bluuebunny/crossref_metadata_2025 huggingface dataset:
Treatment of Hyperglycemia with Empagliflozin, With or Without Metformin, Improves Islet Endothelial Cell Health and Insulin Secretion in db/db Diabetic Mice
Authors: HOGAN MEGHAN F. , HACKNEY DARYL J. , APLIN ALFRED , MUNDINGER THOMAS O. , ZRAIKA SAKENEH , HULL REBECCA L.
Published: June 2018

Islet endothelial dysfunction occurs in diabetic mice and results in impaired insulin release in vitro. Previously, we found that 12-week treatment of db/db mice with the SGLT2 inhibitor empagliflozin (EMPA) improved islet endothelial dysfunction. Here, we determined if this occurs with shorter treatment duration, is associated with increased insulin release and whether EMPA combined with metformin (MET) provides additional benefit. 6-week old male diabetic C57BL/KsJ.db/db mice and littermate controls (db/+;+/+) were treated with EMPA (20 mg/kg/d in diet), MET (0.5 g/kg/d in water), EMPA+MET, or vehicle (VEH) for 6 weeks. A subset of mice (n=3-5) underwent IV glucose tolerance tests to determine insulin release. mRNA levels of cell adhesion molecules E-selectin (Sele) and vascular cell adhesion molecule 1 (Vcam1), proinflammatory cytokine interleukin-6 (Il6) and vasoconstrictor endothelin-1 (Edn1), were assessed. EMPA treatment lowered plasma glucose, increased insulin release and improved islet endothelial health (Table). MET had a similar effect, but was less effective than EMPA, while EMPA+MET was more effective in improving endothelial function but did not further increase insulin release. Improving islet endothelial health with EMPA may have beneficial effects on beta-cell function in human type 2 diabetes.

Disclosure

M.F. Hogan: None. D.J. Hackney: None. A. Aplin: None. T.O. Mundinger: None. S. Zraika: None. R.L. Hull: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company.
Increased Gluconeogenesis Is Not an Early Feature of Whole-Body Insulin Resistance in Black Women—The Federal Women Study
Authors: CHUNG STEPHANIE T. , HAYMOND MOREY W. , ONUZURUIKE ANTHONY , MABUNDO LILIAN , GARRAFFO H. MARTIN , WALTER PETER J. , COURVILLE AMBER B. , GHARIB AHMED M. , CHACKO SHAJI , SUMNER ANNE E.
Published: June 2018

In contrast to white women, fasting hyperglycemia is not an early diabetes risk marker in black women. The etiology of this racial difference in risk is unknown but could be related to differences in rates of gluconeogenesis and hepatic fat in black women. Using stable isotope tracers [2H2O], [6,62H2]glucose, and ([2H5]glycerol), we partitioned basal glucose production into its two components (gluconeogenesis and glycogenolysis) and measured basal whole body lipolysis. Whole body (SI), hepatic (HIS) and adipose tissue insulin sensitivity were calculated in two groups of federally employed pre-menopausal women, without diabetes: 24 black and 19 white; age 37±6 (mean±SD), range 25-50y; BMI 33±6, range 24.9-45.2 kg/m2. Hepatic and visceral fat were measured by proton MRS. The prevalence of prediabetes (17% vs. 26%), age and BMI were similar in blacks and whites, all P>0.4). Black women had less fasting hyperglycemia (4% vs. 22%, P=0.1), lower visceral and hepatic fat (P<0.01) but similar SI, adipose tissue insulin sensitivity and whole body lipolysis (all P>0.5). Basal glucose, insulin and FFA concentrations were similar in both groups (P>0.05). Blacks had lower glucose production (2.52±0.34 vs. 2.75±0.28 mg·kglbm-1·min-1, P<0.05), fractional gluconeogenesis (57.6±10.6 vs. 66.3±11.4%, P=0.02), and absolute gluconeogenesis (1.44±0.28 vs. 1.77±0.31 mg·kglbm-1·min-1, P<0.01). HIS correlated with SI and hepatic fat (both r=0.5, P<0.01) and the relationship did not differ by race (P>0.05). However, at the same SI blacks had higher HIS (intercepts P<0.01).

In summary, black women had relatively preserved HIS and lower hepatic fat, while white women had higher gluconeogenesis and hepatic fat associated with fasting hyperglycemia. Fasting glucose may be a poor early diabetes marker in black women because gluconeogenesis was low despite whole-body insulin resistance.

Disclosure

S.T. Chung: None. M.W. Haymond: Advisory Panel; Self; Zealand Pharma A/S. Other Relationship; Self; Xeris Pharmaceuticals, Inc.. A. Onuzuruike: None. L. Mabundo: None. H. Garraffo: None. P.J. Walter: None. A.B. Courville: None. A.M. Gharib: None. S. Chacko: None. A.E. Sumner: None.
Palmitic Acid Esters of Hydroxy Stearic Acids Are Hepatic Insulin Sensitizers in Chow and High-Fat Diet (HFD)–Fed Mice
Authors: SANTORO ANNA , ZHOU PENG , PERONI ODILE D. , SYED ISMAIL , NELSON ANDREW T. , SIEGEL DIONICIO , SAGHATELIAN ALAN , KAHN BARBARA
Published: June 2018

Palmitic Acid esters of Hydroxy Stearic Acids (PAHSAs), bioactive lipids with potent anti-inflammatory and antidiabetic effects, reduce ambient glycemia and improve glucose tolerance and insulin sensitivity in insulin resistant HFD-fed mice. We aimed to determine the mechanisms by which PAHSAs improve insulin sensitivity. We treated chow- and HFD-fed mice with 5-PAHSA (0.1 mg/day) and 9-PAHSA (0.4 mg/day) by SQ osmotic minipumps for 13 weeks. Serum 5-PAHSA levels increased 3.5 fold and 9-PAHSA 2.5 fold in chow-fed mice, while 5-PAHSA levels increased 17 fold and 9-PAHSA 6 fold in HFD-fed mice. PAHSAs had no effect on weight gain or fat mass. We performed hyperinsulinemic euglycemic clamps (2.5 mU/Kg/min insulin infusion rate). PAHSA treatment increased systemic insulin sensitivity in both chow- [glucose infusion rate (GIR): vehicle 13±2 vs. PAHSA 27±4 mg/kg/min] and HFD-fed mice (GIR: vehicle 2±0.5 vs. PAHSA 10±3 mg/kg/min). Endogenous glucose production (EGP) was suppressed 28% by insulin in vehicle-treated mice and 55% in PAHSA-treated mice on chow diet. Remarkably, while insulin failed to suppress EGP in vehicle-treated HFD-fed mice, PAHSA treatment led to a 37% reduction in EGP. PAHSAs also decreased glycemia during a pyruvate tolerance test, indicating that suppression of hepatic gluconeogenesis contributes to the EGP improvement. Mechanistic studies showed that PAHSAs inhibit basal and glucagon-stimulated EGP and reduce cAMP in isolated hepatocytes. This effect is blocked by pertussis toxin indicating it is mediated by Gα/i protein-coupled receptors. PAHSAs also reduce G6pase activity and phosphorylation of CREB in liver. Sum: PAHSAs are systemic insulin sensitizers and augment insulin action on EGP in vivo. In vitro, PAHSAs reduce EGP through a cAMP dependent pathway involving Gα/i protein-coupled receptors. Thus, PAHSAs could be effective antidiabetic agents and the pathways they engage could provide novel drug targets for type 2 diabetes.

Disclosure

A. Santoro: None. P. Zhou: None. O.D. Peroni: None. I. Syed: None. A.T. Nelson: None. D. Siegel: None. A. Saghatelian: None. B. Kahn: Advisory Panel; Self; Janssen Research & Development. Research Support; Self; Janssen Research & Development. Advisory Panel; Self; Alterna Biotech.
Exenatide Treatment Improves Mitochondrial Metabolism and Hepatic Insulin Sensitivity in Mice with Nonalcoholic Steatohepatitis (NASH)
Authors: KALAVALAPALLI SRILAXMI , BRIL FERNANDO , VERGARA ARIANA , SUNNY NISHANTH , CUSI KENNETH
Published: June 2018

Exenatide (Exe) is a glucagon-like peptide-1agonist (GLP-1) that enhances insulin production, slows gastric emptying and reduces appetite. Nonalcoholic fatty liver disease (NAFLD), and its more severe form known as NASH, are frequent complications of T2DM. As mitochondrial dysfunction and lipotoxicity are central features of NASH, we tested whether Exe improved mitochondrial oxidative function and hepatic insulin resistance in this setting. Mice (C57/BL6) were fed either a high-fructose, high trans-fat (TFD) diet for 24 weeks to induce NASH or a control diet. After 16 weeks, mice were randomly assigned for the final 8 weeks to subcutaneous Exe (30 µg/kg/day) or vehicle. After an overnight fast, mice received an i.v. infusion of [13C3] propionate and [3,4-13C2] glucose to evaluate mitochondrial metabolism by NMR-based 13C-isotopomer analysis. Targeted metabolic analysis of lipids was measured by LC-MS/MS. Exenatide reduced liver weight and intrahepatic triglyceride content (TFD: 287±23 vs. Exe: 198±27 mg/g liver, p=0.03). Fasting plasma glucose, insulin, FFA and TGs were all significantly lower with Exe. Expression of lipogenic genes and inflammatory markers were significantly lowered by Exe. Moreover, Exe reduced hepatic glucose production, TCA cycle flux, anaplerosis and pyruvate cycling. Exenatide administration improved the lipidomic profile, and decreased hepatic byproducts resulting from incomplete fat oxidation (typically associated with insulin resistance/lipotoxicity), such as diacylglycerols (TFD: 111±13 vs. Exe: 64±13 µmol/g protein, p=0.02) and ceramides (TFD: 1.6±0.1 vs. Exe: 1.3±0.1 µmol/g protein, p=0.02).

Conclusion: In an animal model of NASH, Exe improves hepatic insulin sensitivity, mitochondrial TCA cycle flux and reduces toxic lipid metabolites resulting in an improvement of mitochondrial function. Taken together, these results suggest that Exe may be of clinical value in the management of NASH.

Disclosure

S. Kalavalapalli: None. F. Bril: None. A. Vergara: None. N. Sunny: None. K. Cusi: Consultant; Self; Janssen Global Services, LLC., Eli Lilly and Company. Research Support; Self; Cirius Therapeutics. Other Relationship; Self; Nordic Bioscience. Research Support; Self; Novartis Pharmaceuticals Corporation, Novo Nordisk Inc.. Other Relationship; Self; Quest Diagnostics. Research Support; Self; Zydus Pharmaceuticals (USA) Inc.. Other Relationship; Self; OWL metabolomics, Echosens. Research Support; Self; Janssen Global Services, LLC.. Consultant; Self; Tobira Therapeutics, Pfizer Inc..
Hepatic Insulin/Snail1 Axis Protects against Liver Steatosis and Insulin Resistance through Epigenetically Suppressing Lipogenesis
Authors: LIU YAN , IRELAND NICOLE M. , JIANG LIN , RUI LIANGYOU
Published: June 2018

Insulin stimulates hepatic lipogenesis that drives nonalcoholic fatty liver disease (NAFLD). Paradoxically, insulin resistance is associated with increased hepatic lipogenesis in obesity, and the underlying mechanism remains poorly understood. Here, we delineate an insulin-PI 3-kinase-Snail1 pathway that links metabolic signals to epigenetic reprogramming of de novo lipogenesis in the liver. We found that insulin robustly upregulated Snail1 in both primary hepatocytes and liver in a PI 3-kinase-dependent manner. Notably, the hepatic insulin-PI 3-kinase-Snail1 pathway was severely impaired in obesity. Overexpression of Snail1 blocked, whereas deletion of endogenous Snail1 enhanced, insulin-stimulated lipogenesis in hepatocytes. Hepatocyte-specific deletion of Snail1 exacerbated, whereas liver-specific overexpression of Snail1 ameliorated, dietary NAFLD in mice. Ablation of hepatic Snail1 also attenuated NAFLD-associated insulin resistance and glucose intolerance; conversely, liver-specific overexpression had the opposite effect. Mechanistically, Snail1 bound to the fatty acid synthase (Fasn) promoter and repressed Fasn promoter activity through histone modifications. Our data suggest that insulin pathways bifurcate into the canonical lipogenic and the noncanonical Snail1 two arms wherein the Snail1 arm puts a brake on the lipogenic arm, curbing excessive lipogenesis, liver steatosis, and lipotoxicity. Thus, insulin-resistance-induced impairment in the Snail1 arm contributes to increased lipogenesis in obesity.

Disclosure

Y. Liu: None. N.M. Ireland: None. L. Jiang: None. L. Rui: None.
Elevated Serum Cytokeratin-18 Levels in Patients with Type 2 Diabetes Mellitus and Nonalcoholic Fatty Liver Disease
Authors: LEE TAO-CHUN
Published: June 2018

Background: Cytokeratin-18 (CK-18) is a caspase-cleaved fragment released by injured hepatocytes, and serum levels of CK-18 were believed to be a marker of hepatic cell damage such as inflammation. Patients with type 2 diabetes (T2DM) were well known to have a high prevalence of the fatty liver condition. However, rare studies had been reported the serum CK-18 level in patients with T2DM and investigated the association between the levels with metabolic biomarkers.

Material and Methods: Healthy participants and T2DM patients who followed in the specialized diabetes polyclinic were enrolled. Physical and metabolic factors were collected,and NAFLD was screened by abdominal ultrasound and fatty liver index. Cytokeratin 18 level was detected by commercially available immunoassay.

Results: There were 22.8% (29/127) and 35.9%(42/117) participants were diagnosedwith NAFLD in the non-DM group and T2DM group, respectively. Both serum levels of CK-18 M30 and CK-18 M65 were significantly higher in patients with T2DM as compared with those of the non-DM group. Patients with nonalcoholic fatty liver disease (NAFLD) had higher serum CK-18 M30 level whatever withT2DM (205.8±135.1 vs. 108.4±66.19 U/L; P<0.001) or without (177.69±70.53 vs. 87.07±34.57 U/L; P003C0.001). Similarly, serum CK-18 M65 was also higher in patients with NAFLD in theT2DM group (513.9±271.2 vs. 285.4±115.3 U/L; P<0.001)and in the non-DM group (353.5±162.4 vs. 248.51±111.3 U/L; P<0.001).Multi-variate regression analyses demonstrated that fasting plasma glucose was independently and significantly associated with CK-18 M30 (β coefficient: 0.002; P=0.011)) and CK-18 M65 (β coefficient:0.003; P<0.001). These results suggested that CK-18 level was closely connected with diabetes mellitus.

Conclusions: Independent of NAFLD, our result suggests that CK-18 level was closely associated with the hyperglycemic milieu. The association between serum CK-18 and T2DM may worth for further investigation.

Disclosure

T. Lee: None.
The Importance of Metabolic Syndrome in Patient Progression to Type 2 Diabetes Mellitus
Authors: GLASHEEN WILLIAM P. , CORDIER TRISTAN A. , RENDA ANDREW , DONG YANTING , CHIGULURI VINAY , HAUGH GIL
Published: June 2018

Introduction: Type 2 diabetes mellitus (T2DM) is a common chronic condition associated with significant risk of morbidity and mortality. Metabolic syndrome (MetS) is a chronic prevalent condition, but rarely documented in administrative data. A positive MetS finding comes from tracking five clinical dimensions, and employing a minimum three condition threshold. The aim of this study was to validate a new administrative data MetS algorithm by showing its relevance in tracking progression to T2DM.

Methods: A cohort of 1,321,314 persons enrolled for 3 years in a Medicare Advantage health and wellness company with no evidence of diabetes in the first year were tracked in the subsequent 2 years. Demographic information along with evidence of prediabetes from administrative claims and lab data were documented for the first year. Algorithms to detect MetS and Charlson Comorbidity Index (CCI) were applied to the first year data. For the subsequent 2 years, ICD-10 diagnostic information from administrative data was tracked to identify individuals who progressed into T2DM. Descriptive statistics and Poisson regression analysis were used to assess correlation between first year data and subsequent relative risk for T2DM.

Results: The study population was 87.3% white, 56.4% female, mean age of 70.6, and an overall T2DM progression rate of 8.63%. In the first year, 79.3% had no MetS and no prediabetes (NMNP), 6.9% had MetS but no prediabetes (MNP), 6.8% had no MetS but with prediabetes (NMP), and 7.1% had MetS with prediabetes (MP). Compared to NMNP, the MNP group had an elevated risk of progression to T2DM (RR 1.63 | 95% CL: 1.60, 1.67), as did NMP (1.73 | 1.70, 1.76), and MP (2.47 | 2.43, 2.51). Nonwhite races (1.31 to 1.52), male (1.21), and higher CCI scores (1.02 to 1.45) all were associated with elevated and significant relative risk of T2DM.

Conclusion: Our findings reveal that use of MetS diagnosis and consideration of patient demographics and CCI risk score yield valuable information regarding risk of T2DM.

Disclosure

W.P. Glasheen: None. T.A. Cordier: Employee; Self; Humana Inc.. Stock/Shareholder; Self; Humana Inc.. A. Renda: None. Y. Dong: None. V. Chiguluri: None. G. Haugh: None.
Growth Differentiation Factor 15 Ameliorates Nonalcoholic Steatohepatitis Associated with Metabolic Syndrome in Mice
Authors: LEE MYUNG-SHIK , LEE YONG-HO , KIM SEONG HUN , KIM KOOK HWAN
Published: June 2018

Growth differentiation factor 15 (GDF15) is an endocrine hormone belonging TGFβ superfamily member. GDF15 administration or GDF15 overexpression has been reported to have anti-obesity and antidiabetic effects. Although nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH) is frequently associated with metabolic syndrome or diabetes, the functional role of endogenous GDF15 and therapeutic effect of exogenous GDF15 in NASH associated metabolic syndrome have not been addressed. We studied whether GDF15 has therapeutic effects on NASH and associated metabolic syndrome.

To this end, Gdf15-knockout and Gdf15-transgenic mice were employed, and mice were fed Amylin liver NASH (AMLN) diet as a physiologically relevant dietary mouse model of NASH associated with metabolic syndrome.

GDF15 expression was increased in the livers of mice fed AMLN diet and human subjects with NASH. Elevated expression of GDF15 was due to diet-induced endoplasmic reticulum (ER) stress in the liver. Importantly, Gdf15-knockout mice exhibited deteriorated metabolic syndrome and aggravated NASH phenotypes such as increased steatosis, hepatic inflammation, fibrosis and liver injury. Furthermore, we found that GDF15 directly suppresses expression of fibrosis-related genes and osteopontin (OPN) in the liver in vivo. OPN expression was increased in hepatocytes and stellate cells in the liver of Gdf15-knockout mice. Finally, we found that GDF15-transgenic mice showed attenuated NASH phenotypes and improved glucose tolerance in mice fed AMLN diet. Our results suggest that induction of endogenous GDF15 is a compensatory mechanism to protect against NASH associated with metabolic syndrome and that GDF15 could be an attractive therapeutic candidate for treatment of these diseases.

Disclosure

M. Lee: Other Relationship; Self; Jeil Pharmaceutical Co. Ltd.. Y. Lee: None. S. Kim: None. K. Kim: None.
Metabolic Dysfunction within Brown Adipose Tissue and Skeletal Muscle Caused by Complete Hepatic Insulin Resistance Is Reversible by FGF-21 Treatment
Authors: STOEHR OLIVER , TAO RONGYA , COPPS KYLE D. , WHITE MORRIS F.
Published: June 2018

Hepatic Irs1/Irs2 double knockout mice (LDKO) developed severe systemic insulin resistance, glucose intolerance, and dysregulated gluconeogenesis and hyperinsulinemia—which was largely normalized upon deletion of FoxO1. Although hepatic insulin resistance was established genetically in the LDKO mice, altered hepatokine secretion can dysregulate the metabolism of other tissues—including white (WAT) and brown (BAT) adipose tissues, and skeletal muscle. Compared against control mice, glucose uptake into BAT and various skeletal muscles of LDKO-mice was reduced strongly before and during insulin stimulation. Moreover, BAT exhibited higher gene expression levels of muscle and white adipose tissue markers, whereas expression of BAT and thermogenesis markers were decreased. This indicated immature BAT development, and consequently LDKO-mice failed to maintain body core temperature when exposed to a 4°C environment. Among the dysregulated hepatokines in LDKO-mice, FGF-21 (fibroblast growth factor 21) was reduced significantly. As circulating FGF-21 is produced almost entirely in the liver, LDKO-mice treated by hepatic infection with FGF-21AdV (adenoviral FFG-21) displayed better glucose tolerance, insulin sensitivity, and glucose uptake into BAT and skeletal muscle. FGF-21 also restored normal cold sensitivity. On a high fat diet (HFD) LDKO mice developed severe hyperglycemia; however LDKO•FGF-21AdV-mice lost weight and glucose tolerance improved. These results suggest that reduced FGF-21 contributed to BAT and skeletal muscle dysfunction in LDKO-mice, which can dysregulate systemic glucose homeostasis during hepatic insulin resistance. Expression of FGF-21 can restore and improve glucose metabolism despite the persistent hepatic insulin resistance and uncontrolled gluconeogenesis in these mice.

Disclosure

O. Stoehr: None. R. Tao: None. K.D. Copps: None. M.F. White: Advisory Panel; Self; Housey Pharmaceutical Research Laboratories.
Differential Effects of Niacin on High-Fat Diet–Induced Adipose Tissue Inflammation and Nonalcoholic Fatty Liver Disease in C57BL6/J and B6129SF2/J Mice
Authors: FANG HAN , GRAFF EMILY , LI ZHUOYUE , JUDD ROBERT L.
Published: June 2018

Pharmacological doses of niacin improve adipose tissue (AT) inflammation and nonalcoholic fatty liver disease (NAFLD) in rodents chronically fed a high-fat diet (HFD). However, recent mouse studies have demonstrated significant metabolic changes in different strains of mice fed a HFD. Therefore, the aim of this study was to assess the effect of niacin on both AT inflammation and liver steatosis in two mouse strains, C57BL6/J (B6) and B6129SF2/J (B6129), under HFD feeding. Thirty-two male B6 and 32 male B6129 mice were randomized into four groups: Chow/Vehicle (CV), Chow/Niacin (CN), HFD/Vehicle (HV), and HFD/Niacin (HN). They were fed either a chow (10% fat) or HFD (60% fat) for 20 weeks. Niacin (360 mg/kg/day) or vehicle was added to the drinking water from week 5 until the end of the study. As expected, HFD-fed mice gained more weight than chow-fed mice in both strains, with no difference in weight gain between strains. Crown-like structure (CLS) number, a hallmark of AT inflammation, was increased in HV mice of both strains compared to CV mice. In addition, HV B6 mice had higher CLS number than HV B6129 mice. In B6129 mice, niacin decreased CLS number in HN compared to HV mice, while this decrease was not observed in B6 mice. Liver weight to body weight (L/B) ratio, liver triglyceride (TG) content and NASH score was increased in HV compared to CV B6 mice. In contrast, in B6129 mice, only NASH score was increased in HV compared to CV controls. Niacin had no impact on L/B ratio, TG content, or NASH score in B6 mice. However, in B6129 mice, niacin increased all three parameters in HN compared to HV mice.

In conclusion, there are strain differential effects on AT inflammation and NAFLD induced by HFD feeding. Interestingly, liver steatosis is significantly increased in HFD-fed B6129 mice treated with niacin. This increase is potentially due to methyl deficiency, as niacin is a potent hepatic methyl consumer and 129 mice are more sensitive to methyl deficiency.

Disclosure

H. Fang: None. E. Graff: None. Z. Li: None. R.L. Judd: None.
Glutathione Deficiency Induces Epigenetic Alterations of Vitamin D Metabolism Genes in the Liver of High-Fat Diet–Induced Type 2 Diabetic Mice
Authors: PARSANATHAN RAJESH , JAIN SUSHIL K.
Published: June 2018

Vitamin D (VD) deficiency is an epidemic health problem. Over a billion people worldwide are VD-deficient or insufficient. VD-regulates the functions of over 200 genes and is essential for growth and development. VD-deficiency can result in diabetes, hypertension, heart disease and stroke. VD levels correlate positively with GSH and negatively with insulin resistance in type 2 diabetic patients. HFD-fed mice exhibited elevated blood glucose, and decreased blood GSH and 25(OH)VD with reduced GSH and increased oxidative stress in the liver. We investigated whether GSH deficiency induces epigenetic alterations in VD-metabolism genes in the high-fat diet (HFD) fed mice. The levels of VD-metabolism genes (CYP2R1, CYP27A1, CYP27B1, and VDR) and GSH biosynthesis pathway genes (GCLC, GCLM, GSS, and GSR) were significantly (p<0.005) down-regulated in the liver of HFD-fed mice. Conversely, CYP24A1 which metabolizes the active VD was significantly (p<0.005) increased. In vitro mouse hepatocyte GCLC knockdown decreases GSH and alters VD-regulatory genes in the similar pattern of HFD-fed mice. Further, significant increased DNMTs (1, 3a and 3b) and global DNA methylation levels with decreased TET1 (p<0.005) but not TET2/3 were observed in the cultured hepatocytes and liver. Notably, significant (p<0.005) gene-specific hypermethylation of CYP2R1 (1.71, 2.55; Control vs. HFD), CYP27A1 (7.58, 8.68), CYP27B1 (0.11, 0.77), VDR (0.16, 0.46) and hypomethylation of CYP24A1 (0.59, 0.33) was observed in the liver of HFD-fed (16 weeks) mice compared with control (normal diet) group. GSH deficiency-induced epigenetic alterations (methylation status) of VD-metabolism enzyme genes and the VDR gene appear to play a significant role in the circulating VD. These findings indicate that GSH epigenetically regulates VD-metabolism genes in obesity/T2DM and a novel adjuvant therapeutic target for restoring VD-deficiency in susceptible populations.

Disclosure

R. Parsanathan: None. S.K. Jain: None.
MEDI0382, a GLP-1/Glucagon Receptor Dual Agonist, Acutely Inhibits Food Intake in Leptin-Deficient ob/ob Mice and Leptin Receptor–Deficient fa/fa Rats
Authors: HOWARD VICTOR G. , OLDHAM STEPHANIE , KONKAR ANISH , TREVASKIS JAMES , WILL SARAH
Published: June 2018

MEDI0382 is a potent glucagon-like-peptide-1 (GLP-1) and glucagon receptor dual agonist that inhibits feeding and induces weight loss in mice. To test whether MEDI0382-mediated improvements in metabolism require leptin signaling, we determined its acute effects on food intake in rodents lacking either leptin (ob/ob mice) or the leptin receptor (fa/fa rats). An acute dose response study in ob/ob mice was performed with mice dosed subcutaneously (SC) with GLP-1 receptor agonist liraglutide (30 nmol/kg), glucagon receptor agonist G1437 (30 nmol/kg), or MEDI0382 (1, 3, 10 or 30 nmol/kg). At 24 h, liraglutide and G1437 treatment reduced food intake by 50% and 16% vs. controls, respectively. MEDI0382 inhibited food intake in a dose-dependent manner, and exerted superior inhibition of food intake compared to equimolar doses of liraglutide and G1437. The fa/fa rats were also dosed once with liraglutide, G1437 (both at same doses as above), or MEDI0382 (3, 10 and 30 nmol/kg) and food intake measured. Liraglutide and G1437 treatment decreased 24 h food intake by 55% and 27% respectively and MEDI0382 dose-dependently decreased food intake (32%, 67% and 77%). Also, fa/fa rats treated with MEDI0382 had an additional reduction in food intake than equimolar doses of either liraglutide or G1437. Finally, lean C57BL/6 mice housed in the BioDaq food intake monitoring system were dosed SC once with MEDI0382 (0.3, 1, 3, 10 and 30 nmol/kg), and a dose-dependent effect on inhibition of food intake was observed. Further analysis showed MEDI0382 decreased total meal number and increased satiety ratio.

In summary, MEDI0382 exerted potent anorexigenic effects in mice independent of the leptin pathway, characterized by decreased number of meals consumed and increased satiety.

Disclosure

V.G. Howard: Employee; Self; AstraZeneca. Stock/Shareholder; Self; Regeneron Pharmaceuticals, Inc., AstraZeneca. S. Oldham: Stock/Shareholder; Self; AstraZeneca. Employee; Self; MedImmune. A. Konkar: Employee; Self; Sanofi. Other Relationship; Self; MedImmune. J. Trevaskis: Employee; Self; MedImmune. Stock/Shareholder; Self; AstraZeneca. S. Will: Employee; Self; MedImmune. Stock/Shareholder; Self; AstraZeneca.
Dietary Supplementation with Galactooligosaccharide (GOS) Attenuates High-Fat, High-Cholesterol Containing Western Diet (WD)–Induced Glucose Intolerance
Authors: HE HONGLIANG , GHOSH SIDDHARTHA S. , YANNIE PAUL J. , WANG JING , GHOSH SHOBHA
Published: June 2018

WD induces the development of type 2 diabetes (T2DM). In addition to the direct effects on gut dysbiosis, we have shown that WD also affect intestinal barrier function leading to increased release of LPS into circulation resulting in chronic inflammation that underlies the development of T2DM; oral supplementation with curcumin attenuate these WD-induced effects. Herein we examined the effects of GOS supplementation on WD induced changes. C57BL/6 mice were either fed a standard chow or WD or WD+GOS or WD+Curcumin (WD+C) for 16 weeks. Glucose tolerance tests were performed and while no significant differences were seen in female mice (Panels A and C), WD-induced glucose intolerance was significantly attenuated by GOS or curcumin supplementation in male mice (Panels B and D). Although plasma LPS levels were not significantly affected, a significant decrease in circulating macrophages (Panel E) as well as neutrophils (Panel F) was noted with GOS or Curcumin supplementation indicating a reduction in systemic inflammation. Gene expression and histological changes in the ileum and colon indicate GOS or curcumin-mediated increase in mucin production and maintenance of mucous layer integrity. Dietary supplementation with GOS or Curcumin, therefore, represents a simple strategy to ameliorate the metabolic/diabetogenic effects of WD.

Disclosure

H. He: None. S.S. Ghosh: None. P.J. Yannie: None. J. Wang: None. S. Ghosh: None.
Genetically-Regulated Mechanisms of Insulin Resistance in Adipose Tissue
Authors: SHARMA NEERAJ K. , KEY CHIA-CHI C. , COMEAU MARY E. , LANGEFELD CARL D. , PARKS JOHN S. , DAS SWAPAN K.
Published: June 2018

Insulin resistance (IR) is a harbinger of type 2 diabetes (T2D) and partly determined by genetic factors. However, genetically-regulated mechanisms of IR remain poorly understood. Using gene expression, genotype, and insulin sensitivity (SI and Matsuda index from FSIVGT and OGTT) data from an African American cohort (AAGMEx, N=256), we performed transcript-wide association and expression quantitative trait loci (eQTL) analyses to identify IR-associated cis-regulated transcripts (cis-eGenes) in adipose tissue. Data from a European ancestry cohort (METSIM, N=770) was used for testing replication of Matsuda index-associated cis-eGenes. In vitro genetic silencing studies were performed in a human adipocyte model to define the molecular mechanism of selected genes. Expression level of 2724 transcripts in adipose were negatively (1338) or positively (1386) associated (p<0.001) with Matsuda index in both cohorts. In the AAGMEx cohort, we identified cis-eQTLs (FDR<1%) for 786 Matsuda index-associated transcripts. Cis-eQTLs for 510 Matsuda index-associated genes were identified in both the AAGMEx and METSIM cohorts. These genes were enriched for mitochondrial dysfunction, phagosome maturation, and branched-chain amino acid degradation pathways. The enoyl-CoA hydratase domain-containing 3 (ECHDC3) was the top ranked Matsuda index-associated cis-eGene. ECHDC3 expression level was positively correlated with Matsuda index (r=0.47, p=5.3x10-15) and rs200943982 was the top ECHDC3 cis-eSNP (p=1.94x10-9) in AAGMEx. The shRNA-mediated silencing of ECHDC3 in SGBS adipocytes reduced insulin-stimulated Akt phosphorylation. RNA-seq analysis identified 691 differentially-expressed genes in ECHDC3 knockdown cells, which were enriched in γ-linolenate and cholesterol biosynthesis, LXR/RXR activation pathways, and known IR genes.

In summary, variation-based genomic analysis with concurrent in vitro genetic silencing studies identified novel genetic regulatory mechanisms of IR.

Disclosure

N.K. Sharma: None. C.C. Key: None. M.E. Comeau: None. C.D. Langefeld: None. J.S. Parks: None. S.K. Das: None.
National Trends and Outcomes in Patients with Uncontrolled Diabetes and Related Complications
Authors: JAIN NIDHI , AGARWAL MANYOO , KADARIA DIPEN , STEINBERG HELMUT O. , DAGOGO-JACK SAMUEL
Published: June 2018

Introduction: Patients with uncontrolled DM can present to the hospital with life threatening complications: DKA, hyperosmolar syndrome and coma. There is a paucity of data on the contemporary trends and outcomes of uncontrolled diabetes related hospitalizations (UDH).

Methods: We analyzed the 2003 to 2011 Nationwide Inpatient Sample database (https://www.hcup-us.ahrq.gov) to examine the trends and in-hospital outcomes in patients with UDH aged ≥18 years in the U.S. All patients with primary diagnosis of uncontrolled DM were identified using ICD-9 CM codes- 250.02-250.03, 250.10 -250.13, 250.20 -250.23 and 250.3.

Results: From 2003 to 2011; 1,690,457 patients had UDH. The yearly hospitalization increased from 171,408 (2003) to 216,965 (2011)(ptrend <0.001). Breakdown by sex, race, income and insurance status are in Table 1. The in-hospital mortality decreased from 0.8% to 0.4% (adjusted odds ratio [per year] 0.90; 95% confidence interval 0.89 to 0.91; ptrend <0.001). The average hospital charges increased from $14,370 to $22,897 (ptrend <0.001), whereas the average length of stay decreased from 3.9 to 3.4 days (ptrend <0.001).

Conclusion: Hospitalizations with UDH increased by ∼25% in all U.S demographic groups between 2003 and 2011. However, outcomes improved during the same period, with decreases in length of stay and risk-adjusted in-hospital mortality.

Disclosure

N. Jain: None. M. Agarwal: None. D. Kadaria: None. H.O. Steinberg: None. S. Dagogo-Jack: None.
Txnip Deficiency in the Intestinal Epithelial Cells Prevents against the High-Fat Diet-Induced Fructose Uptake
Authors: SHAH ANU , DAGDEVIREN SEZIN , HUNDAL HENNA , LEE RICHARD
Published: June 2018

Thioredoxin interacting protein (Txnip) has emerged as a key player in nutrient sensing and carbohydrate metabolism. Our lab recently discovered that Txnip binds to the fructose transporters, Glut 2 and Glut 5, to increase the fructose uptake by enterocytes. Additionally, we demonstrated that Txnip was required for the streptozotocin-induced diabetic mice to exhibit a significant increase in fructose absorption. In the present study, we explored the effects of high fat diet (HFD) on fructose uptake in mice with fasting euglycemia and elucidated the role of Txnip in this process. We hypothesized that jejunal uptake of fructose would increase in the HFD-fed mice as compared to the regular diet (RD)-fed controls, and this rise would be prevented in mice with intestinal epithelium knockout of Txnip, despite being on a HFD. To test this hypothesis, C57BL/6J wild type mice were kept on HFD for 16 weeks. At the end of this period, the HFD-fed mice not only exhibited a fasting euglycemia, but also displayed significantly higher fructose absorption (hepatic portal vein blood: p<0.01 and liver: p<0.vs. RD-fed mice, n=6-7 mice/group). Interestingly, gene and protein expressions of Txnip were also significantly higher in the jejunal samples from these mice. Since fructose absorption primarily occurs in the jejunum, we generated a Txnip villin-cre mice that lacks Txnip in the intestinal epithelial cells; and then subjected these mice and their cre counterparts to RD/HFD. The preliminary data at one month post-diet shows a reduced trend of fructose absorption in both RD-fed and HFD-fed Txnip villin cre mice compared to the cre-controls. Thus, our data demonstrates that HFD facilitates fructose uptake by the intestine, and the targeted deletion of Txnip at the intestinal level can decrease the absorption of dietary fructose. This outcome implies a molecular mechanism by which high fat diet can promote excess hepatic lipid synthesis via enhanced fructose uptake.

Disclosure

A. Shah: None. S. Dagdeviren: None. H. Hundal: None. R. Lee: None.
Overexpression of Perilipin 2 Induces Cardiac Steatosis and Atrial Fibrillation via Connexin 43 Remodeling
Authors: SATO SATSUKI , SUZUKI JINYA , HIROSE MASAMICHI , NAKAYA TAKAHIRO , YAMADA MIKA , ICHIKAWA MAI , IMAGAWA MICHIKO , ZENIMARU YASUO , TAKAHASHI SADAO , KRAEMER FREDRIC B. , KONOSHITA TADASHI , ISHIZUKA TAMOTSU
Published: June 2018

Atrial fibrillation (AF) is prevalent among diabetic patients. Diabetes is also associated with myocardial lipid droplet accumulation (steatosis), which is thought to be a source of intracellular lipotoxicity. Since the relative contribution of cardiac steatosis per se to AF has not been elucidated, the current study was designed to clarify the causal effect of cardiac steatosis on AF using an aged mouse model of cardiac steatosis. Cardiac-specific perilipin (PLIN) 2-overexpressing mice (PLIN2-Tg) were created and maintained on a chow diet for >12 months. Cardiac steatosis was assessed by electron microscopy and triacylglycerol (TAG) content. AF was induced by transesophageal electrical burst pacing, and the duration of AF was measured. Atrial impulse conduction patterns were analyzed in Langendorff-perfused hearts using optical mapping technique. Myocardial localization of a gap junction protein, connexin (Cx) 43, was analyzed by confocal microscopy. Electron microscopy of the atrium of PLIN2-Tg revealed accumulation of small lipid droplets around clusters of mitochondria, and the atrial TAG content in PLIN2-Tg was 4- to 8-fold higher than that in wild type (Wt) atrium. Electrocardiograms showed significantly longer RR intervals in PLIN2-Tg compared to Wt mice. The prevalence of sustained (>5min) AF was significantly greater in PLIN2-Tg compared with Wt mice (69% vs. 24%). Confocal microscopy revealed that Cx43 was distributed at the intercalated discs in Wt atria, whereas it was heterogeneously redistributed to the lateral side of the cardiomyocytes in PLIN2-Tg atria. Isochrone mapping showed slow and heterogeneous impulse propagation in PLIN2-Tg atria compared with Wt atria. These results indicate that PLIN2-induced steatosis causes Cx43 remodeling, impaired conduction propagation, and a higher incidence of AF. Controlling cardiac steatosis might be an important target for inhibiting AF in patients with cardiac steatosis.

Disclosure

S. Sato: None. J. Suzuki: None. M. Hirose: None. T. Nakaya: None. M. Yamada: None. M. Ichikawa: None. M. Imagawa: None. Y. Zenimaru: None. S. Takahashi: None. F.B. Kraemer: None. T. Konoshita: None. T. Ishizuka: None.
Heart-Specific Overexpression of HMG-CoA Synthase 2 Induces Mitochondrial Stress—Adaptation via the ATF4 Pathway
Authors: ZENIMARU YASUO , SUZUKI JINYA , NAKAYA TAKAHIRO , YAMADA MIKA , ICHIKAWA MAI , SATO SATSUKI , IMAGAWA MICHIKO , KRAEMER FREDRIC B. , KONOSHITA TADASHI , ISHIZUKA TAMOTSU
Published: June 2018

The liver is the central organ for ketone body production, and HMG-CoA synthase2 (HMGCS2) is a rate-limiting enzyme involved in this process. We reported that fasting markedly increased mRNA expression of HMGCS2 in mouse heart, whereas its protein expression was faint and increased only marginally with fasting. To clarify the pathophysiological role of HMGCS2 in the heart, we generated transgenic (Tg) mice with heart-specific HMGCS2 overexpression and analyzed the effects on metabolic status in the myocardium. Plasma concentrations of β-hydroxybutyrate (βOHB), glucose and free fatty acids were comparable between Tg and wild type (Wt) mice, whereas the cardiac βOHB content in Tg mice was increased 10-fold (fed state) or 2-fold (24h-fasted state) over that of Wt mice. Confocal microscopy demonstrated that the HMGCS2 protein co-localized with a mitochondrial protein, ATP5A, in cardiomyocytes of Tg mice. Transmission electron microscopy showed many swollen mitochondria, some of which contained vacuole-like vesicles and decreased cristae in Tg hearts, and ultrasonography showed impaired systolic function. Microarray gene expression analysis revealed that approximately 800 genes were altered >2-fold in Tg hearts compared with Wt hearts. Among the altered genes, ATF4 and its target genes, including PHGDH, MTHFD2 and ASNS which promote glutathione production, were upregulated in Tg hearts. Consistent with this result, a metabolome analysis performed by capillary electrophoresis-mass spectrometry demonstrated that glutathione was increased in Tg hearts, suggesting an adaptation to mitochondrial stress. In addition, 3 branch-chain amino acids were increased in Tg hearts compared with Wt hearts. These results indicate that myocardial HMGCS2 can produce ketone bodies in mitochondria, leading to mitochondrial stress and cardiac dysfunction. The limited induction of HMGCS2 protein upon fasting might constitute a protective mechanism for cardiomyocytes.

Disclosure

Y. Zenimaru: None. J. Suzuki: None. T. Nakaya: None. M. Yamada: None. M. Ichikawa: None. S. Sato: None. M. Imagawa: None. F.B. Kraemer: None. T. Konoshita: None. T. Ishizuka: None.
Foxo1 Corepressor (FCoR) and Foxo1 Regulate a-Cell and ß-Cell Identity
Authors: KODANI NORIKO , NAKAE JUN , ITOH HIROSHI
Published: June 2018

We previously identified a Foxo1 binding protein; Foxo1 CoRepressor (FCoR), in adipocytes. FCoR inhibits Foxo1 activity by enhancing its acetylation. FCoR is expressed in pancreatic α- and β-cells from the embryonic stage. Fcor knockout mice (FcorKO) show impaired glucose tolerance, decreased insulin secretion, increased glucagon secretion and increased α-cell mass. We have shown that FCoR suppresses the expression of master α-cell regulatory transcription factor Aristaless-related homeobox (Arx) by increasing the methylation of the CpG-rich promoter region, while Foxo1 induces Arx expression by hypomethylation of this area. These results suggest that FCoR and Foxo1 have an opposing action in the regulation of Arx expression and α-cell mass. We studied how Foxo1 regulates Arx expression. Arx promoter region contains a conserved forkhead responsive element (FRE). Foxo1 binds to the FRE and induces Arx expression. Binding of Foxo1 to the Arx promoter dissociates binding of DNA methyltransferase 3a (Dnmt3a), and this may result in hypomethylation of the CpG rich region. Our results suggest that Arx is the target gene of Foxo1. Immunohistochemical staining of FcorKO islets showed Foxo1 nuclear localization and decreased Foxo1 in the cytosol. In isolated islets from FcorKO, we observed significant deacetylation of Foxo1, and decreased endogenous Foxo1 protein compared to control. These results are consistent with the previous finding that deacetylated Foxo1 is the active form, and is quickly ubiquitinated and degraded. To study the relationship between FCoR and Foxo1, we generated β-cell-specific Foxo1 knockout mice in the background of FcorKO (DKO). DKO showed further aggravation of glucose tolerance and insulin secretion compared to FcorKO. Foxo1 single knockout has normal glucose metabolism, thus, our results suggest that Foxo1 is activated to maintain β-cell function in FcorKO. We provide evidence that FCoR and Foxo1 function in harmonization to regulate pancreatic α-cell and β-cell identity.

Disclosure

N. Kodani: None. J. Nakae: None. H. Itoh: None.
Hyperglycemia Promotes Muscle Atrophy through the WWP1/KLF15 Pathway
Authors: HIRATA YU , NOMURA KAZUHIRO , SENGA YOKO , IMAMURA MICHIHIRO , TAKEDA SHIN’ICHI , OKADA YUKO , BURDEN STEVEN J. , HOSOOKA TETSUYA , OGAWA WATARU
Published: June 2018

Background: Evidence suggests that diabetes is a promoting factor of sarcopenia. Mechanism how the condition accelerates the development of sarcopenia remains ambiguous.

Results: In streptozotocin (STZ)-induced diabetic mice, skeletal muscle mass was decreased by ∼15 % within 21 days after the STZ treatment. The protein abundance of transcription factor KLF15 as well as the mRNA abundance of proteins related to muscle atrophy (Foxo3a, Atrogin1 and Murf1) were elevated whereas the mRNA of KLF15 was unaltered in skeletal muscle of STZ-diabetic mice. Decrease in skeletal muscle mass and increase in the mRNA abundance of muscle atrophy-related proteins triggered by STZ-induced diabetes were prevented in muscle-specific KLF15 deficient mice suggesting that KLF15 plays a key role in diabetes-induced muscle atrophy. Treatment of C2C12 cells with high concentration of glucose (25 mmol/l) decreased the ubiquitination of and increased the protein abundance of KLF15. The E3 ubiquitin ligase WWP1 was found to be downregulated in skeletal muscle of STZ- diabetic mice and in C2C12 cells treated with glucose. Overexpression and knockdown of WWP1 in C2C12 cells resulted in the decrease and the increase, respectively, of KLF15 protein without affecting its mRNA expression. The mRNA abundance of WWP1 in skeletal muscle was downregulated and the mass of skeletal muscle was reduced in Akita mice, which develop diabetes due to beta-cell dysfunction. Administration of the SGLT2 inhibitor empagliflozin, which lowers glycemia without affecting insulin levels, to Akita mice upregulated the expression of WWP1 in skeletal muscle and increased the skeletal muscle mass of these mice.

Conclusion: Our results suggest that hyperglycemia upregulates the protein abundance of KLF15 via the downregulation of WWP1, which catalyzes the ubiquitination of KLF15. Hyperglycemia-induced upregulation of KLF15 leads to the induction of genes for muscle atrophy-related proteins, which in turn promotes the atrophy of skeletal muscle.

Disclosure

Y. Hirata: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc. K. Nomura: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc. Y. Senga: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc.. M. Imamura: None. S. Takeda: None. Y. Okada: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc.. S.J. Burden: None. T. Hosooka: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc. W. Ogawa: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Abbott.
Mitochondrial Adaptation in Insulin Resistant Human Skeletal Muscle
Authors: AXELROD CHRISTOPHER L. , FEALY CIARAN E. , MULYA ANNY , HUANG EMILY , FUJIOKA HISASHI , BURGUERA BARTOLOME , HOPPEL CHARLES L. , KIRWAN JOHN P.
Published: June 2018

Introduction: The integrity, morphology, structure and functionality of skeletal muscle mitochondria in patients with type 2 diabetes (T2D) is heavily disputed. Thus, the purpose of this investigation was to characterize mitochondrial phenotypes across the insulin resistance spectrum.

Methods: 30 sedentary adults aged 18-55 were grouped as lean (BMI 21±0.6, N=13), obese (BMI 32±0.9, N=12), or T2D (BMI 33±2.5, N=5) based on oral glucose tolerance tests and body mass index (BMI). All subjects completed a 3-day, inpatient stay consisting of cardio-metabolic testing, body composition analysis and a hyperinsulinemic-euglycemic clamp study. On the day of the clamp, muscle specimens were obtained basally and under insulin-stimulated conditions. Mitochondrial membrane potential (δψm) was measured by confocal microscopy using the cationic fluorophore TMRM. Oxidative phosphorylation (OXPHOS) was assessed in permeabilized fibers via high resolution respirometry (Oroboros Oxygraph-2k). Muscle mitochondrial ultrastructure and content were determined by transmission electron microscopy (TEM) of fixed muscle sections.

Results: There was a significant, step-wise decrease in insulin-stimulated glucose uptake across the metabolic spectrum (P<0.001). No differences in resting δψm were observed, however, insulin stimulation depolarized δψm in all groups (P<0.05). Those with T2D exhibited increased basal complex III (P<0.05) and IV (P<0.05) activity vs. lean and obese controls. Furthermore, obese controls and T2D displayed augmented complex IV responsiveness to insulin stimulation (P<0.01). Threshold analysis of muscle sections revealed a progressive loss of mitochondrial content, as well as accumulation of lipid droplets across the metabolic spectrum (P<0.05).

Conclusion: Collectively, these findings provide direct evidence of mitochondrial adaptation in insulin resistant and diabetic skeletal muscle.

Disclosure

C.L. Axelrod: None. C.E. Fealy: None. A. Mulya: None. E. Huang: None. H. Fujioka: None. B. Burguera: None. C.L. Hoppel: None. J.P. Kirwan: None.