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Genome-wide association studies (GWAS) have found few common variants that influence fasting measures of insulin sensitivity. We hypothesized that a GWAS of an integrated assessment of fasting and dynamic measures of insulin sensitivity would detect novel common variants. We performed a GWAS of the modified Stumvoll Insulin Sensitivity Index (ISI) within the Meta-Analyses of Glucose and Insulin-Related Traits Consortium. Discovery for genetic association was performed in 16,753 individuals, and replication was attempted for the 23 most significant novel loci in 13,354 independent individuals. Association with ISI was tested in models adjusted for age, sex, and BMI and in a model analyzing the combined influence of the genotype effect adjusted for BMI and the interaction effect between the genotype and BMI on ISI (model 3). In model 3, three variants reached genome-wide significance: rs13422522 (NYAP2; P = 8.87 × 10−11), rs12454712 (BCL2; P = 2.7 × 10−8), and rs10506418 (FAM19A2; P = 1.9 × 10−8). The association at NYAP2 was eliminated by conditioning on the known IRS1 insulin sensitivity locus; the BCL2 and FAM19A2 associations were independent of known cardiometabolic loci. In conclusion, we identified two novel loci and replicated known variants associated with insulin sensitivity. Further studies are needed to clarify the causal variant and function at the BCL2 and FAM19A2 loci.

Glucokinase (GCK) mediates hepatic glucose uptake during hyperglycemia and is crucial for the regulation of glucose-responsive glycolysis genes. Mutations in GCK are responsible for maturity-onset diabetes of the young, and glucokinase regulatory protein (GKRP) , the regulator of hepatic GCK, is also a diabetes susceptibility locus. Recently in in vivo experiments, we used hepatic lactate export as a surrogate for GCK activity, which was a measure of “glucose effectiveness.” We hypothesized that in vitro GCK and GKRP suppression in hepatocytes promotes a reduction in lactate and pyruvate export from the liver. We used cryopreserved canine hepatocytes and knocked down GCK and GKRP via lipid delivery of siRNA. A control group did not receive siRNA. We measured lactate dehydrogenase D (LDHD) and pyruvate kinase (PK) gene expression and lactate and pyruvate release into the medium 24 and 48 h after siRNA delivery. Selective blocking of GCK and GKRP did not change cell proliferation but reduced the expression of these genes by 90% and 85%, respectively (P Disclosure M.Kabir: None. M.Ader: None. R.N.Bergman: Consultant; Fractyl Health, Inc., Novo Nordisk, Research Support; AstraZeneca, Janssen Research & Development, LLC. Funding National Institutes of Health R01- DK027619-28

Estimating insulin resistance from the oral glucose tolerance test (OGTT) has long been a challenge in diabetes research and practice. Because the time courses of glucose and insulin during the OGTT are similar in shape, the estimates of insulin sensitivity per se may be corrupted by other metabolic responses such as insulin release or glucose absorption. We have previously shown insulin injection during the intravenous test yields accurate values of insulin sensitivity (SI) and glucose effectiveness (SG) . Here we examine an analogous approach during the OGTT - in which exogenous insulin is administered during the test. Methods: For 100 simulated subjects (NGT: n=25, IGT: n=25, T2D: n=25, T1D: n=25) , glucose and insulin data were calculated using Dalla Man Mixed Meal Simulator. For each simulated subject, several protocols were examined by varying the time of insulin bolus (30, 60, or 90 min) and the dose of the bolus (0, 0.01, 0.02, or 0.03 IU/kg) . The resulting 3600 simulated datasets were analyzed with Dalla Man’s OGTT model to estimate SI. Results: When adjusted for the dose of insulin, the time of the dose did not significantly impact the estimation of SI. The 0.IU/kg insulin dose resulted in significantly (P Disclosure D.Stefanovski: None. F.Piccinini: Employee; Medtronic. M.Ader: None. M.Kabir: None. R.N.Bergman: Consultant; Fractyl Health, Inc., Novo Nordisk, Research Support; AstraZeneca, Janssen Research & Development, LLC.

Marking insulin's centennial, we share stories of researchers and clinicians whose seminal work has advanced our understanding of insulin, islet biology, insulin resistance, and diabetes. The past century of pursuing the "hormone of hormones" and advancing diabetes therapies is replete with stories of collaboration, perseverance, and triumph.

AIMS/HYPOTHESIS: Insufficient sleep is increasingly recognised as a major risk factor for the development of obesity and diabetes, and short-term sleep loss in clinical studies leads to a reduction in insulin sensitivity. Sleep loss-induced metabolic impairments are clinically relevant, since reductions in insulin sensitivity after sleep loss are comparable to insulin sensitivity differences between healthy individuals and those with impaired glucose tolerance. However, the relative effects of sleep loss vs high-fat feeding in the same individual have not been assessed. In addition, to our knowledge no diurnal (active during the daytime) non-human mammalian model of sleep loss-induced metabolic impairment exists, which limits our ability to study links between sleep and metabolism.METHODS: This study examined the effects of one night of total sleep deprivation on insulin sensitivity and beta cell function, as assessed by an IVGTT, before and after 9 months of high-fat feeding in a canine model.RESULTS: One night of total sleep deprivation in lean dogs impaired insulin sensitivity to a similar degree as a chronic high-fat diet (HFD)(normal sleep: 4.95 ± 0.45 mU-1 l-1 min-1; sleep deprivation: 3.14 ± 0.21 mU-1 l-1 min-1; HFD: 3.74 ± 0.48 mU-1 l-1 min-1; mean ± SEM). Hyperinsulinaemic compensation was induced by the chronic HFD, suggesting adequate beta cell response to high-fat feeding. In contrast, there was no beta cell compensation after one night of sleep deprivation, suggesting that there was metabolic dysregulation with acute sleep loss that, if sustained during chronic sleep loss, could contribute to the risk of type 2 diabetes. After chronic high-fat feeding, acute total sleep deprivation did not cause further impairments in insulin sensitivity (sleep deprivation + chronic HFD: 3.28 mU-1 l-1 min-1).CONCLUSIONS/INTERPRETATION: Our findings provide further evidence that sleep is important for metabolic health and establish a diurnal animal model of metabolic disruption during insufficient sleep.

BACKGROUND: Mirabegron is a β3-adrenergic receptor (β3-AR) agonist approved only for the treatment of overactive bladder. Encouraging preclinical results suggest that β3-AR agonists could also improve obesity-related metabolic disease by increasing brown adipose tissue (BAT) thermogenesis, white adipose tissue (WAT) lipolysis, and insulin sensitivity. METHODS: We treated 14 healthy women of diverse ethnicities (27.5 ± 1.1 years of age, BMI of 25.4 ± 1.2 kg/m(2)) with 100 mg mirabegron (Myrbetriq extended-release tablet, Astellas Pharma) for 4 weeks in an open-label study. The primary endpoint was the change in BAT metabolic activity as measured by [(18)F]-2-fluoro-d-2-deoxy-d-glucose ((18)F-FDG) PET/CT. Secondary endpoints included resting energy expenditure (REE), plasma metabolites, and glucose and insulin metabolism as assessed by a frequently sampled intravenous glucose tolerance test. RESULTS: Chronic mirabegron therapy increased BAT metabolic activity. Whole-body REE was higher, without changes in body weight or composition. Additionally, there were elevations in plasma levels of the beneficial lipoprotein biomarkers HDL and ApoA1, as well as total bile acids. Adiponectin, a WAT-derived hormone that has antidiabetic and antiinflammatory capabilities, increased with acute treatment and was 35% higher upon completion of the study. Finally, an intravenous glucose tolerance test revealed higher insulin sensitivity, glucose effectiveness, and insulin secretion. CONCLUSION: These findings indicate that human BAT metabolic activity can be increased after chronic pharmacological stimulation with mirabegron and support the investigation of β3-AR agonists as a treatment for metabolic disease. TRIAL REGISTRATION: Clinicaltrials.gov NCT03049462. FUNDING: This work was supported by grants from the Intramural Research Program of the NIDDK, NIH (DK075112, DK075116, DK071013, and DK071014).

BACKGROUND: Discovering the role duodenal exclusion plays in weight loss and resolution of type 2 diabetes (T2D) may help refine the surgical and nonsurgical treatment of obesity and T2D. OBJECTIVES: To assess changes in glucose homeostasis due to duodenal exclusion using a duodenal-jejunal bypass liner (DJBL) in a non-obese canine model. SETTING: Academic laboratory setting. METHODS: An intravenous glucose tolerance test (IVGTT), and a mixed-meal tolerance test (MMTT) at Baseline, 1 and 6 weeks post-DJBL implantation (I1 and I6, respectively), and 1 and 6 weeks post-DJBL removal (R1 and R6, respectively) were done in canines (n=7) fed a normal chow diet. RESULTS: Placement of the DJBL induced weight loss that was maintained until 4 weeks post-removal (R4), despite normal food intake. Total bile acids (TBA) and glucagon-like peptide-1 (GLP-1) during the MMTT were significantly increased at I1 and were associated with increased lactate and free fatty acids. Hypoglycemia counterregulation was blunted during the IVGTT at I1 and I6, returning to Baseline at R1. While there were no changes to insulin sensitivity during the experiment, glucose tolerance was significantly increased following the removal of the DJBL at R1. CONCLUSIONS: These data show that in a normoglycemic, non-obese canine model, duodenal exclusion induces energy intake-independent weight loss and negative metabolic effects that are reversed following reexposure of the small intestine to nutrients.

Differences between sexes contribute to variation in the levels of fasting glucose and insulin. Epidemiological studies established a higher prevalence of impaired fasting glucose in men and impaired glucose tolerance in women, however, the genetic component underlying this phenomenon is not established. We assess sex-dimorphic (73,089/50,404 women and 67,506/47,806 men) and sex-combined (151,188/105,056 individuals) fasting glucose/fasting insulin genetic effects via genome-wide association study meta-analyses in individuals of European descent without diabetes. Here we report sex dimorphism in allelic effects on fasting insulin at IRS1 and ZNF12 loci, the latter showing higher RNA expression in whole blood in women compared to men. We also observe sex-homogeneous effects on fasting glucose at seven novel loci. Fasting insulin in women shows stronger genetic correlations than in men with waist-to-hip ratio and anorexia nervosa. Furthermore, waist-to-hip ratio is causally related to insulin resistance in women, but not in men. These results position dissection of metabolic and glycemic health sex dimorphism as a steppingstone for understanding differences in genetic effects between women and men in related phenotypes., Sex differences in fasting glucose and insulin have been identified, but the genetic loci underlying these differences have not. Here, the authors perform a meta-analysis of genome-wide association studies to detect sex-specific and sex-dimorphic loci associated with fasting glucose and insulin.

Insulin resistance engenders a compensatory increase in plasma insulin. Inadequate compensation is a primary element in the pathogenesis of type 2 diabetes. The signal that heralds developing insulin resistance and initiates hyperinsulinemic compensation is not known. It has often been assumed to be increased glucose. We tested this assumption by determining whether development of fasting and/or glucose-stimulated hyperinsulinemia with diet-induced insulin resistance occurs because of concomitant elevation of glycemia. Male dogs (n = 58) were fed a hypercaloric, fat-supplemented diet for 6 weeks. Dogs underwent magnetic resonance imaging to quantify total and regional (visceral, subcutaneous) adiposity as well as euglycemic hyperinsulinemic clamps. A subset of animals also underwent an insulin-modified intravenous glucose tolerance test to assess insulin sensitivity, acute insulin response (AIRg), and glucose effectiveness. Fat feeding caused modest weight gain, increased visceral and subcutaneous fat, and insulin resistance at both peripheral and hepatic levels. Hyperinsulinemic compensation was observed in fasting levels as well as increased AIRg. However, we observed absolutely no increase in carefully measured fasting, evening (6 to 8 pm) or nocturnal glycemia (2 to 4 am). Insulin resistance and hyperinsulinemia occurred despite no elevation in 24-hour glucose. Compensatory development of hyperinsulinemia during diet-induced insulin resistance occurs without elevated fasting or 24-hour glycemia. These data refute the idea that glucose itself is a requisite signal for β-cell upregulation. Alternative feedback mechanisms need to be identified.

The prodromal prediabetic state is characterized by glucose intolerance. Both insulin-dependent (sensitivity, secretion, clearance) and -independent factors (“glucose effectiveness”) contribute to glucose homeostasis. The relative importance of glucose effectiveness is not known. Healthy dogs (n=18) were tested at baseline (BL), after 6 wk on a high-fat diet (HFD), and 2 wk, after a sub-diabetogenic dose of streptozotocin (STZ, 18.5 mg/kg); HFD was continued. We performed intravenous glucose tolerance tests (IVGTTs)at 0, 6, and 8 wks. Minimal model analysis quantified insulin sensitivity (SI), acute insulin response to glucose (AIRg), disposition index (DI, = SI x AIRg), and glucose tolerance (KG). Glucose effectiveness (SG) was measured both by minimal model analysis and directly by multi-step hyperglycemic clamps at basal insulin in a subset of the dogs (n=5). Consumption of HFD induced 7.7±1.0% weight gain (P Disclosure C. M. Kolka: Research Support; Self; AstraZeneca. J. Porter: None. M. Ader: None. R. N. Bergman: None. Funding National Institutes of Health (DK27619, DK29867)

Sodium glucose cotransporter-2 (SGLT2) inhibitors promote urinary glucose excretion, consequently reducing blood glucose. Previously we showed that 6 weeks of Dapagliflozin (Dapa) treatment improved insulin sensitivity and prevented weight gain. In addition, Dapa promoted adipose tissue (AT) beiging, lipolysis and decreased adipocyte size primarily in the visceral (VIS) depot in a canine model of mild type 2 diabetes. Little is known about the effects of Dapa on AT inflammation and ceramide synthesis in both the subcutaneous (SC) and VIS fat depots. Twelve dogs were fed a high fat diet for 6 weeks, then received a single low dose of streptozotocin (18.5mg/kg) to induce mild type 2 diabetes. Animals were then exposed to Dapa (n=6, 1.25mg/kg) or placebo (n=6) once per day for 6 weeks, while remaining on the high fat diet. At the end of the treatment, fasting plasma parameters were measured, and SC and VIS fat biopsies were obtained for molecular studies. Dapa increased adiponectin, adiponectin receptors 1 and 2 gene expression by 90%, 74% and 69% ( In conclusion, Dapa promoted adiponectin and its receptors, decreased inflammation and enzymes involved in ceramide synthesis mainly in the VIS depot. These changes were independent of fasting glucose and insulin. Thus, for the first time we are beginning to identify mechanisms by which Dapa enhances AT function in regulating energy homeostasis and improving AT inflammation in mild type 2 diabetes canine model. Disclosure M. Kabir: None. R. N. Bergman: None. V. Gopaul: None. H. Yang: Employee; Self; AstraZeneca. C. M. Kolka: Research Support; Self; AstraZeneca. Funding National Institutes of Health

CB1 receptor (CB1R) antagonism improves the deleterious effects of a high-fat diet (HFD) by reducing body fat mass and adipocyte cell size. Previous studies demonstrated that the beneficial effects of the CB1R antagonist rimonabant (RIM) in white adipose tissue (WAT) are partially due to an increase of mitochondria numbers and upregulation thermogenesis markers, suggesting an induction of WAT beiging. However, the molecular mechanism by which CB1R antagonism induces weight loss and WAT beiging is unclear. In this study, we probed for genes associated with beiging and explored longitudinal molecular mechanisms by which the beiging process occurs. HFD dogs received either RIM (HFD+RIM) or placebo (PL) (HFD+PL) for 16 wk. Several genes involved in beiging were increased in HFD+RIM compared with pre-fat, HFD, and HFD+PL. We evaluated lipolysis and its regulators including natriuretic peptide (NP) and its receptors ( NPRs), β-1 and β-3 adrenergic receptor ( β1R, β3R) genes. These genes were increased in WAT depots, accompanied by an increase in lipolysis in HFD+RIM. In addition, RIM decreased markers of inflammation and increased adiponectin receptors in WAT. We observed a small but significant increase in UCP1; therefore, we evaluated the newly discovered UCP1-independent thermogenesis pathway. We confirmed that SERCA2b and RYR2, the two key genes involved in this pathway, were upregulated in the WAT. Our data suggest that the upregulation of NPRs, β-1R and β-3R, lipolysis, and SERCA2b and RYR2 may be one of the mechanisms by which RIM promotes beiging and overall the improvement of metabolic homeostasis induced by RIM.

Normal glucose homeostasis depends on the capacity of pancreatic β-cells to adjust insulin secretion in response to a change of tissue insulin sensitivity. In cold environments, for example, the dramatic increase of insulin sensitivity required to ensure a sufficient supply of glucose to thermogenic tissues is offset by a proportionate reduction of insulin secretion, such that overall glucose tolerance is preserved. That these cold-induced changes of insulin secretion and insulin sensitivity are dependent on the sympathetic nervous system (SNS) outflow suggests a key role for thermoregulatory neurons in the hypothalamic preoptic area (POA) in this metabolic response. As these POA neurons are themselves sensitive to changes in local hypothalamic temperature, we hypothesized that direct cooling of the POA would elicit the same glucoregulatory responses that we observed during cold exposure. To test this hypothesis, we used a thermode to cool the POA area, and found that as predicted, short-term (8-h) intense POA cooling reduced glucose-stimulated insulin secretion (GSIS), yet glucose tolerance remained unchanged due to an increase of insulin sensitivity. Longer-term (24-h), more moderate POA cooling, however, failed to inhibit GSIS and improved glucose tolerance, an effect associated with hyperthermia and activation of the hypothalamic-pituitary-adrenal axis, indicative of a stress response. Taken together, these findings suggest that POA cooling is sufficient to recapitulate key glucoregulatory responses to cold exposure.

We recently reported that in rodent models of type 2 diabetes (T2D), a single intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1) induces remission of hyperglycemia that is sustained for weeks. To clarify the peripheral mechanisms underlying this effect, we used the Zucker diabetic fatty fa/fa rat model of T2D, which, like human T2D, is characterized by progressive deterioration of pancreatic β-cell function after hyperglycemia onset. We report that although icv FGF1 injection delays the onset of β-cell dysfunction in these animals, it has no effect on either glucose-induced insulin secretion or insulin sensitivity. These observations suggest that FGF1 acts in the brain to stimulate insulin-independent glucose clearance. On the basis of our finding that icv FGF1 treatment increases hepatic glucokinase gene expression, we considered the possibility that increased hepatic glucose uptake (HGU) contributes to the insulin-independent glucose-lowering effect of icv FGF1. Consistent with this possibility, we report that icv FGF1 injection increases liver glucokinase activity by approximately twofold. We conclude that sustained remission of hyperglycemia induced by the central action of FGF1 involves both preservation of β-cell function and stimulation of HGU through increased hepatic glucokinase activity.

Glycemic traits are used to diagnose and monitor type 2 diabetes and cardiometabolic health. To date, most genetic studies of glycemic traits have focused on individuals of European ancestry. Here we aggregated genome-wide association studies comprising up to 281,416 individuals without diabetes (30% non-European ancestry) for whom fasting glucose, 2-h glucose after an oral glucose challenge, glycated hemoglobin and fasting insulin data were available. Trans-ancestry and single-ancestry meta-analyses identified 242 loci (99 novel; P-8), 80% of which had no significant evidence of between-ancestry heterogeneity. Analyses restricted to individuals of European ancestry with equivalent sample size would have led to 24 fewer new loci. Compared with single-ancestry analyses, equivalent-sized trans-ancestry fine-mapping reduced the number of estimated variants in 99% credible sets by a median of 37.5%. Genomic-feature, gene-expression and gene-set analyses revealed distinct biological signatures for each trait, highlighting different underlying biological pathways. Our results increase our understanding of diabetes pathophysiology by using trans-ancestry studies for improved power and resolution.A trans-ancestry meta-analysis of GWAS of glycemic traits in up to 281,416 individuals identifies 99 novel loci, of which one quarter was found due to the multi-ancestry approach, which also improves fine-mapping of credible variant sets.

The original version of this Article contained an error in Fig. 2, in which panels a and b were inadvertently swapped. This has now been corrected in the PDF and HTML versions of the Article.

BackgroundSince partial oxygen pressure decreases as altitude increases, environmental hypoxia could worsen COVID-19 patient’s hypoxemia. We compared COVID-19 mortality at different altitudes.MethodsRetrospective analysis of population-level data on COVID-19 deaths in the U.S. (1,016 counties) and Mexico (567 municipalities). Mixed-model Poisson regression analysis of the association between altitude and COVID-19 mortality using individual-level data from 40,168 Mexican subjects with COVID-19, adjusting for multiple covariates.ResultsBetween January 20 and April 13, 2020, mortality rates were higher in U.S. counties located at ≥2,000 m elevation vs. those located below 1,500 m (12.3 vs. 3.2 per 100,000; PConclusionAltitude is associated with COVID-19 mortality in men younger than 65 years.

Glycaemic traits are used to diagnose and monitor type 2 diabetes, and cardiometabolic health. To date, most genetic studies of glycaemic traits have focused on individuals of European ancestry. Here, we aggregated genome-wide association studies in up to 281,416 individuals without diabetes (30% non-European ancestry) with fasting glucose, 2h-glucose post-challenge, glycated haemoglobin, and fasting insulin data. Trans-ancestry and single-ancestry meta-analyses identified 242 loci (99 novel; P-8), 80% with no significant evidence of between-ancestry heterogeneity. Analyses restricted to European ancestry individuals with equivalent sample size would have led to 24 fewer new loci. Compared to single-ancestry, equivalent sized trans-ancestry fine-mapping reduced the number of estimated variants in 99% credible sets by a median of 37.5%. Genomic feature, gene-expression and gene-set analyses revealed distinct biological signatures for each trait, highlighting different underlying biological pathways. Our results increase understanding of diabetes pathophysiology by use of trans-ancestry studies for improved power and resolution.

With the increased prevalence of obesity and related co-morbidities, such as type 2 diabetes (T2D), worldwide, improvements in pharmacological treatments are necessary. The brain- and peripheral-cannabinoid receptor 1 (CB1R) antagonist rimonabant (RIM) has been shown to induce weight loss and improve glucose homeostasis. We have previously demonstrated that RIM promotes adipose tissue beiging and decreased adipocyte cell size, even during maintenance on a high-fat diet. Given the adverse side-effects of brain-penetrance with RIM, in this study we aimed to determine the site of action for a non-brain-penetrating CB1R antagonist AM6545. By using in vitro assays, we demonstrated the direct effects of this non-brain-penetrating CB1R antagonist on cultured adipocytes. Specifically, we showed, for the first time, that AM6545 significantly increases markers of adipose tissue beiging, mitochondrial biogenesis, and lipolysis in 3T3-L1 adipocytes. In addition, the oxygen consumption rate (OCR), consisting of baseline respiratory rate, proton leak, maximal respiratory capacity, and ATP synthase activity, was greater for cells exposed to AM6545, demonstrating greater mitochondrial uncoupling. Using a lipolysis inhibitor during real-time OCR measurements, we determined that the impact of CB1R antagonism on adipocytes is driven by increased lipolysis. Thus, our data suggest the direct role of CB1R antagonism on adipocytes does not require brain penetrance, supporting the importance of focus on peripheral CB1R antagonism pharmacology for reducing the incidence of obesity and T2D.

Insulin clearance has recently been highlighted as a fundamental aspect of glucose metabolism, as it has been hypothesized that its impairment could be related to an increased risk of developing type 2 diabetes. This review focuses on methods used to calculate insulin clearance: from the early surrogate indices employing C-peptide to insulin molar ratio, to direct measurement methods used in animal models, to modeling-based techniques to estimate the components of insulin clearance (hepatic versus extra-hepatic). The methods are explored and interpreted by critically highlighting advantages and limitations.

Selective sodium glucose cotransporter-2 inhibition promotes urinary glucose excretion, thus reducing blood glucose. Previously we showed that 6 weeks of Dapagliflozin (Dapa) treatment improved insulin sensitivity and prevented weight gain in a canine model of mild type 2 diabetes. Little is known about the effects of Dapa on adipose tissue (AT) function. Eleven dogs were fed a high fat diet for 6 weeks, then received a single low dose of streptozotocin (18.5mg/kg) to induce mild type 2 diabetes. Animals were then exposed to Dapa (n=5, 1.25mg/kg) or placebo (n=6) once per day for 6 weeks, while remaining on the high fat diet. At the end of the study, subcutaneous (SC) and visceral (VIS) fat depots were obtained for adipocytes isolation and molecular studies and fasting plasma parameters were measured. Fasting plasma glucose and insulin levels were unaffected by Dapa treatment. Finite mixture modeling linear regression was used for adipocyte distributions. Dapa decreased adipocyte size and bimodal distributions in both SC and VIS by 10% (P In conclusion, Dapa promotes AT beiging, lipolysis, adiponectin and leptin release by adipocytes and decreased adipocyte size and distributions. These changes were independent of glucose and insulin. Thus, for the first time we are beginning to identify mechanisms by which Dapa enhances AT function and improves energy homeostasis in mild type 2 diabetes canine model. Disclosure M. Kabir: None. R.N. Bergman: None. F. Piccinini: Employee; Self; Medtronic. D. Stefanovski: None. V. Gopaul: None. H. Yang: Employee; Self; AstraZeneca. C.M. Kolka: Research Support; Self; AstraZeneca. Funding AstraZeneca (D1690L00045-CSR212966)

Glucose homeostasis involves actions of both insulin and glucose per se (“glucose effectiveness, SG”). While consumption of a high fat diet typically induces insulin resistance, it is unclear whether SG is altered by fat feeding. We quantified SG from the intravenous glucose tolerance test (IVGTT; n=36) or hyperglycemic clamp at basal insulin (HGC; n=5) in dogs, tested before and after 6 weeks fat feeding (+ 6 g/kg per day). SG from IVGTTs were quantified by minimal model (SG-MINMOD). In HGCs, glucose was sequentially raised to 125, 180, and 250 mg/dl during somatostatin (SRIF) and basal insulin replacement; SG-HGC was calculated as the slope of steady state glucose infusion rate and plasma glucose. Insulin sensitivity (SI) was calculated from IVGTTs and from euglycemic clamps. Fat feeding induced weight gain (28.8±0.4 to 31.2±2.5 kg; p Disclosure M. Ader: None. C.M. Kolka: Research Support; Self; AstraZeneca. R.N. Bergman: None. Funding National Institutes of Health (DK29867, DK27619)

AIMS: Adult African American (AA) women have one third of the hepatic insulin clearance of European American (EA) women. This lower hepatic (but not extra-hepatic) insulin clearance in AA individuals is associated with higher plasma insulin concentrations. This study aims to understand whether impairment of hepatic insulin clearance is seen in AA individuals since childhood, possibly suggesting that genetic/epigenetic factors, rather than lifestyle only, contribute to this. MATERIALS AND METHODS: A total of 203 children (105 male and 98 female (55 AA, 88 EA and 60 Hispanic American [HA]; ages, 7–13 years; mean BMI, 19 kg/m(2))) underwent the frequently-sampled intravenous glucose tolerance test (FSIGT) at the University of Alabama at Birmingham, General Clinical Research Center and Department of Nutrition Sciences. Glucose, insulin and C-peptide levels were measured and hepatic and extra-hepatic insulin clearances were calculated using mathematical modelling. RESULTS: Fractional hepatic insulin extraction (FE(L)) was lower in AA than in EA children (mean (SD), 19% (20%) vs 33% (20%); P = 0.0007). Adjusting for age, Tanner stage and body fat, FE(L) was lower in AA than in EA children (P = 0.0012), and there was a slight sex-related difference (FE(L), 24% (10%) vs 29% (10%) in boys vs girls; P = 0.04). Extra-hepatic insulin clearance did not differ with ethnicity (27 (12), 21 (12) and 24 (28) mL/kg/min for AA, HA and EA children, respectively; P > 0.05). CONCLUSIONS: At a young age, FE(L) is lower in AAs than in EAs, which does not rule out genetic/epigenetic factors. These differences are related to hyperinsulinaemia and, over time, could possibly contribute to metabolic disorders in AA individuals.

Diabetes is often thought of as one of two diseases: Type 1 diabetes (T1D), which is caused by immunological destruction of the beta-cells, and Type 2 diabetes (T2D), which is due to a combination of insulin resistance and relative failure of the beta-cells to compensate for the resistance. It is becoming clear, however, that even within these two definitions there may be considerable heterogeneity (1). There are several approaches to examine heterogeneity of T2D. Among these approaches are the use of biomarkers to categorize the disease, or the examination of variants in the genome. A third approach – the one we have been using in our laboratory – is to identify specific phenotypes which may contribute to failure to regulate the glucose level. We have identified a small group of such phenotypes which can be distinguished and measured using clinical protocols and/or mathematical modeling.

High whole-body fat percentage is independently associated with increased mortality. We aimed to identify a simple anthropometric linear equation that is more accurate than the body mass index (BMI) to estimate whole-body fat percentage among adult individuals. National Health and Nutrition Examination Survey (NHANES) 1999–2004 data (n = 12,581) were used for model development and NHANES 2005–2006 data (n = 3,456) were used for model validation. From the 365 anthropometric indices generated, the final selected equation was as follows: 64 − (20 × height/waist circumference) + (12 × sex), named as the relative fat mass (RFM); sex = 0 for men and 1 for women. In the validation dataset, compared with BMI, RFM better predicted whole-body fat percentage, measured by dual energy X-ray absorptiometry (DXA), among women and men. RFM showed better accuracy than the BMI and had fewer false negative cases of body fat-defined obesity among women and men. RFM reduced total obesity misclassification among all women and all men and, overall, among Mexican-Americans, European-Americans and African-Americans. In the population studied, the suggested RFM was more accurate than BMI to estimate whole-body fat percentage among women and men and improved body fat-defined obesity misclassification among American adult individuals of Mexican, European or African ethnicity.

Although the β-cells secrete insulin, the liver, with its first-pass insulin extraction (FPE), regulates the amount of insulin allowed into circulation for action on target tissues. The metabolic clearance rate of insulin, of which FPE is the dominant component, is a major determinant of insulin sensitivity (SI). We studied the intricate relationship among FPE, SI, and fasting insulin. We used a direct method of measuring FPE, the paired portal/peripheral infusion protocol, where insulin is infused stepwise through either the portal vein or a peripheral vein in healthy young dogs (n = 12). FPE is calculated as the difference in clearance rates (slope of infusion rate vs. steady insulin plot) between the paired experiments. Significant correlations were found between FPE and clamp-assessed SI (rs = 0.74), FPE and fasting insulin (rs = −0.64), and SI and fasting insulin (rs = −0.67). We also found a wide variance in FPE (22.4–77.2%; mean ± SD 50.4 ± 19.1) that is reflected in the variability of plasma insulin (48.1 ± 30.9 pmol/L) and SI (9.4 ± 5.8 × 104 dL · kg−1 · min−1 · [pmol/L]−1). FPE could be the nexus of regulation of both plasma insulin and SI.

OBJECTIVE Diets high in saturated fat induce obesity and insulin resistance and impair insulin access to skeletal muscle, leading to reduced insulin levels at the muscle cell surface available to bind insulin receptors and induce glucose uptake. In contrast, diets supplemented with polyunsaturated fat improve insulin sensitivity (SI) and reduce the risk for type 2 diabetes. It was hypothesized that a diet high in polyunsaturated fat would preserve SI and insulin access to muscle, as compared with a diet high in saturated fat. METHODS After 12 weeks of control, saturated (LARD), or polyunsaturated (salmon oil [SO]) high-fat diet feeding, muscle SI and insulin access to skeletal muscle were measured by using lymph, a surrogate of skeletal muscle interstitial fluid. RESULTS Both high-fat diets induced similar weight gain, yet only LARD impaired SI. Hyperinsulinemia in the LARD group did not induce an increase in basal interstitial insulin, suggesting reduced insulin access to muscle after LARD, but not after SO. CONCLUSIONS A diet high in polyunsaturated fat does not impair insulin access to muscle interstitium or induce insulin resistance as observed with a saturated fat diet, despite similar weight gain. Future studies should determine whether dietary SO supplementation improves impairments in insulin access to skeletal muscle.

African Americans (AAs) tend to have higher plasma insulin concentrations than European Americans (EAs); the increased insulin concentrations have been attributed to increased secretion and/or decreased insulin clearance by liver or other tissues. This work characterizes the contributions of hepatic versus extrahepatic insulin degradation related to ethnic differences between AAs and EAs. By using a recently developed mathematical model that uses insulin and C-peptide measurements from the insulin-modified, frequently sampled intravenous glucose tolerance test (FSIGT), we estimated hepatic versus extrahepatic insulin clearance in 29 EA and 18 AA healthy women. During the first 20 min of the FSIGT, plasma insulin was approximately twice as high in AAs as in EAs. In contrast, insulin was similar in AAs and EAs after the 20–25 min intravenous insulin infusion. Hepatic insulin first-pass extraction was two-thirds lower in AAs versus EAs in the overnight-fasted state. In contrast, extrahepatic insulin clearance was not lower in AAs than in EAs. The difference in insulin degradation between AAs and EAs can be attributed totally to liver clearance. The mechanism underlying reduced insulin degradation in AAs remains to be clarified, as does the relative importance of reduced liver clearance to increased risk for type 2 diabetes.

On the basis of studies that investigated the intraportal versus systemic insulin infusion and transendothelial transport of insulin, we proposed the “single gateway hypothesis,” which supposes an indirect regulation of hepatic glucose production by insulin; the rate-limiting transport of insulin across the adipose tissue capillaries is responsible for the slow suppression of free fatty acids (FFAs), which in turn is responsible for delayed suppression of hepatic endogenous glucose production (EGP) during insulin infusion. Preventing the fall in plasma FFAs during insulin infusion either by administering intralipids or by inhibiting adipose tissue lipolysis led to failure in EGP suppression, thus supporting our hypothesis. More recently, mice lacking hepatic Foxo1 in addition to Akt1 and Akt2 (L-AktFoxo1TKO), all required for insulin signaling, surprisingly showed normal glycemia. Inhibiting the fall of plasma FFAs in these mice prevented the suppression of EGP during a clamp, reaffirming that the site of insulin action to control EGP is extrahepatic. Measuring whole-body turnover rates of glucose and FFAs in L-AktFoxo1TKO mice also confirmed that hepatic EGP was regulated by insulin-mediated control of FFAs. The knockout mouse model in combination with sophisticated molecular techniques confirmed our physiological findings and the single gateway hypothesis.

Dynamic adjustment of insulin secretion to compensate for changes of insulin sensitivity that result from alteration of nutritional or metabolic status is a fundamental aspect of glucose homeostasis. To investigate the role of the brain in this coupling process, we used cold exposure as an experimental paradigm because the sympathetic nervous system (SNS) helps to coordinate the major shifts of tissue glucose utilization needed to ensure that increased thermogenic needs are met. We found that glucose-induced insulin secretion declined by 50% in rats housed at 5°C for 28 h, and yet, glucose tolerance did not change, owing to a doubling of insulin sensitivity. These potent effects on insulin secretion and sensitivity were fully reversed by returning animals to room temperature (22°C) for 4 h or by intravenous infusion of the α-adrenergic receptor antagonist phentolamine for only 30 min. By comparison, insulin clearance was not affected by cold exposure or phentolamine infusion. These findings offer direct evidence of a key role for the brain, acting via the SNS, in the rapid, highly coordinated, and reciprocal changes of insulin secretion and insulin sensitivity that preserve glucose homeostasis in the setting of cold exposure.

We thank Dr. Gastadelli and colleagues for their interest in our review article (1) and their comments (2) on the limitations of different approaches to assessing insulin clearance in humans and putative limitations of the hypothesis (3) that lower insulin clearance might cause type 2 diabetes (T2D). In fact, many of their comments relate to a series of articles from our group, and in addition to the article they refer to (1), several others (4–6) are also relevant to their comments. Concerning the Polidori/Bergman model (4), they are correct that the approach used by Polidori et al. (4) assumes either linear or saturable hepatic insulin extraction and chooses the model that best fits the data. We believe these are reasonable assumptions and that there is little support for the claim by Gastadelli et al. (2) that a saturable model of hepatic insulin extraction is not justified based on experimental data. The articles they cite claiming that hepatic insulin extraction does not reach saturability until insulin concentrations are in the 300–500 mU/L range are based on comparisons between groups of …

We evaluated the ability of the Relative Fat Mass (RFM) to estimate whole-body fat percentage among children and adolescents who participated in the National Health and Nutrition Examination Survey from 1999 through 2006 (n = 10,390). The RFM equation for adults (64 − (20 × height/waist circumference) + (12 × sex)) may be used for adolescents 15 to 19 years of age. For children and adolescents 8 to 14 years of age, we suggest a modified RFM equation, named as the RFMp (RFM pediatric): 74 − (22 × height/waist circumference) + (5 × sex). In both equations, sex equals 0 for boys and 1 for girls. RFMp was more accurate than BMI to estimate whole-body fat percentage (measured by dual energy X-ray absorptiometry, DXA) among girls (percentage of estimates that were th percentile or higher) among boys 8 to 14 years of age (6.5% vs. 7.9%; P = 0.018) but not girls (RFMp: 8.2%; BMI-for-age: 7.9%; P = 0.681). Misclassification error of overweight or obesity was similar for RFM and BMI-for-age percentiles among girls (RFM: 8.0%; BMI-for-age: 6.6%; P = 0.076) and boys 15 to 19 years of age (RFM: 6.9%; BMI-for-age: 7.8%; P = 0.11). RFMp for children and adolescents 8 to 14 years of age and RFM for adolescents 15 to 19 years of age were useful to estimate whole-body fat percentage and diagnose body fat-defined overweight or obesity.

We have recently proposed and validated a simple and accurate method to estimate whole-body fat percentage in adults, the relative fat mass (RFM), derived from the ratio of height to waist circumference. We aimed to identify RFM cutoffs to diagnose obesity based on the association between RFM and all-cause mortality.We used data from adult participants (≥20 years of age, n = 43,793) of the National Health and Nutrition Examination Survey (NHANES) 1999-2014 linked with death certificate records from the National Death Index. Optimal RFM cutoffs were determined using receiver-operating characteristic analysis (the Youden's index and the Euclidean minimum distance to the coordinate (0,1)).Final dataset for analyses comprised 31,008 adults. During a median follow-up of 8.3 years (IQR, 7.6-13.7), 2,517 deaths occurred. Youden and Euclidean optimal cutoffs of baseline RFM for all-cause mortality were 40.8% and 41.6% for women, and 30.9% and 28.9% for men, respectively. Similar cutoffs were obtained using measured whole-body fat percentage by dual energy X-ray absorptiometry. Adjusting for age, BMI category, ethnicity, education level, and smoking status, the hazard ratio for mortality using Cox proportional hazard regression was 1.41 (95% CI, 1.02-1.95) among women who had an RFM of 40.0-44.9% compared with women who had an RFM35% (P = 0.035). Among men, the hazard ratio was 1.57 (95% CI, 1.07-2.30) among those with an RFM of 30.0-34.9% compared with men who had an RFM25% (P = 0.020). Similar adjusted hazard ratios for same RFM categories were obtained in our validation population (NHANES III, n = 12,650, median follow-up: 23.3 years): 1.42 (95% CI, 1.01-2.00) among women (P = 0.043) and 1.50 (95% CI, 1.07-2.10) among men (P = 0.021).We suggest rounded RFM cutoffs of 40% for women and 30% for men to diagnose obesity and identify individuals at higher risk of death.

African American (AA) children have lower hepatic insulin clearance than European Americans (EA), as obtained with a 25-sample intravenous glucose tolerance test (FSIGT), over 240 minutes (t=0, 2, 3, 4 5, 6, 8, 10, 12, 15, 19, 20, 21, 22, 24, 26, 28, 30, 35, 40, 50, 60, 70, 180, 240 minutes). C-peptide and insulin concentrations were analyzed with a recent model of hepatic (FEL) and extra-hepatic (CLP) insulin clearance. This model has not been tested in a reduced sampling protocol, easier to be performed in larger cohorts. With data from 203 children of both sexes and 3 ethnic groups [55 AA, 88 EA, 60 HA (Hispanic Americans), age 7-13 years, mean BMI = 19 kg/m2, basal plasma glucose = 99 mg/dL, insulin = 78 pmol/L, C-peptide = 511 pmol/L], a reduced 12-sample schedule was examined (t=0, 2, 4, 8, 19, 22, 26, 30, 40, 50, 70, 180 minutes). FEL and CLP from the reduced protocol were strongly correlated with the full one: FEL: r=0.91, p Disclosure F. Piccinini: None. B. Gower: None. J.R. Fernandez: None. R.N. Bergman: Consultant; Self; Zafgen, Inc. Funding National Institute of Diabetes and Digestive and Kidney Diseases (DK067426); Nutrition Obesity Research Center (DK56336); National Institutes of Health (M01RR00032)

Recent studies showed that hepatic insulin clearance is lower in African Americans (AA) than European Americans (EA). The aim of this work is to elucidate the possible genetic/epigenetic versus lifestyle causes. The frequently-sampled intravenous glucose tolerance test (FSIGT) was performed in 142 children [38 AA, 69 EA, 28 HA (Hispanic Americans), both sexes, age 7-13 years, mean BMI = 19 kg/m2, basal plasma glucose = 99 mg/dL, insulin = 78 pmol/L, C-peptide = 511 pmol/L]. Mathematical modeling provided hepatic (FEL) and extra-hepatic (CLP) insulin clearance values. Two-way analysis of covariance was done (groups: sex and ethnicity, covariates: age, Tanner stage and body fat from DXA). Data are mean (standard error). FEL was lower in AA than EA (18% (2%) vs. 31% (3%), p=0.001), but no difference was observed in HA. CLP was not different among ethnicities. Partial correlation was calculated between FEL, CLP and 1) diet composition (energy intake, % of calories from carbohydrate, fat, protein), 2) physical activity (PA: light, moderate, hard, very hard), 3) admixture scores (African, European, Amerindian). FEL was positively correlated with the European admixture score (r=0.3, p Disclosure F. Piccinini: None. B. Gower: None. J.R. Fernandez: None. R.N. Bergman: Consultant; Self; Zafgen, Inc. Funding National Institute of Diabetes and Digestive and Kidney Diseases (DK067426); Nutrition Obesity Research Center (DK56336); National Institutes of Health (M01RR00032)

ZGN-1061 is a second-generation methionine aminopeptidase 2 (MetAP2) inhibitor in development for treatment of T2D. Full results of a Phase 2 clinical trial of ZGN-1061 vs. placebo (PBO) administered SC for 12 weeks on change from baseline in A1C in subjects with T2D are reported elsewhere. Baseline (ITT population N=175) A1C 8.6±1.1%, BMI 37±7 kg/m2, T2D duration 8±6 y. ZGN-1061 was generally well tolerated; 93% completed the trial. LS mean±SE A1C change was 0.2±0.1% for PBO vs. ±0.4±0.1% and -0.9±0.2% for 0.9 and 1.8 mg ZGN-1061 (both p Disclosure R. Fabunmi: None. R.N. Bergman: Consultant; Self; Zafgen, Inc. F. Piccinini: None. T. Kim: Employee; Spouse/Partner; Celgene Corporation. Employee; Self; Zafgen, Inc. D. Zhuang: Employee; Self; Zafgen, Inc. K. Taylor: Employee; Self; Zafgen, Inc. D.D. Kim: Employee; Self; Zafgen, Inc. Funding Zafgen, Inc.