Hyperinsulinemic-euglycemic Clamp Studies Research Articles

Role of human Kallistatin in glucose and energy homeostasis in mice

ObjectiveKallistatin (KST), also known as SERPIN A4, is a circulating, broadly acting human plasma protein with pleiotropic properties. Clinical studies in humans revealed reduced KST levels in obesity. The exact role of KST in glucose and energy homeostasis in the setting of insulin resistance and type 2 diabetes is currently unknown. MethodsKallistatin mRNA expression in human subcutaneous white adipose tissue (sWAT) of 47 people with overweight to obesity of the clinical trial “Comparison of Low Fat and Low Carbohydrate Diets With Respect to Weight Loss and Metabolic Effects (B-SMART)” was measured. Moreover, we studied transgenic mice systemically overexpressing human KST (hKST-TG) and wild type littermate control mice (WT) under normal chow (NCD) and high-fat diet (HFD) conditions. ResultsIn sWAT of people with overweight to obesity, KST mRNA increased after diet-induced weight loss. On NCD, we did not observe differences between hKST-TG and WT mice. Under HFD conditions, body weight, body fat and liver fat content did not differ between genotypes. Yet, during intraperitoneal glucose tolerance tests (ipGTT) insulin excursions and HOMA-IR were lower in hKST-TG (4.42 ± 0.87 AU, WT vs. 2.20 ± 0.27 AU, hKST-TG, p < 0.05). Hyperinsulinemic euglycemic clamp studies with tracer-labeled glucose infusion confirmed improved insulin sensitivity by higher glucose infusion rates in hKST-TG mice (31.5 ± 1.78 mg/kg/min, hKST-TG vs. 18.1 ± 1.67 mg/kg/min, WT, p < 0.05). Improved insulin sensitivity was driven by reduced hepatic insulin resistance (clamp hepatic glucose output: 7.7 ± 1.9 mg/kg/min, hKST-TG vs 12.2 ± 0.8 mg/kg/min, WT, p < 0.05), providing evidence for direct insulin sensitizing effects of KST for the first time. Insulin sensitivity was differentially affected in skeletal muscle and adipose tissue. Mechanistically, we observed reduced Wnt signaling in the liver but not in skeletal muscle, which may explain the effect. ConclusionsKST expression increases after weight loss in sWAT from people with obesity. Furthermore, human KST ameliorates diet-induced hepatic insulin resistance in mice, while differentially affecting skeletal muscle and adipose tissue insulin sensitivity. Thus, KST may be an interesting, yet challenging, therapeutic target for patients with obesity and insulin resistance.

Impact of Hematopoietic Cell Transplantation on Cardiovascular Risk Factors and Insulin Sensitivity

The use of hematopoietic cell transplantation (HCT) for treating malignant conditions in children has increased over the past five decades, leading to a growing population of long-term survivors.This population of childhood HCT survivors faces increased risks of adverse medical effects due to cancer treatments, including adverse cardiovascular disease (CVD) risk factors such as metabolic syndrome, insulin resistance. but the impact of exposure to HCT preparative conditioning regimen has not been clearly delineated. These risk factors, including obesity, dyslipidemia, hypertension, and insulin resistance (IR), are significant contributors to premature cardiovascular disease and represent a leading cause of non-relapse deaths in childhood cancer and HCT survivors. This study aimed to assess the early development of CVD risk factors and their relationship to insulin resistance in a large population of pediatric and young adult HCT survivors of childhood hematologic malignancies. The study compared their cardiovascular risk profiles, insulin resistance (measured by euglycemic hyperinsulinemic clamp studies), and body composition (determined by dual X-ray absorptiometry - DXA) with a cohort of sibling controls. We enrolled 151 HCT recipients (26.36 ±0.90 years at study enrollment; time since HCT of 2.6-31.5 years) and 92 sibling controls to complete at cardiovascular risk assessment including insulin sensitivity by hyperinsulinemic euglycemic clamp, anthropometry, body composition by dual X-ray absorptiometry, blood pressure, and serum biomarkers. We used linear models to test for mean differences in all continuous outcomes between survivors and siblings, accounting for intra-family correlations with generalized estimating equations. Recipients of HCT were found to have lower insulin sensitivity and more likely to have adverse CVD risk factors in comparison to their healthy siblings. Significantly higher percent fat mass and visceral adipose tissue, and significantly lower lean body mass were noted in HCT recipients than sibling controls despite having a similar body mass index between the two groups. Total body irradiation in the conditioning regimen was one of the strongest factors associated with lower insulin sensitivity, dyslipidemia and abnormal body composition leading to sarcopenic obesity. This study reveals that pediatric and young adult HCT survivors are more insulin resistant and have a higher prevalence of adverse cardiovascular risk factors compared to sibling controls. The presence of cardiovascular risk factors at a relatively young age raises concerns about an escalating trajectory of cardiovascular disease in this population. Therefore, regular monitoring of HCT survivors for cardiometabolic risk factors and early intervention will be crucial for preventing cardiovascular-related complications in the future. The findings underscore the importance of survivorship care for pediatric and young adult HCT survivors, with a focus on managing cardiovascular risk factors and promoting a healthy lifestyle to mitigate long-term adverse effects. Early identification and targeted interventions can significantly improve the long-term health outcomes of this vulnerable population, reducing the burden of cardiovascular disease and related complications.

Study Protocol for the Pleiotropic Effects of Sodium-Glucose Cotransporter2 Inhibitor on Organ-Specific Sympathetic Nerve Activity and Insulin Sensitivity in Participants with Type2 Diabetes.

Hyperinsulinemia and hyperglycemia are associated with exaggerated systemic sympathetic nerve activity (SNA) in patients with type2 diabetes. Sodium-glucose cotransporter2 (SGLT2) inhibitors lower insulin levels, whereas sulfonylureas increase insulin levels. We will test whether these two classes of antidiabetic agents have different effects on SNA. The present study is an ongoing, 24-week, one-center (only Kanazawa University Hospital), open-label, randomized, parallel trial (jRCTs 041200035). Participants with type2 diabetes with multiple atherosclerosis risk factors are randomly assigned in a 1:1 manner to receive 2.5mg luseogliflozin or 0.5mg glimepiride once daily. The sample size was calculated to be 14 in each group, with a significance level of 0.05 and a power of 0.80. The design required 40 evaluable study participants. Our primary endpoint will be the change in muscle SNA (MSNA). The secondary endpoints included organ-specific insulin sensitivity measured by a hyperinsulinemic-euglycemic clamp study using an artificial pancreas combined with a stable isotope-labeled glucose infusion, bioelectrical impedance analysis, and organ-specific (cardiac, renal, and hepatic) 123I-meta-iodobenzylguanidine (MIBG) innervation imaging. Study recruitment started in April 2020 and will end in June 2024, with 40 participants randomized into the two groups. The treatment follow-up of the participants is currently ongoing and is due to finish by March 2025. The study protocol has been approved by the Certified Review Board, Kanazawa University, Ishikawa, Japan, in accordance with the guidelines stipulated in the Declaration of Helsinki (CRB4180005, 2019-001). This trial is registered with the Japan Registry of Clinical Trials, jRCTs 041200035.

Suppression of Endogenous Insulin Secretion by Euglycemic Hyperinsulinemia.

The impact of insulin, particularly exogenous hyperinsulinemia, on insulin secretion in humans is debated. We assessed the effects of exogenous hyperinsulinemia on insulin secretion and whether the response is altered in insulin resistance associated with obesity. Insulin secretion rates (ISRs) during euglycemic hyperinsulinemic clamp studies (52 volunteers) were calculated using a model that employs plasma C-peptide concentrations. One study involved a 2-step insulin clamp and the other study was a single step insulin clamp. For both studies the goal was to achieve plasma glucose concentrations of 95 mg/dL during the clamp irrespective of fasting glucose concentrations. The percent change in ISR from fasting to the end of the insulin clamp interval was the main outcome. Linear regression and analysis of covariance were used to test for the effects of insulin on ISR and to test for group differences. ISR was greater in obese volunteers (P < .001) under fasting and hyperinsulinemic clamp conditions. The change in plasma glucose from baseline to the end of the insulin clamp interval was highly correlated with the change in ISR (r = 0.61, P < .001). From baseline to the end of the clamp we observed a 27% (SD 20) suppression of ISR. The participants who underwent a 2-step insulin clamp had greater suppression of ISR during the second step than the first step (P < .001). The proportional suppression of ISR during euglycemic hyperinsulinemia was not different between nonobese and obese groups (P = .19). Hyperinsulinemia suppresses endogenous insulin secretion and the relative change in insulin secretion produced by exogenous insulin did not differ between nonobese and obese people.

Bariatric surgery improves postprandial VLDL kinetics and restores insulin mediated regulation of hepatic VLDL production.

Dyslipidemia in obesity results from excessive production and impaired clearance of triglyceride (TG)-rich lipoproteins, which is particularly pronounced in the postprandial state. Here, we investigated the impact of Roux-en-Y gastric bypass (RYGB) surgery on the postprandial VLDL1 and VLDL2 apoB and TG kinetics and their relationship with insulin responsiveness indices. 24 obese non-diabetic RYGB surgery patients underwent a lipoprotein kinetics study during a mixed meal test and a hyperinsulinemic-euglycemic clamp study before the surgery, and one year later. A physiologically based computational model was developed to investigate the impact of RYGB surgery and plasma insulin on postprandial VLDL kinetics. After the surgery, VLDL1 apoB and TG production rates were significantly decreased, whereas VLDL2 apoB and TG production rates remained unchanged. TG catabolic rate was increased in both VLDL1 and VLDL2 fractions, but only the VLDL2 apoB catabolic rate tended to increase. Furthermore, post-surgery VLDL1 apoB and TG production rates, but not VLDL2, were positively correlated with insulin resistance. Insulin-mediated stimulation of peripheral lipoprotein lipolysis was also improved after the surgery. In summary, RYGB resulted in a reduced hepatic VLDL1 production that correlated with reduced insulin resistance, an elevated VLDL2 clearance, and improved insulin sensitivity in lipoprotein lipolysis pathways.

Pharmacokinetics, pharmacodynamics, and safety of prandial oral insulin (N11005) in healthy subjects.

To verify whether the oral insulin N11005 is administered as a prandial insulin by assessing the pharmacokinetics (PK), pharmacodynamics (PD), and safety profiles of N11005 with a short-acting biosynthetic human insulin (Novolin R) as reference. This was a randomized, open-label, single-dose, crossover hyperinsulinemic-euglycemic clamp study in healthy Chinese male subjects. A total of 12 subjects were enrolled in the test (T) group (N11005, 300 IU, p.o.) and the reference (R) group (Novolin R, 0.1 IU/Kg, i.h.) with a washout period of 14 days. All subjects were administered on the same day of the clamp study. Glucose Infusion Rates (GIR), serum insulin, and C-peptide concentration were determined during every 8-hour clamp cycle. Trial registration: Clinicaltrials.gov identifier NCT04975022. After administration, the ratios of mean serum C-peptide concentration to baseline concentration in both T and R groups were lower than 50%, which confirmed the stability of the clamp platform. T group (N11005) showed a more rapid onset of action (tGIR10%max≈11 min) and a comparable duration of action to the R group, which was basically in line with the characteristics of prandial insulins. No adverse events (AEs) occurred throughout the study, which demonstrated that N11005 and Novolin R are safe and well-tolerated. The PD profiles of the single-dose N11005 in the human body are similar to those of prandial insulins, with an excellent safety profile. Clinicaltrials.gov, identifier NCT04975022.

The role of postprandial thermogenesis in the development of impaired glucose tolerance and type II diabetes.

Accounting for 5%-15% of total daily energy expenditure, postprandial thermogenesis (PPT) refers to an acute increase in resting metabolic rate (RMR) in the hours after eating. This is largely explained by the energy costs of processing the macronutrients of a meal. Most individuals spend the majority of the day in the postprandial state, thus over one's lifetime even minor differences in PPT may possess true clinical significance. In contrast to RMR, research indicates that PPT may be reduced in the development of both prediabetes and type II diabetes (T2D). The present analysis of existing literature has found that this impairment may be exaggerated in hyperinsulinemic-euglycemic clamp studies compared with food and beverage consumption studies. Nonetheless, it is estimated that daily PPT following carbohydrate consumption alone is approximately 150 kJ lower among individuals with T2D. This estimate fails to consider protein intake, which is notably more thermogenic than carbohydrate intake (20%-30% vs. 5%-8%, respectively). Putatively, dysglycemic individuals may lack the insulin sensitivity required to divert glucose toward storage-a more energy-taxing pathway. Accordingly, the majority of findings has associated an impaired PPT with a reduced "obligatory" energy output (i.e., the energy costs associated with nutrient processing). More recently, it has been reported that "facultative" thermogenesis [e.g., the energy costs associated with sympathetic nervous system (SNS) stimulation] may also contribute to any impairment in PPT among individuals with prediabetes and T2D. Further longitudinal research is required to truly ascertain whether meaningful changes in PPT manifest in the prediabetic state, before the development of T2D.

240-LB: Unrestrained Sympathetic Signaling Is a Key Driver of Obesity-Induced Insulin Resistance

Obesity is the major driver of type 2 diabetes. The mechanisms that account for overnutrition induced insulin resistance are yet to be delineated as impaired cellular insulin signaling may or may not account for the impaired insulin action. We find that overnutrition rapidly impairs insulin action and increase plasma norepinephrine, suggesting heightened activation of the sympathetic nervous system (SNS), possibly due to brain insulin resistance as earlier studies from our group suggested. Here, we hypothesize that unrestrained SNS activity (SNA) is a key driver of obesity induced insulin resistance. To test this hypothesis, we examined a mouse model of inducible and peripherally restricted tyrosine hydroxylase (th) gene deletion (THΔper) that have profoundly lower catecholamine levels in peripheral tissues is protected from high fat diet (HFD) induced insulin resistance. Despite similar food intake, obesity and cellular insulin signaling in liver and adipose tissue, THΔper mice were protected from both short-term and long-term HFD feeding-induced glucose intolerance, increases in plasma catecholamines and hyperglucagonemia. Hyperinsulinemic euglycemic clamp studies revealed that overnutrition induced hepatic insulin resistance was markedly ameliorated in the THΔper mice, even though cellular insulin signaling in liver and adipose tissue was comparable. Moreover, THΔper mice were protected from HFD-induced adipose tissue dysfunction characterized by reduced lipogenic capacity, unrestrained lipolysis and increased low-grade inflammation as well as hepatic steatosis, possibly due to decreased lipolytic flux and hepatic de novo lipogenesis. In addition, fibrosis genes were markedly reduced in the liver of the THΔper mice, suggesting that reducing SNA can prevent metabolic dysfunction-associated fatty liver disease. These results demonstrate that unrestrained SNA is a pivotal driver of insulin resistance, hyperglucagonemia and metabolic dysfunction in obesity. Disclosure K. Sakamoto: None. G. Maurizi: None. B. Chen: None. A. Shawkat: None. L. Patlolla: None. A. Tahiri: None. A. Butera: None. C. Buettner: None.

1514-P: Insulin Sensitivity Initially Worsens but Later Improves with Aging in C57BL/6N Mice

Objective: Aging is believed to induce insulin resistance in humans. However, when and how insulin sensitivity changes with aging remain unclear in both humans and mice. Methods: Groups of C57BL/6N mice at 9-15 weeks (young), 34-67 weeks (one-year-old) and 107-121 weeks (two-year-old) of age underwent hyperinsulinemic-euglycemic clamp studies with somatostatin infusion under awake and non-restrained conditions. Results: The glucose infusion rates for maintaining euglycemia were 17.11±2.94, 5.58±1.28 and 25.30±4.42 mg/kg/min in young, one-year-old and two-year-old mice, respectively. Thus, compared with young mice, one-year-old mice exhibited the expected insulin resistance. In contrast, the insulin sensitivity of two-year-old mice showed significant improvement. These age-related changes were mainly observed in glucose uptake into adipose tissue and skeletal muscle. Epididymal fat weight and hepatic triglyceride levels were highest in one-year-old mice, while being similar in young and two-year-old mice. Conclusions: Our observations indicate that, in mice, insulin resistance appears at one year of age but subsequently improves markedly. These alterations in insulin sensitivity are attributable to changes in visceral fat accumulations and age-related factors. Disclosure H.Kondo: None. H.Ono: None. K.Yokote: None. Funding Japan Society for the Promotion of Science (15K09398, 18K08502, 20H00524); Japan Agency for Medical Research and Development (17GM5010002H0001, 18GM5010002H0002, 19GM5010002H0003, 20GM5010002H0004)

Insulin sensitivity initially worsens but later improves with aging in male C57BL/6N mice.

Aging is believed to induce insulin resistance in humans. However, when and how insulin sensitivity changes with aging remain unclear in both humans and mice. In this study, groups of male C57BL/6N mice at 9-19 weeks (young), 34-67 weeks (mature adult), 84-85 weeks (presenile) and 107-121 weeks (aged) of age underwent hyperinsulinemic-euglycemic clamp studies with somatostatin infusion under awake and non-restrained conditions. The glucose infusion rates for maintaining euglycemia were 18.4±2.9, 5.9±1.3, 20.3±7.2 and 25.3±4.4mg/kg/min in young, mature adult, presenile and aged mice, respectively. Thus, compared with young mice, mature adult mice exhibited the expected insulin resistance. In contrast, presenile and aged mice showed significantly higher insulin sensitivity than the mature adult mice. These age-related changes were mainly observed in glucose uptake into adipose tissue and skeletal muscle (rates of glucose disappearance were 24.3±2.0, 17.1±1.0, 25.5±5.2 and 31.8±2.9mg/kg/min in young, mature adult, presenile and aged mice, respectively). Epididymal fat weight and hepatic triglyceride levels were higher in mature adult mice than those in young and aged mice. Our observations indicate that, in male C57BL/6N mice, insulin resistance appears at the mature adult stage of life but subsequently improves markedly. These alterations in insulin sensitivity are attributable to changes in visceral fat accumulations and age-related factors.

Adipose tissue function and insulin sensitivity in syndromic obesity of Bardet-Biedl syndrome.

Bardet-Biedl syndrome (BBS) is a rare autosomal recessive syndromic obesity of childhood onset among many other features. To date, the excess risk of metabolic complications of severe early-onset obesity in BBS remains controversial. In-depth investigation of adipose tissue structure and function with detailed metabolic phenotype has not been investigated yet. To investigate adipose tissue function in BBS. A prospective cross-sectional study. To determine if there are differences in insulin resistance, metabolic profile, adipose tissue function and gene expression in patients with BBS compared to BMI-matched polygenic obese controls. 9 adults with BBS and 10 controls were recruited from the national centre for BBS, Birmingham, UK. An in-depth study of adipose tissue structure and function along with insulin sensitivity was performed using hyperinsulinemic-euglycemic clamp studies, adipose tissue microdialysis, histology and RNA sequencing, and measurement of circulating adipokines and inflammatory biomarkers. Adipose tissue structure, gene expression and in vivo functional analysis between BBS and polygenic obesity cohorts were similar. Using hyperinsulinemic-euglycemic clamp and surrogate markers of insulin resistance, we found no significant differences in insulin sensitivity between BBS and obese controls. Furthermore, no significant changes were noted in an array of adipokines, cytokines, pro-inflammatory markers and adipose tissue RNA transcriptomic. Although childhood-onset extreme obesity is a feature of BBS, detailed studies of insulin sensitivity and adipose tissue structure and function are similar to common polygenic obesity. This study adds to the literature by suggesting that it is the quality and quantity of adiposity not the duration that drives the metabolic phenotype.

Insulin Resistance in Adolescents and Youth With Type 1 Diabetes: A Review of Problems and Solutions.

Though insulin resistance (IR) was previously considered a feature of only type 2 Diabetes (T2DM), its development in type 1 Diabetes (T1DM) is not an uncommon occurrence, the causes of which are multifactorial (gender, pubertal status, diabetes duration, ethnicity, genetics, adiposity, glycemic control, chronic inflammation). Despite improvements in glucose, blood pressure and lipid profile, vascular complications (coronary artery disease and nephropathy) continue to remain common causes of morbidity and mortality in T1DM. Aggressive glycemic control reduces but does not eliminate the risk of IR. IR accelerates the development of micro and macrovascular complications, many of which can be potentially reversed if diagnosed and managed early. Lack of endogenous insulin production makes estimation of insulin sensitivity in T1DM difficult. As hyperinsulinemic-euglycemic clamp studies are cumbersome and invasive, the use of prediction equations for calculating estimated insulin sensitivity may prove to be useful. Along with intensive insulin therapy, dietary modifications and increasing physical activity, the role of Metformin in managing IR in T1DM is becoming increasingly popular. Metformin adjunct therapy in T1DM has been shown to improve insulin sensitivity, glycemic control, lipid profile, body composition, vascular smooth muscle function, thereby reducing the risk of vascular complications, as well as reversal of early vascular dysfunction. However, further studies to assess long-term efficacy and safety of Metformin use in adolescents and youth with T1DM are needed. This review aims at revisiting the pathophysiology of IR in T1DM and techniques of identifying those at risk so as to put into action various strategies for management of the same.

Independent euglycaemic hyperinsulinaemic clamp studies validate clinically applicable formulae to estimate insulin sensitivity in people with type 1 diabetes

Background and aimLow insulin sensitivity (IS) increases Type 1 diabetes (T1D) complication risk and can be estimated by simple formulae developed from complex euglycemic hyperinsulinaemic clamp studies. We aimed to validate these formulae using independent clamp data. MethodsClamps were performed in 104 T1D adults. Measured glucose disposal rate (GDR) was correlated with eGDR and eLog10 M/I calculated by five IS formulae. ResultsCorrelations ranged between 0.23–0.40. Two IS formulae (by the authors), using age, sex, HDL-C, HbA1c, pulse pressure, BMI, and waist-hip-ratio had the highest correlation with measured GDR and the best performance in detecting low IS.

OR08-4 Endocrine Mechanisms of an Orphan G Protein-Coupled Receptor Regulating Metabolic Homeostasis

Abstract G protein-coupled receptors (GPCRs) are the largest protein family and amenable for pharmacological manipulation. GPCRs in the neuroendocrine and enteroendocrine systems respond to various biological cues and in turn exert crucial roles in maintaining whole body metabolic homeostasis. For example, GPCRs in the gastrointestinal tract are involved in maintaining glucose and energy homeostasis by regulating the release of gut hormones in response to luminal dietary nutrients as well as microbial metabolites. We recently identified that an orphan GPCR, Gpr17, was co-expressed in glucagon-like peptide-1 (GLP-1)-expressing EECs in human and rodent intestinal epithelium. We reported that acute genetic ablation of Gpr17 in intestinal epithelium (iKO) improved oral glucose tolerance and glucose-stimulated insulin secretion (GSIS) in female and male mice through increased secretion of incretin hormone in response to nutrient ingestion (Cell Reports, 2022). In the neuroendocrine system, we previously identified Gpr17 as the transcription target of Forkhead box protein O1 (FoxO1) in the hypothalamus and generated hypothalamic neuron specific Gpr17 knockout animals (Cell, 2012). Our results demonstrated that agouti-related peptide (AgRP) neuron Gpr17 knockout mice had increased satiety during fasting-refeeding challenge (Diabetes, 2015) and that proopiomelanocortin (POMC) neuron Gpr17 knockout mice had increased alpha-melanocyte stimulating hormone (aMSH) processing and increased firing of POMC neurons (Nutrition &amp; Diabetes, 2019). However, how Gpr17 may regulate energy balance especially regarding leptin hormone signaling is unclear. To address this question, we used genetic knockout approach to generate Gpr17 conditional knockout in leptin receptor expressing neurons in the brain. We found Gpr17 deficiency in leptin receptor expressing neurons increased leptin sensitivity in animals and resulted in reduced food intake, which corresponded with reduced inflammatory response in the hypothalamus. Moreover, male Gpr17 whole body knockout mice have improved intraperitoneal glucose tolerance, and increased insulin sensitivity in hyperinsulinemic-euglycemic clamp studies. In conclusion, our collective body of work showed that ablation of Gpr17 signaling in the neuroendocrine and enteroendocrine systems led to improved neurohormonal regulation to maintain metabolic homeostasis, indicating functionally targeting this GPCR may lead to better therapeutic outcome of diabetes and obesity. Presentation: Saturday, June 11, 2022 12:15 p.m. - 12:30 p.m.

1356-P: Investigating the Metabolic Function of an Orphan G Protein–Coupled Receptor in the Intestinal Epithelium

G protein-coupled receptors (GPCRs) in the gastrointestinal tract are involved in maintaining glucose and energy homeostasis by regulating the release of gut hormones in response to luminal dietary nutrients as well as microbial metabolites. We recently identified that an orphan GPCR, Gpr17, was co-expressed in glucagon-like peptide-1 (GLP-1) -expressing EECs in human and rodent intestinal epithelium. However, it is unknown how Gpr17 ablation in the intestinal epithelium affects feeding behavior and satiety regulation. To address this question, we used genetic knockout approach to generate intestinal Gpr17-deficient mice and analyzed their glucose metabolism and feeding behavior. We showed that intestinal Gpr17-deficient mice had similar growth curve, body composition, and ad libitum food intake compared with littermate controls. We found that acute genetic ablation of Gpr17 in intestinal epithelium (iKO) improved oral glucose tolerance and glucose-stimulated insulin secretion (GSIS) in female and male mice. iKO mice responded to glucose or lipid ingestion with increased secretion of GLP-1, but not the other incretin glucose-dependent insulinotropic polypeptide (GIP) . Intestinal Gpr17-deficient mice responded to fasting-refeeding challenge with reduced fasting locomotor activity and less food intake after refeeding, suggesting increased satiety during the phase of rebound hyperphagia. Furthermore, comprehensive studies including gut morphology, gut epithelium heterogeneity by scRNA-seq, and microbiome analysis were examined but none of them was changed because of intestinal Gpr17 ablation. In addition, male Gpr17 whole body knockout mice have improved intraperitoneal glucose tolerance, and increased insulin sensitivity in hyperinsulinemic-euglycemic clamp studies. In conclusion, our work showed that ablation of intestinal Gpr17 signaling led to improved neurohormonal regulation to maintain metabolic homeostasis. Disclosure H.Ren: None. Funding National Institute of Health (R01DK120772)

1336-P: FoxO1-ATGL-Sirt1-FoxO1 Feed Forward Signaling Mediates Effects on Hepatic Gene Expression and Glucose Homeostasis in LIRKO Mice

FoxO1 is a major target of insulin action and regulates expression of ATGL and its inhibitor, G0S2, in the liver (Cell Rep 15:349, 2016) . Here, we asked whether ATGL mediates effects of FoxO1 in liver-specific insulin receptor knockout (LIRKO) mice. Studies in LIRKO and IR/FoxO1 double knockout (LIRFKO) mice show ATGL expression is increased and G0S2 is suppressed in a FoxO1-dependent fashion when insulin signaling is disrupted in the liver. ATGL knockdown (KD) reduces high plasma glucose and insulin concentrations, HOMA-IR and fatty acid oxidation, and improves glucose tolerance in LIRKO mice. Hyperinsulinemic-euglycemic clamp studies show ATGL KD improves insulin sensitivity due to effects on both HGP and glucose utilization, reflecting changes in WAT lipolysis, BAT metabolism and hepatic gene expression. The expression of FoxO1-regulated gluconeogenic genes (PEPCK, G6Pase, PCO, PGC-1α) , hepatokines (e.g., follistatin) and IGFBP-1 is increased in LIRKO mice and reduced by ATGL KD, reflecting changes in acetylation of FoxO1 and its co-activator, PGC-1α (decreased in LIRKO, increased by ATGL KD) . Rictor acetylation, which promotes activation of Akt and phosphorylation of FoxO1, also is increased by ATGL KD in LIRKO mice and isolated hepatocytes. Since ATGL promotes activation of Sirt1 (Mol Cell 77:810, 2020) and Sirt1 deacetylates FoxO1, PGC-1α and rictor, these studies suggest FoxO1 promotes increased ATGL and Sirt1 activity, and that Sirt1 activation enhances the effects of FoxO1 on gene expression and glucose homeostasis in a feed forward fashion (FoxO1 -&amp;gt; ATGL -&amp;gt; Sirt1 -&amp;gt; FoxO1) . Targeting the FoxO1/ATGL/Sirt1 pathway may provide a useful strategy for improving glucose homeostasis when insulin signaling is impaired in the liver. Disclosure I.O-sullivan: None. S.Bhattacharyya: None. C.Liew: None. S.M.Lee: None. J.Cordoba-chacon: None. R.J.Perry: None. G.I.Shulman: Advisory Panel; 89bio, Inc., AstraZeneca, Equator Therapeutics, Inc., Janssen Research &amp; Development, LLC, Merck &amp; Co., Inc., Consultant; DiCerna Pharmaceuticals, Inc., Novo Nordisk, Other Relationship; Generian Pharmaceuticals, iMetabolic Biopharma Corporation, Maze Therapeutics, The Liver Company, Stock/Shareholder; Levels Health, Inc. . T.Unterman: None. Funding Department of Veterans Affairs (5I01BX001968)

An Atypical Form of Diabetes Among Individuals With Low BMI.

Diabetes among individuals with low BMI (<19 kg/m2) has been recognized for >60 years as a prevalent entity in low- and middle-income countries (LMICs) and was formally classified as "malnutrition-related diabetes mellitus" by the World Health Organization (WHO) in 1985. Since the WHO withdrew this category in 1999, our objective was to define the metabolic characteristics of these individuals to establish that this is a distinct form of diabetes. State-of-the-art metabolic studies were used to characterize Indian individuals with "low BMI diabetes" (LD) in whom all known forms of diabetes were excluded by immunogenetic analysis. They were compared with demographically matched groups: a group with type 1 diabetes (T1D), a group with type 2 diabetes (T2D), and a group without diabetes. Insulin secretion was assessed by C-peptide deconvolution. Hepatic and peripheral insulin sensitivity were analyzed with stepped hyperinsulinemic-euglycemic pancreatic clamp studies. Hepatic and myocellular lipid contents were assessed with 1H-nuclear magnetic resonance spectroscopy. The total insulin secretory response was lower in the LD group in comparison with the lean group without diabetes and the T2D group. Endogenous glucose production was significantly lower in the LD group than the T2D group (mean ± SEM 0.50 ± 0.1 vs. 0.84 ± 0.1 mg/kg · min, respectively; P < 0.05). Glucose uptake was significantly higher in the LD group in comparison with the T2D group (10.1 ± 0.7 vs. 4.2 ± 0.5 mg/kg · min; P < 0.001). Visceral adipose tissue and hepatocellular lipids were significantly lower in LD than in T2D. These studies are the first to demonstrate that LD individuals in LMICs have a unique metabolic profile, suggesting that this is a distinct entity that warrants further investigation.

Metabolic state switches between morning and evening in association with circadian clock in people without diabetes.

Aims/Introduction Understanding morning–evening variation in metabolic state is critical for managing metabolic disorders. We aimed to characterize this variation from the viewpoints of insulin secretion and insulin sensitivity, including their relevance to the circadian rhythm. Materials and Methods A total of 14 and 10 people without diabetes were enrolled, and underwent a 75‐g oral glucose tolerance test (OGTT) and hyperinsulinemic‐euglycemic clamp study, respectively. Participants completed the OGTT or hyperinsulinemic‐euglycemic clamp at 08.00 hours and 20.00 hours in random order. Before each study, hair follicles were collected. In mice, phosphorylation levels of protein kinase B were examined in the liver and muscle by western blotting. Results Glucose tolerance was better at 08 .00 hours, which was explained by the higher 1‐h insulin secretion on OGTT and increased skeletal muscle insulin sensitivity on hyperinsulinemic‐euglycemic clamp. Hepatic insulin sensitivity, estimated by the hepatic insulin resistance index on OGTT, was better at 20.00 hours. The 1‐h insulin secretion and hepatic insulin resistance index correlated significantly with Per2 messenger ribonucleic acid expression. The change (evening value – morning value) in the glucose infusion rate correlated significantly with the change in non‐esterified fatty acid, but not with clock gene expressions. The change in non‐esterified fatty acid correlated significantly with E4bp4 messenger ribonucleic acid expression and the change in cortisol. In mice, phosphorylation of protein kinase B was decreased in the liver and increased in muscle in the beginning of the active period as, expected from the human study. Conclusions Glucose metabolism in each tissue differed between the morning and evening, partly reflecting lipid metabolism, clock genes and cortisol levels. Deeper knowledge of these associations might be useful for ameliorating metabolic disorders.

Pharmacological Inhibition of Cullin Neddylation Improves Hepatic Insulin Sensitivity

Background and significanceHepatic insulin resistance is a hallmark feature of non‐alcoholic fatty liver disease (NAFLD) and type‐2 diabetes (T2D) and significantly contributes to systemic insulin resistance. Abnormal activation of nutrient and stress‐sensing kinases leads to serine/threonine phosphorylation of insulin receptor substrate (IRS) and subsequent IRS proteasome degradation, which is a key underlying cause of hepatic insulin resistance. Recently, members of the cullin‐RING E3 ligases (CRLs) have emerged as mediators of IRS protein turnover. CRLs are activated upon cullin neddylation, a process of covalent conjugation of a ubiquitin‐like protein called Nedd8 to a cullin scaffold.AimTo investigate the role of CRLs in regulating hepatic insulin signaling in NAFLD.MethodsNeddylation inhibitor MLN4924 was used to study its effect on insulin sensitivity in liver cells. Hyperinsulinemic‐euglycemic clamp was used to assess MNL4924 effect on insulin sensitivity in vivo. siRNA‐mediated screening was used to identify the Cullin members that mediates IRS protein turnover in liver cells. Co‐immunoprecipitation was performed to study Cullin and IRS interaction. Glucose homeostasis and insulin sensitivity were studied in mice with AAV‐mediated liver specific knockdown of Cul1 or Cul3.ResultsNeddylation inhibition delays CRL‐mediated IRS protein turnover to prolong insulin action in cultured liver cells. Consistently, acute MLN4924 treatment rapidly decreases hepatic glucose production without causing hypoglycemia in mice, while chronic MLN4924 treatment completely normalizes hyperglycemia in obese mice independent of obesity. Hyperinsulinemic‐euglycemic clamp study revealed that MLN4924 significantly increased insulin sensitivity and improved hepatic glucose output. In vitro siRNA based screening showed that knockdown of Cul1 or Cul3, but not other cullin family members, stabilized IRS protein and increased insulin sensitivity in AML12 cells. Consistently, Co‐immunoprecipitation demonstrated IRS interaction with Cul1 and Cul3. AAV‐mediated liver specific knockdown of Cul1 in mice improved insulin sensitivity, lowered fasting glucose, and prevented Western diet‐induced obesity and NAFLD. In contrast, AAV‐mediated liver Cul3 knockdown only decreased fasting glucose in chow‐fed mice but not Western diet‐fed mice.ConclusionThese findings suggest that pharmacological inhibition of cullin neddylation is a novel therapeutic strategy for hepatic insulin sensitization in NAFLD and T2D. This effect may be at least in part mediated by hepatic Cul1 and Cul3 neddylation.

Insulin sensitivity and obstructive sleep apnea in adolescents with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) in adults is linked with insulin resistance (IR) and obstructive sleep apnea (OSA). However, less is known about these associations in adolescents. We studied 3 groups of adolescents: 27 obese PCOS (OPCOS) (ages 13-21)11 normal-weight PCOS (NPCOS) (ages 13-21 years), and 8 healthy controls (ages 18-21 years). A hyperinsulinemic euglycemic clamp study was performed in all groups to determine IR by insulin sensitivity (M/I). Polysomnography was performed to assess for OSA in OPCOS and NPCOS groups. We compared indices of IR among all groups and OSA among OPCOS, and NPCOS. We noted that OPCOS and NPCOS and controls differed significantly in their IR. M/I was significantly lower in OPCOS vs. controls (p=0.0061), and also lower for NPCOS vs control but this approached but did not reach statistical significance (p=0.084). In addition, none of the NPCOS subjects had OSA compared to 42% of OPCOS (p=0.03). Our study suggests OPCOS adolescents have increased IR compared to controls and NPCOS subjects. Higher IR for NPCOS vs controls approached but did not reach statistical significance. Larger studies are needed. In addition, adolescents with OPCOS are at a high risk for OSA.