bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessment of Fasting Glucagon Levels in Subjects with Diabetes and Healthy Controls Using ELISA and RIA
Authors: BRUNNER MARTINA , HABERLANDER MAXIMILIAN , SVEHLIKOVA EVA , EBERHARD KATHARINA , STACH ELISABETH , LIPP ROBERT , OBERMAYER-PIETSCH BARBARA M. , PIEBER THOMAS R. , SOURIJ HARALD
Published: June 2018

Based on the lack of accurate methods for glucagon analysis, it has been suggested that glucagon levels are elevated in subjects with type 1 diabetes. The availability of more specific methods for the determination of glucagon has indicated that actual glucagon levels are lower than previously assumed. This study aimed to characterize fasting glucagon levels in subjects with type 1 diabetes (T1D) and type 2 diabetes (T2D) compared to their nondiabetic counterparts using two different assays. We recruited 20 healthy, 20 T1D and 20 T2D subjects for the trial. Glucagon samples were obtained using BD P800 blood collection tubes and measured with a double-antibody sandwich ELISA (Mercodia) and a RIA (MP Biomedicals). Using the ELISA, healthy subjects had similar glucagon levels compared to T1D. ELISA based glucagon measurements demonstrated significantly higher fasting glucagon levels in T2D as compared to healthy subjects, while RIA measurements tend to be numerically higher, albeit not statistically different (p=0.201). Median glucagon values obtained by RIA were significantly (p=0.0001) higher than those measured by ELISA in a pooled analysis.

In conclusion, T2D show fasting hyperglucagonaemia, whereas T1D present with similar fasting glucagon levels compared to nondiabetic subjects. Furthermore, this study highlights the importance of selective analysis methods in endocrine research.

Disclosure

M. Brunner: None. M. Haberlander: None. E. Svehlikova: None. K. Eberhard: None. E. Stach: None. R. Lipp: None. B.M. Obermayer-Pietsch: None. T.R. Pieber: Consultant; Self; Arecor, AstraZeneca, Eli Lilly and Company, Novo Nordisk A/S, Sanofi. Employee; Self; CBmed. Research Support; Self; Novo Nordisk A/S, AstraZeneca. H. Sourij: Speaker's Bureau; Self; Boehringer Ingelheim GmbH, Novo Nordisk A/S, Amgen Inc., Sanofi, MSD K.K.. Research Support; Self; AstraZeneca, Boehringer Ingelheim GmbH, MSD K.K., GI Dynamics Inc..
Metabolomics Shows an Association of Betatrophin with Bile Acids, Suggesting an Involvement of Betatrophin in Bile Acid–Mediated Metabolic Control
Authors: LEIHERER ANDREAS , MUENDLEIN AXEL , GEIGER KATHRIN , SAELY CHRISTOPH H. , BRANDTNER EVA-MARIA , EBNER JANINE , LARCHER BARBARA , MADER ARTHUR , FRAUNBERGER PETER , DREXEL HEINZ
Published: June 2018

Betatrophin, also known as ANGPTL8, is secreted by liver and adipose tissue and plays an important role in glucose and lipid metabolism, but the mechanisms behind this remain unclear. Betatrophin is highly activated by active triiodothyronine (T3), a key player in metabolic control, which in turn is activated by bile acids (BA) via the BA-G-protein-coupled receptor TGR5-cAMP-deiodinase (D2), mediating the conversion of prohormone thyroxine to T3. We here measured betatrophin by ELISA and also applied GWAS and targeted metabolomic profiling in 233 serum samples of coronary angiographied patients. We found a correlation between betatrophin and BMI (r=0.142, p<0.001), fasting glucose (r=0.133, p=0.002), insulin (r=0.221, p<0.001), triglycerides (r=0.233, p<0.001), HDL (-0.122, p=0.004), and oxLDL (r=0.247, p<0.001). GWAs also found a genome wide-association between betatrophin and genomic variants in sterol transporter genes ABCG5/G8 and ABCA1. In the metabolomic assay we identified BA as the top metabolites to be associated with betatrophin concentration in serum (CDCA, r=0.234, p<0.001; GDCA, r=0.247, p<0.001; GCA, r=0.269, p<0.001; GCDCA, r=0.268, p<0.001; GLCA, r=0.211, p=0.002; TDCA, r=0.184, p=0.006).

In conclusion, this study for the first time describes the association between betatrophin and BA. As (i) T3 is activated by BA via the BA-TGR5-cAMP-D2 signalling pathway and (ii) T3 is a strong activator of betatrophin, the link between betatrophin concentration and BA suggests that, at least in part, the impact of BA on energy homeostasis and metabolic control may be mediated via betatrophin. Moreover, TGR5 receptors have also been identified on pancreatic islet α- and β-cells and are stimulating insulin secretion in β-cells and able to restore β-cell mass and function under hyperglycemic conditions. Altogether, this may elucidate the impact of betatrophin on lipid and glucose metabolism.

Disclosure

A. Leiherer: None. A. Muendlein: None. K. Geiger: None. C.H. Saely: None. E. Brandtner: None. J. Ebner: None. B. Larcher: None. A. Mader: None. P. Fraunberger: None. H. Drexel: None.
Lactobacillus Gasseri in the Upper Small Intestine Impacts an ACSL3-Dependent Fatty Acid Sensing Pathway That Regulates Whole-Body Glucose Homeostasis
Authors: BAUER PAIGE V. , DUCA FRANK , ZAVED WAISE T.M. , DRANSE HELEN J. , RASMUSSEN-SMALL BRITTANY A. , PURI AKSHITA , RASTI MOZHGAN , LAM TONY K.T.
Published: June 2018

Long chain acyl CoA synthetase (ACSL)-dependent upper small intestinal lipid metabolism activates preabsorptive pathways to regulate metabolic homeostasis, but whether changes in the upper small intestinal microbiota alter specific fatty acid-dependent pathways to impact glucose homeostasis remains unknown. We here first find that upper small intestinal infusion of Intralipid, oleic acid, or linoleic acid preabsorptively increases glucose tolerance and lowers glucose production in rodents. High-fat feeding impairs preabsorptive fatty acid sensing and reduces upper small intestinal Lactobacillus gasseri levels and ACSL3 expression. Transplantation of healthy upper small intestinal microbiota to high-fat fed rodents restores Lactobacillus gasseri levels and fatty acid sensing via increased ACSL3 expression, while Lactobacillus gasseri probiotic administration to non-transplanted high-fat fed rodents is sufficient to restore upper small intestinal ACSL3 expression and fatty acid sensing.

In summary, we unveil a glucoregulatory role of upper small intestinal Lactobacillus gasseri that impacts an ACSL3-dependent glucoregulatory fatty acid sensing pathway.

Disclosure

P.V. Bauer: None. F. Duca: None. T. Waise: None. H.J. Dranse: None. B.A. Rasmussen-Small: None. A. Puri: None. M. Rasti: None. T.K.T. Lam: None.
Roles of Adipose Tissue VLDL-VLDLR Axis in Energy Metabolism
Authors: KIM JAE BUM , SHIN KYUNG CHEUL , HWANG INJAE , CHOE SUNG SIK
Published: June 2018

Elevated plasma triglyceride-rich lipoprotein, such as very low-density lipoprotein (VLDL), is considered a risk factor for prevalence of obesity, type 2 diabetes mellitus, and atherosclerosis. VLDL receptor (VLDLR), one of the lipoprotein receptor family proteins, is involved in clearance of circulating VLDL. In obese animals, adipose tissue macrophages (ATMs) contain high level of lipid metabolites, which seems to be linked to pro-inflammatory responses. However, it has not been thoroughly understood whether lipid uptake via VLDLR in macrophages is involved in obesity-induced inflammatory responses and insulin resistance. In this study, we demonstrate that elevated VLDLR in ATMs could accelerate adipose tissue inflammation and glucose intolerance in obesity. Expression of VLDLR was increased in ATMs from obese mice. In VLDL-treated macrophages, the level of ceramides was elevated through VLDLR, which potentiates pro-inflammatory responses and augments M1-like macrophage polarization. Moreover, adoptive transfer of VLDLR knockout bone marrow to wild type mice relieved adipose tissue inflammation and improved insulin resistance in diet-induced obesity. Taken together, these findings suggest that increased VLDL-VLDLR axis in ATMs would aggravate adipose tissue inflammation and insulin resistance in obesity.

Disclosure

J. Kim: None. K. Shin: None. I. Hwang: None. S. Choe: None.
Fasting Glucagon Concentrations Predict Longitudinal Changes in ß-Cell Function of Nondiabetic Humans
Authors: ADAMS J.D. , LAURENTI MARCELLO C. , HURTADO MARIA D. , MAN CHIARA DALLA , COBELLI CLAUDIO , RIZZA ROBERT A. , VELLA ADRIAN
Published: June 2018

The diabetes-associated allele at rs7903146 in the TCF7L2 locus predisposes to type 2 diabetes (T2DM). Recent work has shown that this variant is associated with impaired postprandial glucagon suppression. Since defects in α-cell function occur independently of impaired β-cell function, we hypothesized that impaired glucagon suppression could accelerate progression to T2DM. Therefore, we recruited 73 nondiabetic subjects with prior (6.7 ± 0.3 years) measurement of β-cell function using the oral minimal model (Baseline) and compared these indices to those obtained during a repeat Experiment (Present). Weight increased slightly, but significantly (2.8 ± 0.7 Kg, p < 0.01) over the period of study. Overall Disposition Index (DI) which expresses β-cell responsivity (ϕ) as a function of insulin action (Si) did not change (1234±160 vs. 1143±116 10-14, dl/kg/min2 per pmol/l, DIBaseline vs. DIPresent, p=0.55). However, there was considerable heterogeneity with a decline in DI associated with impaired Si Baseline after adjusting for age and weight changes (R2 = 0.22, p = 1.2 x 10-3). Of note, fasting glucagon concentrations at the time of initial study ([Glucagon]Baseline) were also significantly and inversely related to DIPresent (R2 = 0.13, p = 8 x 10-3). Baseline nadir and integrated (Area Under Basal-AUB) glucagon concentrations did not predict (p > 0.05) changes in DI. Fasting [Glucagon]Baseline, adjusted for age and weight changes interacts with Si Baseline to predict the longitudinal decline in DI (DIBaseline-DIPresent: (R2 = 0.29, p < 1 x 10-4). Taken together, these data suggest that defects in α-cell function, manifest as elevated fasting glucagon, drive subsequent decline in β-cell function. It remains to be ascertained if defects in postprandial-glucagon suppression attributable to or independent of, diabetes-associated genetic variation in TCF7L2 also interact with defects in fasting glucagon secretion to drive decline in β-cell function in nondiabetic humans.

Disclosure

J. Adams: None. M.C. Laurenti: Other Relationship; Self; GlySens Incorporated. M.D. Hurtado: None. C. Dalla Man: None. C. Cobelli: Research Support; Self; Sanofi-Aventis. Advisory Panel; Self; Novo Nordisk Inc.. R.A. Rizza: None. A. Vella: Research Support; Self; Novo Nordisk Inc., XOMA Corporation. Advisory Panel; Self; VTV Therapeutics, Bayer AG.
Exendin-4, a Glucagon-Like Peptide-1 Receptor Agonist, Attenuates Prostate Cancer Cell Proliferation via Phosphorylation of MKP-1
Authors: KAWANAMI TAKAKO , NOMIYAMA TAKASHI , HAMAGUCHI YURIKO , TANAKA TOMOKO , YANASE TOSHIHIKO
Published: June 2018

Pleiotropic benefits of incretin therapy beyond glycemic control have been reported and receiving much attention. Cancer is one of major causes of death in patients with type 2 diabetes. We have previously reported Exendin-4(Ex-4), a glucagon-like peptide-1 receptor agonist, attenuates prostate cancer growth by the inhibition of ERK-MAPK phosphorylation (Diabetes 2014). On the other hand, it is well known that MAPK is inactivated and tightly regulated by MAPK phosphatase (MKP) family. Then, we next examined whether MKP could be involved in the anti-proliferative effect of Ex-4 in the prostate cancer cell. In a human prostate cancer cell line, LNCaP cell, MKP-1 was abundantly expressed. Knocking down of MKP-1 using siRNA abolished Ex-4 induced-reduction of LNCaP cell number in growth curve. In addition, Ex-4 treatment significantly increased MKP-1 mRNA expression. To activate its phosphatase activity, Ser359 in the C terminus of MKP-1 is specifically phosphorylated. Ex-4 significantly phosphorylated Ser359 of MKP-1along with time course of dephosphorylating ERK-MAPK. Using Immunoprecipitation assay, we further confirmed that Ex-4 exactly phosphorylated Ser359 of MKP-1. In addition, if we transiently transfected GFP conjugated MKP-1 into LNCaP cells, nuclear translocation of MKP-1 induced by Ex-4 treatment was observed in early phase stimulation. These data suggest that Ex-4 attenuates prostate cancer cell proliferation via phosphorylation of MKP-1 followed by inhibition of ERK-MAPK activation.

Disclosure

T. Kawanami: None. T. Nomiyama: Research Support; Self; MSD K.K., Sanofi-Aventis, Boehringer Ingelheim Pharmaceuticals, Inc., Takeda Pharmaceuticals, Japan. Y. Hamaguchi: None. T. Tanaka: None. T. Yanase: Research Support; Self; MSD K.K., Boehringer Ingelheim Pharmaceuticals, Inc., Takeda Pharmaceuticals, Japan.
The Hyperinsulinemic Isoglycemic Hyperlactemic Clamp
Authors: ZARETSKY DMITRY , YOUNG ANDREW A.
Published: June 2018

The Cori Cycle (glucose-lactate interchange) dominates carbohydrate fluxes. Glucose-to-lactate flux follows peripheral disposal; lactate-to-glucose occurs during gluconeogenesis. As glucose flux can be assessed by the change in an exogenous glucose infusion rate (GIR) required to maintain a constant concentration (glucose clamp), so too can lactate flux be assessed from an exogenous lactate infusion rate (LIR) required to maintain isolactemia. Simultaneous assessment of both fluxes will enable estimation of lactate-derived gluconeogenesis (GNG), and its contribution to the rate of glucose appearance (Ra) without using radiolabeled tracers, the mainstay of flux measurement. Anesthetized Sprague-Dawley rats were intubated, catheterized via artery and vein, and partially pancreatectomized to eliminate endogenous glucagon secretion. Establishment of a hyperinsulinemic (100mU/kg/h), hyperglycemic (7mM) clamp was followed by a hyperlactemic (4mM) clamp via independent variable infusions of glucose/Na-lactate in response to 5-min measures of plasma concentrations (YSI 2950D Analyzer with glucose/lactate electrodes). Hyperlactemia (∼10x resting) rendered it the dominant gluconeogenic substrate. After at least 30 min of isolactemia, glucagon was infused for 60 min (0, 1, 3, 10 or 50 µg/kg/h) to enable dose/concentration response analysis. Glucagon administration dose-dependently reduced the GIR and increased the LIR necessary to maintain isoglycemia/isolactemia (respective ED50’s 1.33, 0.98 µg/kg/h). δ-GIR of up to -12.2 mmol/kg/h potentially represented increased glucose Ra. δ-LIR of up to +8.6 mmol/kg/h potentially represented GNG. Since 2 lactate moieties are required per glucosyl, GNG contributed ∼35% of glucagon-stimulated glucose Ra (∼65% being glycogenolysis).

In conclusion, the hyperinsulinemic isoglycemic hyperlactemic clamp enables a quantitative assessment of hepatic processes, complementing that of peripheral processes revealed with the glucose clamp.

Disclosure

D. Zaretsky: Employee; Self; Intarcia Therapeutics, Inc. A.A. Young: Employee; Self; Intarcia Therapeutics, Inc.. Stock/Shareholder; Self; GlaxoSmithKline plc..
Autoimmunity in Patients with Type 2 Diabetes in Newfoundland and Labrador
Authors: IQBAL SAHAR J.
Published: June 2018

Latent Autoimmune Diabetes of the Adult (LADA) is a slowly progressive form of diabetes associated with autoantibody positivity. LADA shares characteristics with type 1 and 2 diabetes (T1D, T2D) but is often misdiagnosed as T2D. Epidemiologic data shows patients with LADA may account for 2 to 12% of all cases of diabetes.

The aim of the study was to collect information on prevalence of autoimmunity in patients with T2D and describe their characteristics in Newfoundland and Labrador, Canada.

A cross- sectional study design was used to collect data in 140 patients. The frequency of GAD 65 antibody, ICA, Insulin and C-peptide levels was examined by radioimmunoassay and enzyme-linked immunosorbent assays. We collected data on height, weight, Body Mass Index (BMI) and tested for Thyroid Stimulating Hormone and Glycosylated Hemoglobin levels. Data collected included age at diagnosis, family history and number of co-morbidities. Adults 18 years and above with T2D were included. Pregnant and lactating females were excluded. SPSS version 24 was used to analyze data.

Results obtained showed 13 (8 males and 5 females, p 0 .181) of the 132 patients tested were GAD 65 positive thus placing prevalence of LADA in NL at 9.7%. Baseline characteristics were similar in both GAD 65 positive and negative patients. None of the patients tested were positive for ICA antibodies thereby improving our confidence that the sample did not contain any patients with T1D. Prevalence of GAD 65 was higher in the patients requiring Insulin whereas the C-peptide levels were lower. BMI was high in both GAD 65 positive and negative group with data trending towards higher BMI in the GAD 65 positive group.

GAD 65 positivity was associated with older age group, higher BMI which is different from reported literature indicating that older age and obesity have a predictive value in diagnosis of T2D but not in LADA.

Disclosure

S.J. Iqbal: None.
Role of ROCK1 in Pancreatic ß Cells
Authors: SUNG BYUNG-JUN , HUR KYU YEON , KIM JAE HYEON , KIM YOUNG-BUM , LEE MOON-KYU
Published: June 2018

Insulin signaling in pancreatic β-cell is critical for normal insulin secretion. ROCK1 plays an important role in the regulation of insulin and leptin signaling in the context of glucose and energy metabolism. However, the role of ROCK1 in pancreatic β-cell remains unknown. The current study was to determine whether deleting ROCK1 from β-cell affects insulin secretion and glucose metabolism in mice and cultured cell lines. Here we show that β-cell-specific deletion of ROCK1 results in a significant decrease insulin secretion in mice fed a normal chow diet, leading to hyperglycemia. These effects were independent of changes in adiposity. Consistent with this, in vitro studies with INS-1 and MIN6 cells indicated that inhibition of ROCK1 decreased insulin secretion in response to glucose without changes in β-cell proliferation and apoptosis. In addition, β-cell-ROCK1 deficient mice displayed glucose intolerance, as evidenced by the fact that the area under the glucose curve was increased 53% in β-cell-ROCK1 deficient mice compared with control mice. However, neither body weight, food intake nor insulin sensitivity was changed in theses mice. Upon insulin stimulation, proximal insulin signaling components, including p-IRS2, p-PI3K and p-Akt were impaired when ROCK1 expression was inhibited in INS-1 cells, suggesting a role for ROCK1 in insulin signaling. These data indicate that activation of ROCK1 in pancreatic β-cell is required for the maintenance of normal insulin secretion. Thus, pancreatic ROCK1 might be a key regulator of glucose homeostasis.

Disclosure

B. Sung: None. K. Hur: None. J. Kim: None. Y. Kim: None.M. Lee: None.
The Simultaneous Changes of Endogenous Glucose Production, Postprandial Glucagon, and Fasting Glutamine during Weight Loss in Type 2 Diabetes
Authors: OTTEN JULIA , STOMBY ANDREAS , WALING MARIA , CHORELL ELIN , RYBERG MATS , SVENSSON MICHAEL B. , HOLST JENS J. , OLSSON TOMMY
Published: June 2018

In type 2 diabetes, high blood glucose levels are associated with increased glucagon levels and insufficient suppression of endogenous glucose production (EGP). Notably, glutamine, a gluconeogenic amino acid, is decreased compared to healthy individuals.

We randomized 32 overweight patients with type 2 diabetes to either a Paleolithic diet (PD) or a Paleolithic diet combined with supervised exercise (PD-EX). Study duration was 12 weeks. Subjects conducted a solid mixed meal test: glucagon, insulin and glucose were measured at 0, 30, 60, 120 and 180 min with calculation of the total area under the curve for the response. On another study day, fasting glutamine was measured with GC-MS and suppression of EGP was examined with the hyperinsulinemic euglycemic clamp technique with [6,6-2H2]glucose as a tracer and with an insulin infusion of 40 mIU/m2/min.

Median weight loss was 7 kg in both study groups. Fasting glucose and fasting insulin decrease significantly only in the PD-EX group. Postprandial glucose decreased by 13% in the PD group (P<0.05) and by 26% in the PD-EX group (P<0.01), without difference between groups. Fasting glucagon tended to decrease in both study groups. Postprandial glucagon decreased by 22% in the PD-group (P<0.01) and by 21% in the PD-EX group (P=0.13). Fasting glutamine increased non-significantly in both groups. Suppression of EGP increased by 26% in the PD group (P<0.05) and by 10% in the PD-EX group (P=0.75). During the intervention, the increased suppression of EGP was associated with a) reduction of postprandial glucagon levels (rS=-0.65, P<0.01), b) decreasing fasting glucagon (rS=-0.51, P<0.05) and c) the increasing fasting glutamine levels (rS=0.51, P<0.05).

We conclude that glucagon, an activator of EGP, decreases in concert with improved suppression of EGP during weight loss in type 2 diabetes patients. An increase in plasma glutamine may be due to decreased gluconeogenesis.

Disclosure

J. Otten: None. A. Stomby: None. M. Waling: None. E. Chorell: None. M. Ryberg: None. M.B. Svensson: None. J.J. Holst: Advisory Panel; Self; Novo Nordisk A/S. Board Member; Self; Zealand Pharma A/S. Speaker's Bureau; Self; Merck Sharp & Dohme Corp., AstraZeneca. Research Support; Self; Danish Diabetes Academy, Novo Nordisk Foundation. Other Relationship; Self; Antag Therapeutics. Other Relationship; Spouse/Partner; Antag Therapeutics. T. Olsson: None.
GLP-1 Secretion Is Increased upon Blockade of the Somatostatin Receptor Subtype 2 and 5 Resulting in GLP-1 Receptor-Mediated Lowering of Blood Glucose in Mice
Authors: JEPSEN SARA L. , WEWER ALBRECHTSEN NICOLAI J. , PEDERSEN JENS , ENGELSTOFT MAJA S. , DEACON CAROLYN F. , HOLST JENS J.
Published: June 2018

Somatostatin (SS) is a hormone inhibiting the secretion of the incretin hormone, glucagon-like peptide-1 (GLP-1) by binding to 5 SS receptors (SSTr). However, which SSTrs involved is still unclear. We hypothesized that antagonizing the SSTrs on enteroendocrine L-cells may increase secretion of GLP-1 and via GLP-1 receptor mediated mechanism lead to lowering of blood glucose. To investigate the level of the SSTrs we did expression analysis on L-cells isolated from throughout the murine intestine (n=3). We perfused the proximal small intestine from mice (n=6-7) to investigate the impact of SSTr2 and 5 on GLP-1 secretion using intra-arterial administration of specific antagonists. Lastly we performed oral glucose tolerance tests (OGTT) on mice (n=5-8) after giving 1mg/ml SSTr2 or 5 antagonist with and without the GLP-1 receptor antagonist exendin(9-39) to investigate the role of these receptors on the glucose profile and to elucidate if their effect was mediated via GLP-1. The expression levels of the SSTrs showed that SSTr2 and especially 5 had the highest expression on L-cells throughout the intestine. The SSTr2 antagonist resulted in a increase in GLP-1 secretion from the perfused intestine (100nM, 4.96pM±0.56; 1µM, 14.55pM±1.4; 10µM, 16.46pM±1.18pM; all p<0.0001). The same doses of SSTr5 antagonist gave a more potent GLP-1 secretion in this setup (100nM, 5.82pM±0.7 p=0.0004; 1µM, 19.79pM±1.2 p<0.0001; 10µM, 48pM±2.6 p<0.0001). Both SSTr2 and SSTr5 antagonists significantly reduced the blood glucose during an OGTT compared to the control (p=0.00and p=0.0072 respectively). This reduction in blood glucose was significantly increased when adding exendin(9-39) together with either the SSTr2 or 5 antagonist (p<0.0001 for SSTr2, p=0.0036 for SSTr5). Our results demonstrate that inhibiting the SSTr2 and SSTr5 increases GLP-1 secretion which in turn may cause the lowering in blood glucose during an OGTT.

Disclosure

S.L. Jepsen: None. N.J. Wewer Albrechtsen: Speaker's Bureau; Self; Merck Sharp & Dohme Corp.. Other Relationship; Self; Mercodia, Alpco. J. Pedersen: None. M.S. Engelstoft: None. C.F. Deacon: Other Relationship; Self; Merck & Co., Inc., Eli Lilly and Company. Employee; Spouse/Partner; Merck Sharp & Dohme Corp.. Stock/Shareholder; Spouse/Partner; Merck & Co., Inc. J.J. Holst: Advisory Panel; Self; Novo Nordisk A/S. Board Member; Self; Zealand Pharma A/S. Speaker's Bureau; Self; Merck Sharp & Dohme Corp., AstraZeneca. Research Support; Self; Danish Diabetes Academy, Novo Nordisk Foundation. Other Relationship; Self; Antag Therapeutics. Other Relationship; Spouse/Partner; Antag Therapeutics.
In Insulin-Resistant Subjects, Islet Functional Changes Might Represent an Attempt to Increase the Incretin Effect
Authors: MEZZA TERESA , MOFFA SIMONA , SORICE GIAN PIO , ASSUNTA CEFALO CHIARA MARIA , CINTI FRANCESCA , MARI ANDREA , GIACCARI ANDREA
Published: June 2018

Nondiabetic insulin-resistant subjects (InsRes) display an increased α-cell mass, suggesting a key role of α-cells in the compensatory mechanisms to cope with insulin resistance states. The mechanism contributing to greater GLP-1 secretion detected in healthy patients following pancreatoduodenectomy (PD), is not fully understood and suggests other potential sources of the incretin hormone, such as α-cells. To investigate the dynamic changes of in-vivo GLP-1 secretion, insulin secretion (IS), and incretin effect (IE), 16 nondiabetic patients (10 F/6 M, 51±15 years.) scheduled for PD underwent a mixed meal test (MMT), a 2-h hyperglycemic clamp (HC), and a hyperinsulinemic euglycemic clamp (HEC) before and after surgery. β-cell glucose sensitivity (GS) was calculated as the ratio of IS and glucose increments, both during HC and MMT. The IE was estimated as the ratio of GS during the HC and MMT. InsRes displayed a reduction in the 1st IS at baseline (p=0.04), which did not decrease following PD. Both groups experienced a reduction of 2nd phase ISR (p<0.for both groups) and arginine-induced IS (p<0.for both groups) after surgery, with no difference observed in the reduction delta; while a significant reduction in basal ISR and GS was observed only in InsRes after surgery (respectively p=0.03 and p=0.06). Following PD, GLP-1 secretion significantly increased in both groups (P=0.and P=0.02 respectively), but the percentage of increase was greater in InsRes compared to insulin-sensitive subjects (InsSen) (respectively 54.4% vs. 36.2%). Further, the IE was significantly decreased in the InsSen (p=0.05), whereas no changes were observed in InsRes. Our data suggest that acute islet mass reduction leads to different functional effects in InsRes compared to InsSen. Impaired β-cell GS and reduced 1stIS are relatively compensated by preserved IE following PD in InsRes. This may be driven by an increased α-mass in InsRes, that might be the source of the increased GLP-1 secretion.

Disclosure

T. Mezza: None. S. Moffa: None. G. Sorice: None. C. Cefalo: None. F. Cinti: None. A. Mari: None. A. Giaccari: Advisory Panel; Self; Sanofi-Aventis. Speaker's Bureau; Self; AstraZeneca, Merck Sharp & Dohme Corp., Amgen Inc..
The Family Coriobacteriaceae Is a Potential Contributor to the Beneficial Effects of Roux-en-Y Gastric Bypass on Type 2 Diabetes
Authors: HU CHENG , JIA WEIPING , LIU HAIJUN
Published: June 2018

Background: Changes in gut microbiota induced by bariatric surgery have been associated with metabolic benefits.

Objectives: Our aim was to identify specific gut microbiota that may contribute to the improvement of type 2 diabetes (T2D) after RYGB.Setting: Laboratories of Shanghai Diabetes Institute and Shanghai Sixth People’s Hospital.

Methods: Diabetic rats induced via a high-fat diet and low-dose streptozotocin administration were randomized to RYGB or sham surgery (SHAM), and stool samples were collected at baseline and at postoperative week 8. The faecal microbiota was profiled using 16S ribosomal RNA (rRNA) gene sequencing. Additionally, we performed a case-control study of the gut microbial community profiles of T2DM patients compared to those of healthy individuals (CONT) via 16S rRNA gene sequencing of mucosal-luminal interface samples collected from the ascending colon during colonoscopy.

Results: RYGB significantly reduced the body weight and improved glucose tolerance and insulin sensitivity in diabetic rats. Principal coordinate analysis showed that RYGB caused marked alterations in the gut microbiota. The RYGB group was postoperatively enriched for Bacteroidetes, Proteobacteria, Fusobacteria and Actinobacteria; whereas the SHAM group was enriched for Firmicutes and Verrucomicrobia. Based on the gut microbial patterns in the T2D patients, we found that the family Coriobacteriaceae within Actinobacteria might contribute to the beneficial effects of RYGB on T2D.

Conclusions: RYGB significantly improves glucose metabolism and alters the gut microbiota. Moreover, the family Coriobacteriaceae may partly mediate the beneficial effects of RYGB on T2D and thus possibly contribute to the development of novel bacteria-based therapeutic approaches.

Disclosure

C. Hu: None. W. Jia: None. H. Liu: None.
The Regulation of BRD7 in the Insulin Signaling Pathway
Authors: PARK SANG , HAN YOUNGAH , SALAZAR HERNANDEZ MARIO A.
Published: June 2018

Elevated endoplasmic reticulum (ER) stress plays crucial roles in the development of insulin resistance, type 2 diabetes, and obesity. X-Box binding protein 1 (XBP1) is one of the master regulators of ER folding function and is involved in the regulation of ER stress. Haploinsufficiency of XBP1 leads to the development of obesity, insulin resistance, and type 2 diabetes even in mice on a background that is resistant to the development of obesity. To date, our understanding of the regulation of XBP1 in metabolic processes is still limited. In our previous work, we demonstrated that bromodomain containing protein 7 (BRD7) regulates glucose homeostasis by increasing the nuclear translocation of XBP1s through its interaction with the regulatory subunits of phosphatidyl inositol 3-kinase (PI3K), p85s. We also showed that BRD7 protein levels are significantly reduced in the liver of genetically obese ob/ob and high fat diet (HFD)-induced obese mouse models, and over-expression of BRD7 in the liver of ob/ob and HFD fed mice ameliorates hyperglycemia and glucose intolerance through increasing the activity of XBP1s. In our current unpublished work, we show that BRD7 heterozygous knockout mice are prone to developing obesity when they are challenged with a HFD. They display increased hepatic triglyceride levels with moderately severe steatosis. In addition, we generated a liver-specific BRD7 knockout mouse model (LBKO) to examine the effect of BRD7 specifically in the liver. LBKO mice fed on a HFD exhibit increased body weights compared to their wild type control mice. Downregulation of BRD7 in the liver led to lack of nuclear XBP1s in the liver in both the knockout mouse model and adenovirus-mediated knockdown system. Our results provide the first evidence that BRD7, which was originally reported as a tumor suppressor, is involved in the development of obesity as well as in the regulation of unfolded protein response (UPR) and glucose metabolism.

Disclosure

S. Park: None. Y. Han: None. M.A. Salazar Hernandez: None.
Berberine Hydrochloride Protects Cytokine-Induced Inflammation through Multiple Pathways
Authors: POUDEL ANIL , ZHOU JOSEPH , MASSEY SCOTT , LI LIXIN
Published: June 2018

Berberine hydrochloride (BBR) has been reported to improve insulin resistance. However, the underlying mechanism remains largely elusive. We therefore investigate the anti-inflammatory role of BBR and the cell signaling pathways that involved.

C2C12 myoblasts were treated with IL-1β (10ng/mL) alone or in combination with TNF-α (50 ng/mL) in the presence or absence of BBR (10 µM) for 24 hours. Western blot analysis was used to assess the protein level. In vivo study is conducted using C57BL/6 mice fed with high fat high sucrose (HFHS) diet to induce insulin resistance. Skeletal muscle was collected after 4 weeks treatment with either 5 mg/kg BBR or vehicle.

BBR reduced activity of iNOS, and stress-related kinases, including p38 MAPK, NF-kB, SAP/JNK, MMK4 and C-JUN that was induced by cytokines in C2C12 myoblast. Furthermore, BBR reversed cytokine mediated suppression of AMPK-α, augmented activity of SIRT-1 and PGC-1α. Antioxidant enzyme or transcription factor, nuclear factor like 2 (NRF-2) were also elevated after BBR treatment. In addition, BBR improved insulin signaling in C2C12 myoblast. HFHS diet induced iNOS expression was reversed by BBR in skeletal muscle. Augmentation of the activity of uncoupled protein-3 (UCP-3) and PPARα in HFHS diet skeletal muscle was observed.

Our results demonstrate that BBR ameliorates cytokine induced inflammation and oxidative stress. BBR improves mitochondrial function, promotes insulin sensitivity. The anti-inflammatory effect of BBR in skeletal muscle may mediated through multiple pathways including activation of AMPKα-SIRT-1-PGC-1α pathway, and inhibition of MMK4- SAP/JUNK-C-JUN pathway. BBR is a potential medication in treating obesity induced insulin resistance.

Disclosure

A. Poudel: None. J. Zhou: None. S. Massey: None. L. Li: None.
Endothelial ULK1 Regulates High-Fat Diet–Induced Obesity via FGF-21
Authors: LI MANNA , QIAN MING , XU JIAN
Published: June 2018

Fibroblast grow factor (FGF) 21 is a hormone with huge potential for pharmaceutical development for treatment of metabolic disease. The role of endothelial cell (EC)-derived FGF-21 in obesity remains largely unknown, although we detected lower EC-FGF-21 levels in visceral fat of Ob/Ob and high-fat diet (HFD)-fed mice than the control lean mice. This study was to identify the role and mechanism of EC-FGF-21 in HFD-induced obesity using cell culture and mouse models. Mimicking HFD, long-term incubation with palmitate (100 µM, 16h) reduced FGF-21 levels in HUVEC that were co-cultured with 3T3-L1-adipocytes. The reduction was prevented in ULK1-siRNA-treated HUVEC, via ATF4-promoted mRNA expression and increased protein stability of FGF-21. Thus, FGF-21-induced expression of FGF-R1c, β-Klotho, UCP1 and GLUT1 was enhanced in co-cultured adipocytes. To confirm these findings in vivo, we used a Tie2/Cre-lox system to generate mice with EC-knockout of FGF-21 (FKO), ULK1 (UKO), or both (DKO). In 8-to-10-week-old male mice (n=15-20/group) fed an HFD (TestDiet: 60% energy from fat) for 3 months, wild type (WT, C57BL/6J) mice developed obesity and insulin resistance, which was worsened in FKO mice. In contrast, UKO mice gained less body weight and visceral fat mass and had improved glucose tolerance, without significant differences in food and water intake. Levels of adipose EC-FGF-21 was normalized in UKO mice, as determined by immunohistochemistry of FGF-21 and EC markers (CD31 and lectin). Expression of adipose FGF-R1c, β-Klotho, UCP1, and GLUT1 was restored in UKO, but not WT, mice. Concomitant with UCP1 induction, the visceral fat showed beige-cell-like deposits with fewer expanded adipocytes. However, this favorable phenotype was absent in DKO mice.

In summary, EC-ULK1 mediated HFD-induced obesity and insulin resistance through loss of EC-FGF-21, at least in part, by blunting local FGF-21-signaling in adipose tissue. Therapeutic targeting of the EC-ULK1-FGF-21 pathway may be a new treatment for obesity and insulin resistance.

Disclosure

M. Li: None. M. Qian: None. J. Xu: None.
Loss of Fractalkine-CX3CR1 Signaling Exacerbates Obesity-Induced Adipose Tissue Inflammation and Insulin Resistance through M1 Dominant Shift in Macrophages
Authors: NAGASHIMADA MAYUMI , NI YINHUA , OTA TSUGUHITO
Published: June 2018

Adipose tissue macrophages (ATMs) accumulation through CCR2 and its ligand MCP-1 is considered pivotal in the development of insulin resistance. Recently, we found that CX3C chemokine, fractalkine (CX3cl1) mRNA expression was persistently downregulated in epididymal white adipose tissue (eWAT) of HF diet (HFD)-induced obese (DIO) mice, as compared to lean controls. Furthermore, we demonstrated that loss of fractalkine receptor, CX3CR1, exacerbated HFD-induced insulin resistance and glucose intolerance. Here, we show that fractalkine-CX3CR1 signaling plays a crucial role in the inflammatory response to HF feeding by regulating macrophage M1/M2 status. To determine the effect of fractalkine-CX3CR1 signaling on ATM subsets, we performed flow cytometry analysis to quantify M1/M2 ATMs in eWAT of CX3CR1-/- (KO) and WT mice. Immunofluorescence study of eWAT in DIO mice revealed that CX3CR1 was predominantly expressed by F4/80+ macrophages in crown-like structures (CLS). On an HFD, KO mice had increased macrophage infiltration and formation of CLS in eWAT compared with those of WT mice, despite that weight and adipocyte size were similar. KO mice had 40% or 39% more CD11c+CD206- (M1) ATMs on normal chow (NC) or HFD, and 17% or 49% fewer CD11c-CD206+ (M2) ATMs on NC or HFD, respectively, than WT mice, resulting in predominance of the M1 over M2 ATM population. Furthermore, the predominance of the Ly6Chi over the Ly6C- monocyte population was observed in the peripheral blood and bone marrow (BM) of KO mice on NC or HFD. Importantly, HFD-induced adipose inflammation and hyperinsulinemia were aggravated in chimeric mice lacking CX3CR1 only in myeloid cells (BM transplant of KO to WT) as compared to control mice (BM transplant of WT to WT). Thus, loss of fractalkine-CX3CR1 signaling causes M1 dominant phenotypic shift in ATMs, which contributes to exacerbation of obesity-induced adipose tissue inflammation and insulin resistance.

Disclosure

M. Nagashimada: None. Y. Ni: None. T. Ota: None.
Glycolytic Activation Promotes PDGF-B Production in Adipose Tissue Macrophages during Obesity Development
Authors: ONOGI YASUHIRO , WADA TSUTOMU , MATSUZAWA TAKATOSHI , OKEKAWA AKIRA , WATANABE ERI , TSUNEKI HIROSHI , SASAOKA TOSHIYASU
Published: June 2018

The vascular network develops with remodeling in adipose tissue (AT) during obesity. We have found that PDGF-B detaches pericytes from the vasculature, which plays a crucial role in angiogenesis during obesity. The expression of PDGF-B was significantly elevated in obesity-associated infiltrated CD11c-positive (+) AT macrophages (ATMs). Interestingly, the expression was low in both CD11c+ ATMs and CD206+ ATMs of lean mice. Since glycolytic and oxidative metabolism are key metabolic programs that regulate ATMs’ polarity and function, we hypothesized that hyperglycemia and inflammation synergistically drive PDGF-B production by reprogramming metabolic pathways in ATMs.

The expression of PDGF-B was increased in the AT of high-fat diet (HFD)-fed mice and STZ-induced hyperglycemic mice. In accordance, exposure to high glucose led to increase the expression of PDGF-B in association with that of Itgax (CD11c) in RAW264.7 macrophages. In peritoneal macrophages, LPS but not IL-4 provoked PDGF-B expression, which was completely blocked by pretreatment with 2-DG, a glycolysis inhibitor. Moreover, LPS effectively augmented high glucose-induced its expression, and that was abolished by MG132, an NFκB inhibitor. Since macrophages highly accumulated in obese AT, where pericytes were detached from vasculature, we examined directly whether PDGF-B from macrophage involves in AT expansion during obesity. To this end, we intraperitoneally injected liposome-encapsulated clodronate (CL) to mice twice a week for 6 weeks during HFD feeding. ATMs were effectively depleted by CL with reduced PDGF-B expression in AT of HFD-induced obese mice. Importantly, HFD-induced obesity was effectively prevented by CL.

In conclusion, PDGF-B from ATMs has a crucial function in AT expansion during obesity. Hyperglycemia and chronic inflammation reprogram metabolic pathways toward glycolysis, and subsequent activation of NFκB plays a pivotal role in the production of PDGF-B in ATMs during obesity.

Disclosure

Y. Onogi: None. T. Wada: Research Support; Self; Sanwa Kagaku Kenkyusho Co., Ltd.. T. Matsuzawa: None. A. Okekawa: None. E. Watanabe: None. H. Tsuneki: None. T. Sasaoka: None.
Deficiency of Melanocortin 4 Receptor Promotes Vascular Vulnerability in Mice
Authors: MORI KENTARO , TSUCHIYA KYOICHIRO , NAKAMURA SUGURU , MIYACHI YASUTAKA , SHIBA KUMIKO , OGAWA YOSHIHIRO , KITAMURA KENICHIRO
Published: June 2018

Background: Obesity is known as a risk factor of hypertension and abdominal aortic aneurysm (AAA) in humans. Melanocortin 4 receptor (MC4R) is primarily expressed in brain, and mediates most of the anorectic effects of leptin; in humans, deficiency or mutation of MC4R is the most common monogenic form of obesity. However, it remains unknown whether MC4R signaling affects the development of obesity-associated hypertension, vascular vulnerability and aortic aneurysm.

Methods and Results: MC4R-knockout (KO) mice fed a high-fat/high-sucrose diet (HF/HS) for 20 weeks displayed obesity and insulin resistance associated with hyperleptinemia compared to HF/HS-fed wild type mice. HF/HS-fed MC4R-KO mice exhibited hypertension, and showed increase of medial thickness and elastic lamina destruction in aortae. In aortae of HF/HS-fed MC4R-KO mice, genes of MCP-1, TNF-α, and F4/80 were markedly upregulated relative to HF/HS-fed wild type mice. Following the administration of angiotensin II (Ang II, 500 ng/kg/min), HF/HS-fed MC4R-KO mice showed significantly lower survival rate and higher incidence of AAA than HF/HS-fed wild type mice, with increased expression of F4/80- and osteopontin-positive cells in aortae. Furthermore, HF/HS-fed ob/ob mice exhibited significantly lower incidence of Ang II-induced AAA with a trend of higher survival rate than HF/HS-fed MC4R-KO mice. Leptin (100 ng/ml) significantly upregulated osteopontin gene expression in primary cultured vascular smooth muscle cells (VSMCs), but not in primary cultured macrophages.

Conclusion: Our observation clearly demonstrates that deficiency of melanocortin 4 receptor promotes the development of hypertension, vascular vulnerability, and Ang II-induced AAA in mice. It also suggests that hyperleptinemia caused by central leptin resistance could contribute to the development of these vascular phenotypes in MC4R-KO mice, at least partly via induction of osteopontin in VSMCs.

Disclosure

K. Mori: None. K. Tsuchiya: None. S. Nakamura: None. Y. Miyachi: None. K. Shiba: None. Y. Ogawa: Research Support; Self; AstraZeneca. Advisory Panel; Self; Gilead Sciences, Inc.. Research Support; Self; Japan Agency for Medical Research and Development, Japan Society for the Promotion of Science, Mitsubishi Tanabe Pharma Corporation, Mochida Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd.. K. Kitamura: None.
Continuous Subcutaneous Delivery of a Glucagon Analog, ICA6150349, to Rats and Nonhuman Primates Demonstrates Safety and Tolerability in a 14-Day Toxicity Study
Authors: ZANE DORIS , SINGH ROSHNI , SHELTON JOSEPHINE , ROLLER SHANE , PAULIK MARK , WAY JAMES , BLACKWELL WILLIAM C. , SRIVASTAVA VED , HODGE REBECCA , FELDMAN PAUL L. , ALESSI THOMAS R.
Published: June 2018

ICA6150349, a 38-amino-acid peptide, is a glucagon analog that is highly selective for the human glucagon receptor. In a 14-day toxicity study, rats and cynomolgus monkeys received ICA6150349 at doses of 0, 0.875, 8.75, 87.5 mcg/day and 0, 17.92, 179.2, 3584 mcg/day, respectively. Animals were evaluated for reversibility of findings for an additional 14 days. The TK characteristics of ICA6150349 were also determined. In rats, there was a pharmacologically expected dose-dependent decrease in body weight (BW) gains and food consumption, which demonstrated a recovery trend. Clinical pathology and anatomic pathological changes, largely restricted to the exaggerated pharmacological effect of glucagon at high exposures, had mostly recovered during the dose-free period. ICA6150349 exposures (AUC0-24) increased dose-proportionally in male and female rats on D2. On D14, exposure was 40-60% lower than on D2 in the 0.875 and 8.75 mcg/day dose groups and similar to D2 in the 87.5 mcg/day dose group after adjustment for changes in BW. ADAs were observed in rats from all dose groups, but did not impact the TK of ICA6150349. In cynomolgus monkeys, the effects of ICA6150349 were limited to minimally decreased RBC mass (hemoglobin, red blood cell count, and hematocrit) at 3584 mcg/day with recovery. ICA6150349 exposure (AUC0-24) increased dose-proportionally on D2. On Day 14, exposures were 14-65% lower than on D2 in all groups except the 3584 mcg/day females. ADA were observed in 46.7% of treated monkeys; however, ADA status only affected TK at the 3584 mcg/day dose level. Overall, ICA6150349 was well tolerated. Nonclinical safety studies identified no adverse effects (based on the minimal magnitude of change, severity grading and reversibility) at any dose level tested with a NOAEL of 87.5 mcg/day in rats and 3584 mcg/day in monkeys. All observed effects have been non-adverse, reversible, and consistent with known actions of glucagon.

Disclosure

D. Zane: None. R. Singh: None. J. Shelton: None. S. Roller: Employee; Self; Intarcia Therapeutics, Inc. M. Paulik: Employee; Self; Intarcia Therapeutics, Inc. J. Way: Employee; Self; Intarcia Therapeutics, Inc. W.C. Blackwell: Employee; Self; Intarcia Therapeutics, Inc. V. Srivastava: Employee; Self; Intarcia Therapeutics, Inc. R. Hodge: Employee; Self; Intarcia Therapeutics, Inc.. Stock/Shareholder; Self; Intarcia Therapeutics, Inc. P.L. Feldman: Employee; Self; Intarcia Therapeutics, Inc. T.R. Alessi: Employee; Self; Intarcia Therapeutics, Inc..
Cost-Related Insulin Underuse Is Common and Associated with Poor Glycemic Control
Authors: HERKERT DARBY M. , VIJAYAKUMAR PAVITHRA , LUO JING , SCHWARTZ JEREMY , RABIN TRACY L. , DEFILIPPO EUNICE M. , LIPSKA KASIA J.
Published: June 2018

Insulin is an essential medicine for people with diabetes (DM), but increasing prices have threatened its affordability. We examined the prevalence of and factors associated with cost-related insulin underuse.

We administered a cross-sectional survey to patients with DM prescribed insulin at Yale Diabetes Center (YDC). Our primary outcome was cost-related underuse in the past 12 months, defined by a positive response to any 1 of 6 questions: Did you ⋯ 1) Use less insulin than prescribed 2) Try to stretch out your insulin 3) Take smaller doses of insulin than prescribed 4) Stop insulin 5) Not fill an insulin prescription 6) Not start insulin ⋯ because of cost? We examined the association of cost-related underuse with HbA1c >9% using logistic regression controlling for age, sex, age, DM duration, and income.

Out of 354 patients prescribed insulin who had YDC visit in July 2017, 199 (56.2%) completed the survey (50.8% female, 60.8% white, 41.7% type 1). Of these patients, 51 (25.5%) reported cost-related insulin underuse. Patients with cost-related underuse had lower income levels, variable drug coverage and employment (Figure), and 3-fold higher odds of HbA1c >9% (p = 0.03) than patients who did not report underuse.

One in four patients at our urban diabetes center reported cost-related insulin underuse, and this was associated with poor glycemic control. These results highlight an urgent need to address high insulin prices in the U.S.

Disclosure

D.M. Herkert: None. P. Vijayakumar: None. J. Luo: Consultant; Self; Alosa Health, Inc.. J. Schwartz: None. T.L. Rabin: None. E.M. DeFilippo: None. K.J. Lipska: Other Relationship; Self; Centers for Medicare and Medicaid Services. Research Support; Self; National Institutes of Health.