bluuebunny/crossref_metadata_2025 huggingface dataset:
The Molecular Role of WNT5A and Its Antagonist SFRP5 in Gluconeogenesis and Inflammation in Human Hepatocytes
Authors: CARSTENSEN-KIRBERG MAREN , NIERSMANN CORINNA , ROEHRIG KARIN , RODEN MICHAEL , HERDER CHRISTIAN
Published: June 2018

The wingless-related MMTV integration site (WNT)5A has pro-inflammatory effects and induces insulin resistance in mice. The secreted frizzled-related protein 5 (SFRP5) blocks the deleterious effects of WNT5A in mice. In humans, the interaction of WNT5A and SFRP5 in the pathogenesis of type 2 diabetes is almost unknown. Therefore, the aim of this study was to characterize the interplay of WNT5A and SFRP5 in glucose metabolism and inflammation in human hepatocytes. We treated the human HepaRG cell line without or with (i) 5 ng/ml WNT5A, (ii) 5000 ng/ml SFRP5 as well as (iii) 5 ng/ml WNT5A and 5000 ng/ml SFRP5 for 24 h. The mRNA levels of key enzymes of the gluconeogenesis were measured using real-time PCR. We analyzed 92 inflammatory proteins in the supernatant using the OLINK inflammation panel. Total and phosphorylated protein content of markers of inflammatory pathways were analysed using Western blotting. WNT5A increased mRNA levels of phosphoenolpyruvate carboxykinase (PCK)2 and glucose-6-phosphatase catalytic subunit (G6PC) by 50% and 196% compared to control (p<0.05). Interestingly, the co-treatment with SFRP5 decreased PCK2 and G6PC mRNA by 36% and 48% compared to control (p<0.05). In addition, WNT5A reduced the secretion of protein levels of 23 pro-inflammatory proteins in the supernatant on average by 56% compared to control (p<0.05). Most of these proteins were chemokines (e.g., CXCL5, CCL2) and cytokines (e.g., IL-6, IL-18). WNT5A also decreased the phosphorylation level of NF-κB by 39% (p<0.01). The co-treatment with SFRP5 led to a secretion profile similar to control.

In conclusion, contrary to mouse models, WNT5A has anti-inflammatory properties in human HepaRG which might be mediated by inactivation of the NF-κB signaling pathway. Nevertheless, WNT5A adversely affects the expression of gluconeogenic enzymes. SFRP5 antagonizes both beneficial and harmful hepatic effects of WNT5A.

Disclosure

M. Carstensen-Kirberg: None. C. Niersmann: None. K. Roehrig: None. M. Roden: Speaker's Bureau; Self; Boehringer Ingelheim GmbH. Research Support; Self; Boehringer Ingelheim GmbH. Consultant; Self; Poxel SA. Research Support; Self; Danone Nutricia Early Life Nutrition, GlaxoSmithKline plc., Nutricia Advanced Medical Nutrition, Sanofi. C. Herder: Other Relationship; Self; Sanofi, Eli Lilly and Company.
Hypothalamic Orexin Is Essential for Estrogenic Action on Glucose and Energy Metabolism in Diet-Induced Obese Mice
Authors: KON KANTA , TSUNEKI HIROSHI , TAKATA SHINJIRO , MAEDA TAKAHIRO , WADA TSUTOMU , SASAOKA TOSHIYASU
Published: June 2018

Decreased estrogen is a crucial pathogenic factor for type 2 diabetes in postmenopausal women; however, the precise mechanism remains unknown. Orexin is a major hypothalamic neuropeptides regulating energy/glucose metabolism, and its anti-obese effect is more evident in females. We therefore investigated whether estrogen prevents obesity and glucose intolerance through interaction with the orexin system. Ovariectomized (OVX) mice and female orexin knockout (OXKO) mice were prepared to examine the impact of estrogen and orexin deficiency, respectively. When maintained on 60% high fat diet (HFD) for 16-24 weeks, body weight gain, adipose tissue inflammation, and hepatic triglyceride contents were markedly increased in OXKO, compared to wild type (WT) mice. Ovariectomy did not significantly affect the body weight in WT and OXKO mice after prolonged HFD feeding. Importantly, the levels of proinflammatory markers (e.g., TNFα mRNA) in the white adipose tissues were increased mildly in sham-operated OXKO (Sham-OXKO) mice and remarkably in OVX-OXKO mice. The rank order of the severity was OVX-OXKO>Sham-OXKO>OVX-WT>Sham-WT. Moreover, Sirius-Red staining to evaluate the fatty liver disease (i.e., NAFLD/NASH) demonstrated mild and severe fibrosis in the liver of Sham-OXKO and OVX-OXKO mice, respectively, whereas there was no abnormality in OVX-WT and Sham-WT mice. In glucose tolerance test, 1) metabolic activity was greater in Sham-WT than OVX-WT mice, whereas no difference was observed between Sham-OXKO and OVX-OXKO mice on HFD, 2) intracerebroventricular injection of estrogen improved glucose tolerance in OVX-WT but not OVX-OXKO mice on HFD, suggesting that estrogen prevents glucose intolerance on HFD in an orexin-dependent manner. Taken together, orexin may play a fundamental role in the anti-obese and antidiabetic effects of estrogen. Therefore, orexin appears to be a novel therapeutic target to prevent metabolic disorders in postmenopausal women.

Disclosure

K. Kon: None. H. Tsuneki: None. S. Takata: None. T. Maeda: None. T. Wada: Research Support; Self; Sanwa Kagaku Kenkyusho Co., Ltd.. T. Sasaoka: None.
Metabolic Activity of the Liver during Exercise—A Metabolomics Approach
Authors: PLOMGAARD PETER , HU CHUNXIU , HANSEN JAKOB S. , ZHAO XINJIE , HOENE MIRIAM , WANG XIAOLIN , SECHER NIELS , HAERING HANS-ULRICH , LEHMANN RAINER , XU GUOWANG , WEIGERT CORA
Published: June 2018

The liver plays a pivotal role in substrate metabolism during fasting and exercise. Chances in the hepatic uptake or release of metabolites regulate the hepatic energy supply for other organs. Here we evaluated substrate flow over the hepato-splanchnic bed by assessing arterial-to-venous differences based on UPLC and CE-MS metabolomics analysis at rest and during exercise. Catheterized healthy young male subjects (n = 10) performed 2 h of ergometer cycling followed by 4 h of recovery. In response to exercise there was pronounced hepatic uptake of lactate, pyruvate, various amino acids and dicarboxylic acids, indicating high demand for gluconeogenic substrates and increase in anaplerotic reactions of the citric acid cycle. Medium chain fatty acids e.g., caproic acid showed a clear hepatic uptake during exercise, underlining their potential role as regulators of gluconeogenesis and mitochondrial substrate oxidation. While most long chain fatty acids showed increased hepatic uptake, saturated fatty acids with carbon chain > 18 were released. In contrast to other amino acids, branched chain amino acids were not taken up or released from the liver at any time point. Notably, the hepato-splanchnic bed showed pronounced uptake of oxygen and release of CO2 during exercise indicating up-regulation of energy consuming metabolic processes.

In conclusion, the data provide novel insight in the regulation of hepatic metabolism at rest and during exercise.

Disclosure

P. Plomgaard: None. C. Hu: None. J.S. Hansen: Employee; Self; Novo Nordisk A/S. X. Zhao: None. M. Hoene: None. X. Wang: None. N. Secher: None. H. Haering: None. R. Lehmann: None. G. Xu: None. C. Weigert: None.
Characterization of the G2L1 Interactome Leads to the Discovery That CLIP2, G2L1, and EB1 Undergo Insulin-Stimulated Phosphorylation
Authors: BARKER NATALIE K. , KRANTZ JAMES L. , PARKER SARA , MOUNEIMNE GHASSAN , LANGLAIS PAUL R.
Published: June 2018

G2L1 is a member of the plus-end tracking (+TIP) microtubule (MT)-associated protein family. G2L1 coordinates cooperativity between MTs and actin and was recently detected in an adipocyte CLASP2 interactome. Since CLASP2 has been linked to insulin action, and both actin and MTs play an unknown role in insulin action, we chose to study G2L1 within 3T3-L1 adipocytes by performing the first-ever G2L1 interactome. Using a commercially available antibody for G2L1 and two antibodies for epitope-tagged, overexpressed G2L1, we performed multiple affinity purification coupled with mass spectrometry experiments in combination with label-free quantitative proteomics and analysed the data with the bioinformatics tool Significance Analysis of Interactome. In concordance with findings in other cell types, we discovered that G2L1 co-IPs the master integrators of +TIP assembly, the end-binding (EB) proteins, with a strong preference for EB1 over EB3 in adipocytes. Subsequent EB1 interactome analysis confirmed reciprocal co-IP of G2L1. We discovered that G2L1 co-IPs the interaction partners CLASP2 and CLIP2, both of which are also +TIP family members, data which was confirmed by IP and western blot. Real-time total internal reflection fluorescence microscopy revealed colocalization of G2L1 and CLASP2 in adipocytes at growing MT ends. Since CLASP2 undergoes insulin-regulated phosphorylation in adipocytes, we hypothesized that the G2L1/CLIP2/EB1 network of +TIPs are also candidates for involvement in insulin-regulated control of MT dynamics. Using targeted quantitative phosphoproteomics, we discovered that insulin stimulates the phosphorylation of G2L1 at Ser597/599, CLIP2 at Ser552, and EB1 at Ser155. This now expands the novel network of insulin-affected +TIP family members to CLASP2, G2L1, CLIP2, and EB1 and introduces the new hypothesis that a coordinated network of MT-associated proteins cooperate to mediate insulin-regulated MT dynamics.

Disclosure

N.K. Barker: None. J.L. Krantz: None. S. Parker: None. G. Mouneimne: None. P.R. Langlais: None.
Regulation of Hepatic Insulin Sensitivity and Hepatic Steatosis by ULK1
Authors: KOO YOUNGDO , GARNEAU MICHAEL P. , ZHANG QUANJIANG , DALE ABEL E.
Published: June 2018

Hepatic insulin resistance (IR) plays a central role in the pathophysiology of impaired glucose tolerance, type 2 diabetes and nonalcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH), which is emerging as a major cause of liver failure and hepatocellular carcinoma. Impaired autophagy may also contribute to hepatic insulin resistance, but the interactions between insulin sensitivity and autophagy are incompletely understood. The Unc-51 Like Autophagy Activating Kinase 1 (ULK1) is an early stage autophagosome formation related kinase that is activated in selective autophagy and may be independent of nutrient or energy status. We observed that in addition to its role in autophagy regulation, ULK1 may also directly regulate insulin signaling by modulating AKT dephosphorylation. Specifically, silencing ULK1 significantly impairs insulin-stimulated activation of AKT and GSK3β in hepatocytes in the absence of significant changes in autophagy. To understand the role of ULK1 in hepatic insulin action ULK1 liver specific knock-out mice were generated. Body weight increased in L-ULK1KO relative to wild type mice on normal chow diet or 60% high fat diet. L-ULK1 KO mice exhibited impaired glucose tolerance and insulin resistance. Serum concentrations of insulin, triglyceride, cholesterol, AST and ALT were increased. Phosphorylation levels of FOXO1 and FOXO3a were decreased in concert with increased expression levels of PEPCK and G6pase and increased hepatic glucose production. In contrast, L-ULK2 KO mice were phenotypically normal. Thus, ULK1 independently and directly regulates hepatic insulin sensitivity and glucose homeostasis.

Disclosure

Y. Koo: None. M.P. Garneau: None. Q. Zhang: None. E. Abel: None.
Liver-Specific Overexpression of 17ß-HSD13 Causes Hepatic Steatosis in Mice
Authors: SU WEN , GUAN YOUFEI
Published: June 2018

According to our previous study, considerable evidences showed that a liver lipid droplet-associated protein 17β-HSD13 contribute to the development of liver disease. To understand the basis for this, we generated and studied transgenic mice that express human hsd17b13 specifically in hepatocytes (L-h17B13 mice).We examined the hepatic expression of several genes involved in fatty acid synthesis and oxidation in L-h17B13 mice. mRNA levels of three enzymes involved in fatty acid synthesis (SREBP-1c, fatty acid synthase [FAS], and ACC) were significantly increased L-h17B13 mice. The mRNA levels of peroxisome proliferator-activated receptor α (PPARα), a transcription factor that promotes fatty acid oxidation and its target gene, acyl-CoA oxidase (ACOX) and medium-chain acyl-CoA dehydrogenase (MCAD), also higher in L-h17B13 mice. mRNA levels of carnitine palmitoyltransferase (CPT1a), a protein involved in translocating fatty acids into the mitochondria for oxidation, was higher in L-h17B13 mice. ApoB, a lipid secretion related gene, was markedly reduced in L-h17B13 mice. Strikingly, male liver-specific h17β-HSD13 mice fed a standard chow diet display significantly elevated hepatic triglyceride levels at 3-4 months of age compared to matched littermate controls. When mice were fed a high-fat diet, this hepatic phenotype, as well as other metabolic phenotypes (obesity and glucose intolerance), worsened. Our investigations establish that 17β-HSD13 specifically overexpressed in mice hepatocytes induces hepatic steatosis and metabolic disorders.

Disclosure

W. Su: None. Y. Guan: None.
Simultaneous 2H and 13C Metabolic Flux Analysis of Liver Metabolism Using NMR and GC-MS—Methods Validation and New Applications
Authors: DEJA STANISLAW , FLETCHER JUSTIN A. , KUCEJOVA BLANKA , FU XIAORONG , YOUNG JAMEY , BURGESS SHAWN C.
Published: June 2018

Hepatic metabolism is critically altered by the pathology of many diseases, including obesity, diabetes and NAFLD. Quantitative estimation of gluconeogenic flux and TCA cycle turnover are clinically useful parameters as they represent major biosynthetic and energetic pathways that are perturbed by these diseases. NMR spectroscopy is an important technique used to study hepatic fluxes since it can precisely distinguish between 2H and 13C metabolic tracers, and provide position-specific enrichment. However, the throughput of NMR for flux analysis is hindered by its low sensitivity, making microscale experiments that have limited biological mass (e.g., primary hepatocytes, tissue biopsies or mouse blood analysis) often infeasible. Thus, we investigate an alternative approach based on GC-MS measurements of glucose mass isotopomers and mathematical modeling of metabolic fluxes. In contrast with NMR, MS approaches are very sensitive, but cannot directly distinguish nuclei or positional enrichment information. Hence, we validated a GC-MS method in isolated perfused mouse liver and in vivo infusions in rats against NMR measurements. A medium-scale metabolic network containing atom transitions for both hydrogen and carbon atoms in the gluconeogenic pathway and TCA cycle was able to deconvolute 2H and 13C labeling information and estimate metabolic fluxes. Although some fluxes were significantly different between the NMR and GC-MS, there was excellent correlation between the two approaches. Finally, we applied GC-MS based simultaneous 2H and 13C approach in primary mouse hepatocytes cultured in 60 mm dishes, an experiment not feasible using standard NMR equipment. This opens future possibilities for drug screening and translational flux studies.

Disclosure

S. Deja: None. J.A. Fletcher: None. B. Kucejova: None. X. Fu: None. J. Young: None. S.C. Burgess: None.
A Novel Mechanism by Foxo1 Phosphorylation Mediates Glucagon Signaling in Control of Glucose Homeostasis
Authors: WU YUXIN , YAN HUI , PAN QUAN , ZHENG HONGTING , DAHANAYAKA SUDATH A. , SUN YUXIANG , ZHOU AIMIN , ZHANG LIN , GUO SHAODONG
Published: June 2018

Glucagon increases hepatic glucose production (HGP) to maintain blood glucose homeostasis. The forkhead transcription factor Foxo1 promotes HGP through increasing expression of genes encoding the rate-limiting metabolic enzymes for gluconeogenesis. We previously established that insulin suppresses Foxo1 by Akt-mediated phosphorylation at Ser256 in human hepatocytes. Here we demonstrated glucagon promotes Foxo1 nuclear translocation and stability via protein kinase A (PKA)-dependent phosphorylation of Foxo1 at Ser276, serving as a novel mechanism of glucagon action in control of glucose homeostasis. In vitro protein kinase assay showed a direct phosphorylation of Foxo1 at Ser276 by PKA. Replacing Foxo1-Ser276 with alanine (S276A) or aspartate (S276D) reduced or increased Foxo1 stability in human hepatocytes, respectively. To establish the in vivo function of Foxo1-Ser276 phosphorylation in glucose metabolism, we generated Foxo1-S273A and Foxo1-S273D knock-in mice, revealing the novel mechanism by which glucagon, via PKA-dependent phosphorylation of Foxo1 at Ser276, promotes Foxo1 nuclear localization, stability, and HGP. Thus, Foxo1-Ser276 is a potential target site for the control of Foxo1 bioactivity and associated metabolic diseases.

Disclosure

Y. Wu: None. H. Yan: None. Q. Pan: None. H. Zheng: None. S.A. Dahanayaka: None. Y. Sun: None. A. Zhou: None. L. Zhang: None. S. Guo: None.
Protection of Progression of Nonalcoholic Steatohepatitis in Diet-Induced Obese Mice by a Low-Iron Diet
Authors: SALAYE LIPIKA , BYCHKOVA IELIZAVETA , LORENZO FELIPE , SINK SANDY T. , MCCLAIN DON
Published: June 2018

Iron is a risk factor for type 2 diabetes, but its relationship to other aspects of metabolic syndrome is less clear. To investigate iron’s relation to nonalcoholic steatohepatitis (NASH), mice were fed a “fast food” (FF)-diet (40% energy as fat, 12% SFA, 0.2% cholesterol, and 18.9 g/L glucose and 23.1 g/L fructose in their drinking water) for 10 or 24 weeks. The diets contained either 4 (low iron, LI), 35 (normal iron, NI) or 2000mg iron/kg chow (high iron, HI). HOMA-IR values were 2.2-fold higher (p<0.001) in the FF-groups at 10 weeks, but did not increase further with HI (1.48-fold, p=0.5). At 10 weeks all groups had similar glucose excursions during glucose and pyruvate tolerance testing. There was no significant difference in IP-GTT or IP-PTT area under the glucose curve. Liver triglycerides (TG) were significantly increased by FF (4.2-fold, p<0.01) but not iron at 10 weeks. The effect of FF was greater at 24 weeks (22.4 fold p<0.001), and at 24 weeks iron further increased liver TG (3.3 fold HIFF vs. LIFF, p<0.001). A marker of liver injury (ALT) increased 10.3-fold (p<0.001) on the HIFF diet compared to LI normal chow, but only 3.2-fold (p<0.05) on LIFF. The fibrotic gene collagen1alpha increased 14.3-fold (p<0.001) in HIFF but not at all (0.8-fold) on LIFF compared to LI normal chow. Similar protection was afforded by the LI diet to increases in expression of the inflammatory genes TGF-beta and TNF-alpha seen in HIFF. RNA sequencing data revealed transcriptional regulation of a large family of fat metabolic genes by FF, whereas only the TGF-beta signaling pathway was significantly altered by iron content. We conclude that dietary iron restriction can protect from NASH induced by a high-fat and -carbohydrate diet. A likely candidate mechanism is through a known mediator of hepatic fibrosis, TGF-beta.

Disclosure

L. Salaye: None. I. Bychkova: None. F. Lorenzo: None. S.T. Sink: None. D. McClain: None.
Pyruvate Carboxylase Is Required for Hepatic Gluconeogenesis and TCA Cycle Function
Authors: CAPPEL DAVID , DEJA STANISLAW , FU XIAORONG , BURGESS SHAWN C.
Published: June 2018

Production of glucose by the liver is crucial for maintaining whole-body gluconeogenesis and is dependent on carbon substrates provided by anaplerotic pathways into the TCA cycle. In the liver, the large majority of anaplerotic flux is through the conversion of pyruvate to oxaloacetate by the action of pyruvate carboxylase (PC). To investigate the role of PC in hepatic gluconeogenesis, we produced a liver-specific PC knockout mouse (LPKCO). LPCKO mice have impaired ability to convert pyruvate into glucose. Surprisingly, they display no difference in blood glucose under fed and overnight fasted conditions. LPCKO mice, however, have elevated concentrations of ketone bodies in plasma under both fed and fasted conditions, suggesting a switch from gluconeogenesis to ketogenesis. Additionally, we observed changes in kidney gene expression that suggest a compensatory upregulation of renal gluconeogenesis. Using stable isotope tracers, we analyzed anaplerotic and TCA cycle fluxes in LPCKO livers and found that LPCKO livers are deficient in anaplerotic flux and have a large reduction but not total ablation of hepatic gluconeogenesis. The LPCKO livers also show a significant reduction in TCA cycle turnover and reduced concentrations of several TCA cycle metabolites. On a high-fat diet (HFD), LPCKO mice gain slightly less weight than their non-transgenic littermates. After 12 weeks of HFD the LPCKO mice have significantly lower blood glucose and plasma insulin compared to non-transgenic littermates. When subjected to a glucose tolerance test, HFD-fed LPCKO mice show significantly better glucose tolerance than non-transgenic littermates. Overall, we demonstrate that flux through hepatic PC is required for anaplerosis and TCA cycle function in the liver and that restricting hepatic gluconeogenesis and TCA cycle turnover may help to protect against the deleterious metabolic effects of high fat diet.

Disclosure

D. Cappel: None. S. Deja: None. X. Fu: None. S.C. Burgess: None.
Foxo1-Ser253 Phosphorylation Regulates Glucose Homeostasis in Mice
Authors: YAN HUI , WU YUXIN , PAN QUAN , SHEN ZHENG , ZHENG HONGTING , WHITE MORRIS F. , SUN YUXIANG , GUO SHAODONG
Published: June 2018

The forkhead transcription factor Foxo1 is a key mediator in insulin signaling pathway that controls hepatic glucose production (HGP) and pancreatic beta-cell function. Upon activation of the protein kinase Akt, insulin promotes glycogen synthesis and inhibits gluconeogenesis in the liver, reducing blood glucose. We previously demonstrated human Foxo1-Ser256, an equivalent to mouse Foxo1-Ser253, is a key phosphorylation site for insulin and Akt suppression, promoting Foxo1 nuclear export and suppressing the expression of the target gene for liver gluconeogenesis. Here we investigated the role of Foxo1-Ser253 phosphorylation in control of glucose hemostasis in vivo, by generating Foxo1-S253A/A knock-in (KI) mice, in which Foxo1-Ser253 replaced by alanine (A mutation) that blocks phosphorylation. Foxo1-S253A/A mice displayed mild increases in feeding blood glucose and insulin level, but reductions in fasting blood glucose and glucagon concentration, as well as a decrease in the ratio of the number of pancreatic α-cells/β-cells per islet. Foxo1-S253A/A mice exhibited slight increases in insulin sensitivity but barely changed glucose uptake among tissues, and an enhanced energy expenditure. Further analyses indicate that Foxo1-S253A/A enhanced Foxo1 ability and promoted the effect of glucagon on HGP. This is the first report demonstrating that Foxo1-Ser253 phosphorylation status itself is sufficient to affect HGP and glucose homeostasis, as well as regulate the synthesis of insulin and glucagon in in vivo.

Disclosure

H. Yan: None. Y. Wu: None. Q. Pan: None. Z. Shen: None. H. Zheng: None. M.F. White: Advisory Panel; Self; Housey Pharmaceutical Research Laboratories. Y. Sun: None. S. Guo: None.
Meal Intake Is Suppressive of Ketogenesis in Patients with Type 1 Diabetes
Authors: GHANIM HUSAM , GREEN KELLY , HEJNA JEANNE M. , KUHADIYA NITESH D. , BATRA MANAV , CHAUDHURI AJAY , DANDONA PARESH
Published: June 2018

Aim: To investigate whether the intake of a meal suppresses ketogenesis in patients with type 1 diabetes.

Methods: 30 patients with type 1 diabetes with no C-peptide, fasted overnight, were divided into 3 groups of 10 patients each. The first (group 1) continued the fast for 5 hours after arrival in our clinical research unit while the second (group 2) was administered a high fat high calorie (HFHC) meal without preprandial insulin and the third (group 3) was administered the meal with preprandial insulin. Blood samples were obtained prior to and after the meal at hourly intervals for 5 hours.

Results: In group 1, glucose and glucagon concentrations remained unchanged while FFA concentrations increased (by 74±16%) as did acetoacetate (AcAc) and beta-hydroxybutyrate (BHB) concentrations (by 52±13% and 64±16%, respectively). In group 2, the intake of the meal without preprandial insulin induced marked increase in glucose, glucagon and FFAs (by 235±32%, 81±18% and 75±17%, respectively) concentrations. However, the meal induced significantly lower increase in AcAc or BHB concentrations (increased by 29±9% and 35±11%, respectively) compared to group 1. In group 3, given preprandial insulin before the meal, there was a significant reduction in glucose and glucagon concentrations as compared to group 2 and a fall in FFA concentrations (by 41±12%) below baseline levels. There was no significant increase in AcAc and BHB.

Conclusions: While a prolonged fast was associated with maintenance of glucose and increases in FFA, AcAc and BHB concentrations, the intake of the meal prevented increases in AcAc and BHB in spite of increases in glucose, glucagon and FFA concentrations. These actions are consistent with an anti-ketogenic effect of the meal intake even in type 1 diabetes with an absence of β cell function.

Disclosure

H. Ghanim: None. K. Green: None. J.M. Hejna: None. N.D. Kuhadiya: Speaker's Bureau; Self; AstraZeneca, Novo Nordisk A/S, Janssen Scientific Affairs, LLC.. Advisory Panel; Self; AstraZeneca. Consultant; Self; Dexcom, Inc. M. Batra: Speaker's Bureau; Self; Eli Lilly and Company. A. Chaudhuri: Speaker's Bureau; Self; AstraZeneca, Eli Lilly and Company, Boehringer Ingelheim Pharmaceuticals, Inc., Merck & Co., Inc., Novo Nordisk Inc. P. Dandona: Advisory Panel; Self; AstraZeneca. Consultant; Self; AstraZeneca. Research Support; Self; AstraZeneca.
Mfge8 Binding of the avß5 Integrin Promotes Insulin Resistance in Mice
Authors: DATTA RITWIK , KHALIFEH-SOLTANI AMIN , HA ARNOLD , ATABAI KAMRAN
Published: June 2018

Milk fat globule EGF factor 8 (Mfge8) promotes obesity by inducing fatty acid uptake through ligation of the αvβ5 integrin. Global deletion of Mfge8 in mice increases insulin sensitivity through effects on lean body mass. The primary objective of the present study was to assess whether acute disruption of the Mfge8 signaling pathway in wild type mice modulates insulin sensitivity independent of body composition. We therefore evaluated insulin sensitivity in age and sex-matched wild type mice with insulin (ITT) and glucose tolerance tests (GTT) in the presence of a blocking antibody (ALULA) targeting the β5 integrin. Intraperitoneal administration of 5mg/kg ALULA 1h prior to ITT resulted in significantly lower blood glucose levels compared to control antibody treated mice. ALULA treated mice had a significantly lower blood glucose levels in GTT. Serum insulin level measured during GTT in ALULA treated mice was lower compared to control group, suggesting enhanced sensitivity to insulin. We validated our findings in vitro using differentiated 3T3L1 adipocytes and hepG2 liver cells. Both cell types had significantly higher uptake of the glucose analog 2NBDG after insulin treatment in presence of ALULA as compared with insulin treatment with control antibody. Insulin is known to induce glucose uptake in adipocytes through a PI3K/Akt signaling pathway. β5 integrin blockade followed by insulin administration augmented insulin mediated phosphorylation of Akt and As160 in white adipose tissue (WAT) and skeletal muscle (SM) in mice indicating that the 㬥 integrin normally dampens insulin signaling. In preliminary data we have found that after insulin treatment, the β5 integrin associates with the mature insulin receptor 㬥 (IR㬥) in WAT and SM as shown in coimmunoprecipitation studies. Further mechanistic insight to this novel regulation of IR㬥 by 㬥5 integrin will direct us towards testing the therapeutic potential of blocking 㬥5 integrin producing insulin resistance in diabetes.

Disclosure

R. Datta: None. A. Khalifeh-Soltani: None. A. Ha: None. K. Atabai: None.
Diabetes Prevention Using a Simulation Model That Explains Individual Variability in Response to Diet Change
Authors: CHEN JULIA H. , FUKASAWA MOMOKO , CHEN QIAN , BURNS SAMUEL P. , KUMAR KEI , NIRENGI SHINSUKE , TAKAHASHI KAORU , SUGANUMA AKIKO , KUZUYA HIDESHI , SAKANE NAOKI , DWIVEDI GAURAV
Published: June 2018

Objectives: Limited understanding of the causes of variability in individual response to similar lifestyle changes makes it challenging to design individualized diet plans for diabetes prevention. We previously reported a simulation model of lifestyle disease and tested it using data from DPP and a low carb-low fat diet crossover study. Here we used the model to analyze data from J-DOIT1 to understand the causes of variability and to optimize diet therapy.

Methods: J-DOIT1 is a large-scale intervention study of 2,607 subjects with a 5.5 year follow-up. We selected 60 baseline-matched individuals from placebo and intervention arms. Model was calibrated to each subject’s body weight, FPG, HbA1c time courses to estimate physiology and lifestyle parameters (e.g., RMR, diet intake). Simulation analysis was used to optimize individual diet.

Results: Individuals with the most and least improved biomarkers showed no significant difference in model-estimated energy balance or in self-reported lifestyle at baseline. Neither caloric deficit nor linear combinations of model-estimated parameters were predictive of outcomes, showing the value of our complex physiological model for prediction. The model suggests that unique sets of optimal diets exist for achieving individual health goals (Fig. 1).

Fig. 1. Optimized changes in fat and carbohydrate intake for two individuals to reduce body weight by 5-7%. Fig. 1. Optimized changes in fat and carbohydrate intake for two individuals to reduce body weight by 5-7%.

Disclosure

J.H. Chen: None. M. Fukasawa: None. Q. Chen: None. S.P. Burns: None. K. Kumar: None. S. Nirengi: None. K. Takahashi: None. A. Suganuma: None. H. Kuzuya: None. N. Sakane: Research Support; Spouse/Partner; Pwc. G. Dwivedi: None.
Comparison of Dietary Fat Intake Using Food Records vs. RBC Fatty Acid Biomarkers in a Multiethnic Population—The Federal Women Study
Authors: COURVILLE AMBER B. , BERNSTEIN SHANNA , GALVAN-DE LA CRUZ MIRELLA , ONUZURUIKE ANTHONY , MATTHAN NIRUPA R. , LICHTENSTEIN ALICE H. , SUMNER ANNE E. , CHUNG STEPHANIE T.
Published: June 2018

Erythrocyte membrane fatty acids (RBC FA) may be a useful biomarker of dietary fat type to assess its impact on health and disease. Our objective was to examine the association of dietary fat type and RBC FA composition in 2 groups of women of African descent (blacks) compared to whites. Dietary fat type was assessed with food records (5±2 days [mean±SD]) and RBC FA composition (mol%) by gas chromatography in 1federally employed women (53% African American, 14% African immigrants, 33% white; age 44±9y, range 24-62y, BMI 30.5±5.9 kg/m2, range 20.3-45.2 kg/m2). Dietary fat type (SFA, MUFA, n-3 PUFA, n-6 PUFA, and trans FA) was calculated as a proportion of total dietary fat. Blacks reported consuming higher SFA and n-6 PUFA (all P<0.05). Dietary report of MUFA, trans FA or n-3 PUFA intake were similar by race (all P≥0.20). In contrast, when RBC FA composition was measured, blacks had higher MUFA and n-3PUFA (both P<0.05). RBC composition of SFA, n-6 PUFA and trans FA were similar by race (all P>0.05). Spearman correlations were calculated to determine the relationship between dietary FA and RBC FA. There was a positive correlation between dietary FA and RBC n-6 PUFA (r=0.3, P<0.02).

In summary, RBC FA was a good marker of n-6 PUFA intake, however, food records appeared to provide a more informative assessment of overall dietary fat type exposure.

Disclosure

A.B. Courville: None. S. Bernstein: None. M. Galvan-De La Cruz: None. A. Onuzuruike: None. N.R. Matthan: None. A.H. Lichtenstein: None. A.E. Sumner: None. S.T. Chung: None.
White- and Brown-Rice Supplementation Improves Insulin Tolerance, Brown Fat Activation, Energy Expenditure, and Changes the Gut Microflora in Diet-Induced Obesity
Authors: YOKOMIZO HISASHI , ISHIKADO ATSUSHI , SHINJO TAKANORI , PARK KYOUNGMIN , MAEDA YASUTAKA , WU I-HSIEN , POBER DAVID M. , DEVKOTA SUZANNE , MATSUMOTO MOTONOBU , KOSTIC ALEKSANDAR , KING GEORGE L.
Published: June 2018

Increased consumption of white rice (WR) with parallel decreased use of brown rice (BR) have been associated with increased prevalence of type 2 diabetes (T2DM), suggesting WR may elevate the risk of T2DM. To test whether WR or BR have differential effects on glucose metabolism in diet-induced obesity, C57BL/6 mice were fed 6 different diets: control diet (CD, 10%), high fat diet (HFD, 46%) and CD and HFD with either WR or BR. Diets with WR or BR were controlled with comparable carbohydrate and protein contents. After 18 weeks, the median weights of HFD mice were greater (36%) than those on CD. Surprisingly, adding WR or BR to HFD decreased weight gains equally by 53% with parallel reduction of abdominal, visceral, subcutaneous, and total fat vs. HFD. Activity and food intake did not change among mice on HFD. However, HFD decreased heat, VO2, and VCO2 production vs. CD which were normalized by adding WR or BR, per indirect calorimetry with metabolic cages. Intraperitoneal glucose tolerance test on HFD were improved by either WR or BR, although mice on BR+HFD exhibited greater improvements than mice on WR+HFD (P<0.05). Adding WR or BR to HFD decreased HFD-induced elevations of plasma leptin and MCP-1 levels. HFD decreased brown fat tissue mass and markers, UCP-1 and PGC1α vs. CD mice which improved when WR or BR was added to HFD. Gut microbiota composition, assessed by 16S rRNA sequencing and metagenomic analysis, showed clear differences between HFD vs. CD or vs. the addition of BR or WR. The ratio of Firmicutes to Bacteroidetes was increased significantly in HFD vs. CD diet, which has been associated with obesity and metabolic syndrome, but decreased by the addition of WR (63%) or BR (81%). Thus, BR and, surprisingly, WR may activate BAT, increase energy expenditure to reduce body weight and improve insulin sensitivity while on HFD, possibly by altering gut microbiome, indicating that common substances may exist in BR and WR to lower the risks for T2DM.

Disclosure

H. Yokomizo: None. A. Ishikado: Employee; Self; Sunstar Inc.. Employee; Spouse/Partner; Sunstar Inc.. T. Shinjo: None. K. Park: None. Y. Maeda: None. I. Wu: None. D.M. Pober: None. S. Devkota: None. M. Matsumoto: Employee; Self; Sunstar Inc.. A. Kostic: None. G.L. King: Research Support; Self; Sanofi-Aventis.
Angiotensin-Converting Enzyme 2 Deficiency Increases Translocation of Gut Bacteria by Depletion of Bone Marrow-Derived Circulating Angiogenic Cells
Authors: DUAN YAQIAN , FENG DONGNI , BELI ELENI , CALZI SERGIO LI , LONGHINI ANA LEDA F. , LAMENDELLA REGINA , WRIGHT JUSTIN R. , MARKEL TROY A. , OBUKHOV ALEXANDER G. , GRANT MARIA B.
Published: June 2018

A healthy microbiome is central to good nutrition and efficient metabolism both of which can be altered in diabetes. Disruption of gut-vascular integrity may lead to translocation of microbial antigens and bacteria into the circulation. Bone marrow-derived circulating angiogenic cells (CACs) are essential to maintain vascular integrity. We tested the hypothesis that angiotensin-converting enzyme 2 (ACE2), the key enzyme in the vasoprotective axis of renin-angiotensin system (RAS), acts to regulate the infiltration of bone marrow-derived CACs in the gut and to maintain gut-vascular integrity in diabetes and could influence the systemic translocation of pathogenic antigens and bacteria from the gut.

Diabetic ACE2 knockout (ACE2-/y)/C57BL/6-Ins2WT/C96Y (Akita) mice were examined at 9 months after the onset of diabetes and compared to age-matched controls. Beta diversity analysis of the gut microbiome revealed a distinct bacterial profile among the different genotypes (n=9-17 per group, p=0.002). The fecal samples from ACE2-/y-Akita group compared to Akita group were enriched with a great diversity of bacterial taxa that have been reported to contribute to the pathogenesis of diabetes and showed activation of peptidoglycan biosynthesis pathways by metatranscriptome analysis (n=4 per group, p=0.005). Flow cytometry analysis showed that loss of ACE2 led to reduced infiltration of bone marrow-derived CACs (CD45+Flk-1+CD31+) into the gut (n=8-14 per group, p<0.0001), without modifying levels of proinflammatory or immune cell phenotypes. The CAC depletion mediated by ACE2 deficiency was associated with increased levels of gut bacteria in the blood as assessed by SourceTracker analysis (n=9 per group). These data suggested a loss of the protective arm of RAS contributes to the depletion of CACs and disruption of gut-vascular barrier that may promote bacterial translocation in diabetes.

Disclosure

Y. Duan: None. D. Feng: None. E. Beli: None. S. Li Calzi: None. A.F. Longhini: None. R. Lamendella: None. J.R. Wright: None. T.A. Markel: None. A.G. Obukhov: None. M.B. Grant: None.
Prokineticin Receptor 1 Activation Induces Myogenesis and Ameliorates Insulin Resistance in Mouse Skeletal Muscle Cells
Authors: JONGSOO MOK , PARK JOONGHOON
Published: June 2018

Type 2 diabetes is manifested by progressive metabolic impairments especially in skeletal muscle which becomes resistant to insulin. From KO mouse phenotype analyses (http://www.mousephenotype.org), we identified that loss of function of Melanocortin 2 Receptor Accessory Protein 2 (Mrap2) could induce metabolic disorders through Prokineticin receptor 1 (Prokr1/Gpr73). In this study, we investigated the possible role of Prokr1 in myogenesis and insulin resistance using murine myoblast cell line (C2C12) and primary mouse muscular satellite cells. We examined the expression levels of Prokr1 in C2C12 and muscular satellite cells during myogenic differentiation, and Prokr1 was upregulated during myogenic differentiation at mRNA and protein levels. Prokineticin 2 (Pk2), a natural ligand of Prokr1, induced intracellular calcium mobilization and cAMP generation in dose-dependent manner. Activation of Prokr1 using Pk2 led to increase the number of myoblast and myotube formation. It also induced Akt1 phosphorylation throughout the myogenesis without insulin stimulation. In myotube, Prokr1 activation increased Slc2a4/Glut4 expression, resulted in 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose uptake as well. Prokr1-mediated insulin resistance was attenuated in palmitate-induced insulin resistant myotube. These effects had been abolished in Prokr1 KO C2C12 cells. These results suggest that Prokr1 could be a crucial player in myogenesis and insulin resistance. Our data support the emerging concept that Prokr1 is a potential target for treatment of insulin resistance in type 2 diabetes mellitus.

Disclosure

M. Jongsoo: None. J. Park: None.
Mechanism Underlying the Biphasic Effect of ADP on Succinate-Energized Muscle Mitochondrial Respiration
Authors: FINK BRIAN , YU LIPING , SIVITZ WILLIAM
Published: June 2018

Recently, we reported a novel phenomenon involving basic mitochondrial physiology. When ADP was sequentially added to mouse skeletal muscle mitochondria energized by succinate (in the absence of rotenone, commonly used to block complex I), we found a biphasic (increasing then decreasing) respiratory dose-response to clamped concentrations of ADP. Here we studied the mechanism. First, we confirmed observations that oxaloacetate (OAA) inhibits succinate dehydrogenase (SDH) by assessing the activity of the extracted complex. OAA is known to be difficult to measure by mass spectroscopy. Here we used a nuclear magnetic resonance method to assess OAA as well as malate and fumarate in mitochondria respiring on succinate. When isolated muscle mitochondria were incubated at varying clamped [ADP], we again observed the biphasic response to [ADP], but also found that the decrease in respiration was strongly associated with OAA accumulation. In addition, when a low (0.5 mM) concentration of pyruvate was added to metabolize OAA to citrate, the loss of succinate-supported respiration was reversed. Pyruvate (0.5 mM), added alone (without succinate), was essentially unable to support respiration. The effect of pyruvate to clear OAA and rescue succinate-supported respiration was blocked by inhibition of pyruvate uptake. Further, OAA accumulation with increasing [ADP] corresponded to a decrease in NADH consistent with utilization electrons by the transport system as malate was converted to OAA and consistent with OAA inhibition of SDH. We also found that the dynamic relationship of [ADP] to succinate-energized respiration varies according to muscle fiber type and specific body tissues in addition (as we reported in the past) to being affected in a rodent model of type 2 diabetes.

In summary, the biphasic respiratory response to succinate as respiration progresses from state 4 to 3 is initially triggered by [ADP]-dependent effects on membrane potential followed by inhibition of SDH by OAA.

Disclosure

B. Fink: None. L. Yu: None. W. Sivitz: None.
Real-World Distribution of Hematocrit Values in the Netherlands and the Czech Republic—A Cross-Sectional Study
Authors: ZIMA TOMAS , STORM HUIBERT , SPACEK MARTIN , WARDAT SAMI , HAUSS OLIVER , HINZMANN ROLF
Published: June 2018

Many self-monitoring of blood glucose (SMBG)-systems demonstrate positive glucose bias at lower and negative bias at higher hematocrit (HCT) levels. Variations can thus lead to measurement errors and possibly to over- or underdosage of insulin. ISO 15197:2013 demands that manufacturers publish HCT-ranges within which BG-meters perform within certain tolerance limits. But how wide should these ranges be? In this cross-sectional study, we collect real-world data from hospital outpatients to assess the frequency of out-of-range HCT-values compared to healthy individuals.

Data from more than 1 million patients were collected from hospitals in Leeuwarden (NL) and Prague (CZ) and stratified by outpatient status, sex and age. Data were compared to common HCT labelled range limits and data from 1780 healthy Czech subjects to assess differences in distribution.

Compared to healthy subjects, outpatients from Prague were distributed with significantly higher dispersion of values. Independent of the location, low HCT-values are common in age groups known to also have high prevalence of diabetes.

Real-world data indicate that BG-meters labelled to perform only within the frequently used 30-55% range would leave thousands of patients at risk of falsely high or low results. Adequate SMBG-systems should be chosen carefully, particularly for patients with increased and decreased HCT-values.Age groupLocationTotal number Female / MaleNumber <30% HCT  F / MPercentage <30% HCT  F / MPrevalence of Diabetes (Eurostat)  F / M55-64NL27329 / 335461043 / 13243.8% / 3.9%6.2% / 10.3%55-64CZ22212 / 19035748 / 9703.4% / 5.1%10.9% / 11.6%65-74NL32004 / 441811958 / 28496.1% / 6.4%12.0% / 15.0%65-74CZ33598 / 313211987 / 19245.9% / 6.1%18.1% / 19.5%75+NL42435 / 450803431 / 45358.1% / 10.1%14.4% / 19.7%75+CZ26645 / 227061923 / 20417.2% / 9.0%27.3% / 25.3%

Disclosure

T. Zima: Research Support; Self; Roche Diabetes Care Health and Digital Solutions. H. Storm: None. M. Spacek: None. S. Wardat: Employee; Self; Roche Diagnostics Corporation. O. Hauss: Consultant; Self; Roche Diabetes Care GmbH. R. Hinzmann: Employee; Self; Roche Diagnostics Corporation.
Serum Creatinine Progressively Decreases with Obesity and Type 2 Diabetes in Nonhuman Primates
Authors: CHAUDHARI UDDHAV K. , NEWCOMB JENNIFER D. , HANSEN BARBARA C.
Published: June 2018

Low muscle mass has been associated with insulin resistance and the development of type 2 diabetes (T2DM). Serum creatinine is a metabolite of creatine present in the skeletal muscle. The creatinine concentration is stable and directly proportional to skeletal muscle mass. Low levels of creatinine have been shown to be associated with the development of T2DM. The underlying mechanism of low creatinine and its risk for the development of diabetes is not known. Nonhuman primates (NHPs), maintained on a healthy diet, frequently develop spontaneous middle-aged obesity, metabolic syndrome (MetSyn), and T2DM similar to humans. In the present study, longitudinal evaluation of the associations between creatinine and the development of obesity, MetSyn, and T2DM in 125 male rhesus monkeys were studied. In addition, 7 male monkeys with long term calorie-restriction (CR) to maintain a healthy lean mass were used as a comparison group. Creatinine decreased as age (r=-0.39, p\0.001), body fat percent (r=-0.27, p\0.001), HbA1c% (r=-32, p\0.001) increased. Decrease in insulin sensitivity was significantly (r=0.07, p\0.001) associated with a decrease in percent fat free mass. Creatinine also declined with decreasing insulin sensitivity (r=0.23, p\0.05) and fat free mass (FFM) (r=0.26, p\001) indicating that monkeys with higher lean muscle mass exhibited better insulin sensitivity. Creatinine was significantly decreased in monkeys with T2DM compared to both MetSyn and metabolically normal monkeys (p\0.001). Longitudinal data analysis showed progressive decline of creatinine with MetSyn and the development of T2DM. CR monkeys showed no significant change in creatinine throughout the lifelong duration of calorie restriction. Also unchanged during CR were fasting plasma glucose, body fat, FFM, or body weight. This is the first longitudinal study showing the association of creatinine with the development of T2DM. Serum creatinine levels declined with decrease in fat free mass and insulin sensitivity.

Disclosure

U.K. Chaudhari: None. J.D. Newcomb: None. B.C. Hansen: None.
Metabolomic Signatures of Insulin Resistance in Human Skeletal Muscle Are Exacerbated with Insulin Stimulation
Authors: NEWSOM SEAN A. , PERREAULT LEIGH , KEREGE ANNA , HARRISON KATHLEEN A. , KAHN DARCY E. , NEMKOV TRAVIS , D’ALESSANDRO ANGELO , BERGMAN BRYAN
Published: June 2018

Metabolomic profiling has identified signatures of insulin resistance in skeletal muscle; however, the direct effect of insulin stimulation on these metabolite signatures in humans remains largely unresolved. To address this, we investigated the effect of insulin stimulation on skeletal muscle metabolites in a well characterized cohort of humans spanning the spectrum of insulin sensitivity. Vastus lateralis muscle samples were obtained from endurance athletes, sedentary lean and obese adults, and individuals with type 2 diabetes (n=16,15,15,12, respectively) under basal conditions and 1-hour into a hyperinsulinemic-euglycemic clamp. Metabolomic analysis was performed using UPLC-MS/MS. Basal concentrations of TCA cycle intermediates, including citrate, alpha-ketoglutarate, succinate, fumarate, malate and oxaloacetate, were positively related to insulin sensitivity (steady-state clamp Rd glucose in mg/kg/min; all P<0.01). Most of these relationships strengthened during insulin stimulation, with the strongest relationships observed for malate (r2 = 0.453, P<0.01) and fumarate (r2 = 0.501, P<0.01) that reflected a blunted increase in TCA intermediates during insulin stimulation with insulin resistance. Insulin stimulation lowered most medium- and long-chain acyl-carnitines compared with basal levels; however, this effect was also attenuated as a function of insulin resistance, signaling impaired metabolic flexibility. Notably, basal levels of several acyl-carnitines, including C12:0, C14:0, C16:0 and C18:1, were increased in endurance athletes compared with other groups (all P<0.05). Several amino acids, including leucine, isoleucine and lysine, were negatively related to insulin sensitivity in the basal and insulin stimulated state (all P<0.01). Together these findings indicate that physiologic insulin stimulation exacerbates metabolic signatures of skeletal muscle insulin resistance in humans.

Disclosure

S.A. Newsom: None. L. Perreault: Advisory Panel; Self; Novo Nordisk A/S. Speaker's Bureau; Self; Novo Nordisk A/S. Advisory Panel; Self; Merck & Co., Inc.. Speaker's Bureau; Self; Merck & Co., Inc., AstraZeneca, Janssen Pharmaceuticals, Inc., Boehringer Ingelheim Pharmaceuticals, Inc.. Consultant; Self; Boehringer Ingelheim Pharmaceuticals, Inc.. Advisory Panel; Self; Sanofi. Speaker's Bureau; Self; Sanofi. A. Kerege: None. K.A. Harrison: None. D.E. Kahn: None. T. Nemkov: None. A. D’Alessandro: None. B. Bergman: Research Support; Self; Eli Lilly and Company. Advisory Panel; Spouse/Partner; Novo Nordisk Inc., Merck & Co., Inc., AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company.