tolerance test) and incident type 2 diabetes in a 5-year follow-up study of a population-based cohort of Finnish men (METSIM Study, N = 1,050) having non-cholesterol sterols measured at baseline. Additionally we determined the association of 538,265 single nucleotide polymorphisms (SNP) with non-cholesterol sterol levels in a cross-sectional cohort of non-diabetic offspring of type 2 diabetes (the Kuopio cohort of the EUGENE2 Study, N = 273). We found that in a cross-sectional METSIM Study the levels of sterols indicating cholesterol absorption were reduced as a function of increasing fasting glucose levels, whereas the levels of sterols indicating cholesterol synthesis were increased as a function of increasing 2-hour glucose levels. A cholesterol synthesis marker desmosterol significantly predicted an increase, and two absorption markers (campesterol and avenasterol) a decrease in the risk of hyperglycemia and incident type 2 diabetes in a 5-year follow-up of the METSIM cohort, mainly attributable to insulin sensitivity. A SNP of ABCG8 was associated with fasting plasma glucose levels in a cross-sectional study but did not predict hyperglycemia or incident type 2 diabetes. In conclusion, the levels of some, but not all non-cholesterol sterols are markers of the worsening of hyperglycemia and type 2 diabetes.

BACKGROUND: Development of Type 2 diabetes, like obesity, is promoted by a genetic predisposition. Although several genetic variants have been identified they only account for a small proportion of risk. We have asked if genetic risk is associated with abnormalities in storing excess lipids in the abdominal subcutaneous adipose tissue. METHODOLOGY/PRINCIPAL FINDINGS: We recruited 164 lean and 500 overweight/obese individuals with or without a genetic predisposition for Type 2 diabetes or obesity. Adipose cell size was measured in biopsies from the abdominal adipose tissue as well as insulin sensitivity (HOMA index), HDL-cholesterol and Apo AI and Apo B. 166 additional non-obese individuals with a genetic predisposition for Type 2 diabetes underwent a euglycemic hyperinsulinemic clamp to measure insulin sensitivity. Genetic predisposition for Type 2 diabetes, but not for overweight/obesity, was associated with inappropriate expansion of the adipose cells, reduced insulin sensitivity and a more proatherogenic lipid profile in non-obese individuals. However, obesity per se induced a similar expansion of adipose cells and dysmetabolic state irrespective of genetic predisposition. CONCLUSIONS/SIGNIFICANCE: Genetic predisposition for Type 2 diabetes, but not obesity, is associated with an impaired ability to recruit new adipose cells to store excess lipids in the subcutaneous adipose tissue, thereby promoting ectopic lipid deposition. This becomes particularly evident in non-obese individuals since obesity per se promotes a dysmetabolic state irrespective of genetic predisposition. These results identify a novel susceptibility factor making individuals with a genetic predisposition for Type 2 diabetes particularly sensitive to the environment and caloric excess.

A meta-analysis combining results from three genome-wide association studies and followed by large-scale replication identified six novel type 2 diabetes loci. Subsequent studies of the effect of these variants on estimates of the beta-cell function and insulin sensitivity have been inconclusive. We examined these variants located in or near the JAZF1 (rs864745), THADA (rs7578597), TSPAN8 (rs7961581), ADAMTS9 (rs4607103), NOTCH2 (rs10923931) and the CDC123/CAMK1D (rs12779790) genes for associations with measures of pancreatic beta-cell function and insulin sensitivity.Oral and intravenous glucose stimulated insulin release (n = 849) and insulin sensitivity (n = 596) estimated from a hyperinsulinemic euglycemic clamp were measured in non-diabetic offspring of type 2 diabetic patients from five European populations. Assuming an additive genetic model the diabetes-associated major C-allele of rs4607103 near ADAMTS9 associated with reduced insulin-stimulated glucose uptake (p = 0.002) during a hyperinsulinemic euglycemic clamp. However, following intravenous and oral administration of glucose serum insulin release was increased in individuals with the C-allele (p = 0.003 and p = 0.01, respectively). A meta-analyse combining clamp and IVGTT data from a total of 905 non-diabetic individuals showed that the C-risk allele associated with decreased insulin sensitivity (p = 0.003) and increased insulin release (p = 0.002). The major T-allele of the intronic JAZF1 rs864745 conferring increased diabetes risk was associated with increased 2(nd) phase serum insulin release during an IVGTT (p = 0.03), and an increased fasting serum insulin level (p = 0.001). The remaining variants did not show any associations with insulin response, insulin sensitivity or any other measured quantitative traits.The present studies suggest that the diabetogenic impact of the C-allele of rs4607103 near ADAMTS9 may in part be mediated through decreased insulin sensitivity of peripheral tissues.

Hypertension is a major health problem of largely unknown genetic origins. To identify new genes responsible for hypertension, genetic analysis of recombinant inbred strains of mice followed by human association studies might prove powerful and was exploited in our current study. Using a set of 27 recombinant BXD strains of mice we identified a quantitative trait locus (QTL) for blood pressure (BP) on distal chromosome 9. The association analysis of markers encompassing the syntenic region on human chromosome 3 gave in an additive genetic model the strongest association for rs17030583 C/T and rs2291897 G/A, located within the UBP1 locus, with systolic and diastolic BP (rs17030583: 1.3+/-0.4 mmHg p

HYPOTHESIS:Serum- and Glucocorticoid-inducible Kinase 1 (SGK1) is involved in the regulation of insulin secretion and may represent a candidate gene for the development of type 2 diabetes mellitus in humans. METHODS:Three independent European populations were analyzed for the association of SGK1 gene (SGK) variations and insulin secretion traits. The German TUEF project provided the screening population (N = 725), and four tagging SNPs (rs1763527, rs1743966, rs1057293, rs9402571) were investigated. EUGENE2 (N = 827) served as a replication cohort for the detected associations. Finally, the detected associations were validated in the METSIM study, providing 3798 non-diabetic and 659 diabetic (type 2) individuals. RESULTS:Carriers of the minor G allele in rs9402571 had significantly higher C-peptide levels in the 2 h OGTT (+10.8%, p = 0.04; dominant model) and higher AUC(C-Peptide)/AUC(Glc) ratios (+7.5%, p = 0.04) compared to homozygous wild type TT carriers in the screening population. As interaction analysis for BMIxrs9402571 was significant (p = 0.04) for the endpoint insulin secretion, we stratified the TUEF cohort for BMI, using a cut off point of BMI = 25. The effect on insulin secretion only remained significant in lean TUEF participants (BMI< or =25). This finding was replicated in lean EUGENE2 rs9402571 minor allele carriers, who had a significantly higher AUC(Ins)/AUC(Glc) (TT: 226+/-7, XG: 246+/-9; p = 0.019). Accordingly, the METSIM trial revealed a lower prevalence of type 2 diabetes (OR: 0.85; 95%CI: 0.71-1.01; p = 0.065, dominant model) in rs9402571 minor allele carriers. CONCLUSIONS:The rs9402571 SGK genotype associates with increased insulin secretion in lean non-diabetic TUEF/EUGENE2 participants and with lower diabetes prevalence in METSIM. Our study in three independent European populations supports the conclusion that SGK variability affects diabetes risk.

The characteristics of human resistin (RETN) are unclear and controversial despite intensive adipose-focused research. Its transcriptional and functional similarity with the murine myeloid-specific and CCAAT/enhancer binding protein epsilon (Cebpe)-dependent gene, resistin-like gamma (Retnlg), is unexplored. We examined the human CEBPE-regulatory pathway by unbiased reference and custom gene expression assays. Real-time RT-PCR analysis demonstrated lack of both the transcriptional factor CEBPE and RETN expression in adipose and muscle cells. In contrast, primary myelocytic samples revealed a concerted CEBPE-RETN transcription that was significantly elevated in inflammatory synoviocytes relative to intact peripheral blood mononuclear cells (PBMC). Mouse Cebpe and Retnlg were predictably expressed in macrophages, whereas Retn was abundant in adipocytes. Quite the opposite, a low and inconsistent RETN transcription was seen in some human white adipose tissue (WAT) biopsies without any relationship to body mass index, insulin sensitivity, or fat depot. However, in these cases, RETN was co-detected with CEBPE and the leukocyte-specific marker, EMR1, indicating the presence of inflammatory cells and their possible resistin-mediated effect on adipocytes. Indeed, addition of human resistin to WAT in culture induced, like in PBMC, the inflammatory cytokines IL6, IL8 and TNF. Importantly, the expression of the adipose-specific markers CEBPA, FABP4 and SLC2A4 was unchanged, while the expected inhibitory effect was seen with TNF. Both cytokines increased the mRNA level of CCL2 and MMP3, which may further promote inflammation in WAT. Thus, the myeloid-restricted nature of CEBPE precludes the expression of RETN in human adipocytes which, however, are targeted by this innate immune-derived proinflammatory cytokine.

The lipolytic response to catecholamines and the antilipolytic effect of insulin were studied as a function of adipose cell size and number. The results show that cellular enlargement is associated with an increase in the basal lipolysis as well as the release of glycerol induced by salbutamol (a β2-receptor agonist), noradrenaline, adrenaline, and isopropylnoradrenaline. The glycerol release induced by all these agents seems to be more favorably correlated with cell surface area than with cell volume or diameter.Under the incubation conditions used with glucose in the medium, the antilipolytic effect of insulin on the basal as well as on the adrenaline- and isopropylnoradrenaline-stimulated lipolysis was not consistent at any cell size studied. However, in the presence of noradrenaline and salbutamol, insulin exerted a consistent antilipolytic effect. The results show that the larger adipose cells are at least as sensitive to the antilipolytic effect of insulin as the smaller cells.The results imply that the previously reported diminished responsiveness to insulin shown by large adipose cells is exerted only on the side of lipid accumulation. It is suggested that the negative correlation between cell size and responsiveness to insulin on the side of lipid accumulation may be one way to control adipose cell enlargement.

When adipose tissue cells were incubated with collagenase for different periods of time, cell populations with different mean cell sizes were obtained from the same tissue sample. Lipid synthesis from glucose was studied as a function of adipose cell size and number. The incubations were performed in Parker medium 199, which is suitable for tissue culture of human adipose tissue. The results show that the larger cells of a specimen have a greater rate of lipid synthesis than the smaller cells of the same specimen. This is mainly due to an increase in the synthesis of glyceride-glycerol. Addition of insulin stimulated lipid synthesis. However, the larger adipose cells were less sensitive to the stimulating effect of insulin than the smaller cells.

The mean cell sizes of specimens of human adipose tissue were determined on sectioned slices according to the method described by Sjöström et al. (J. Lipid Res. 1971. 12: 521–530) and on adipocytes isolated after treatment of the tissue with collagenase. The average mean cell sizes from 11 biopsy specimens were 94.4 and 94.0 μm, respectively (r = 0.964; P(tb) < 0.001; y = 0.90x + 9.74), for the two methods. There was no indication of an increased rupture of isolated large human adipose cells. Thus, with precautions (freshly siliconized glassware and omitting the centrifugation of the isolated cells), the collagenase method may be used for metabolic as well as morphologic studies of human adipose tissue.