bluuebunny/crossref_metadata_2025 huggingface dataset:
Liver-Specific siRNA Inhibition of Class 2a Histone Deacetylases (HDACs) Reduces Expression of Genes Regulating Gluconeogenesis in Primary Human and Mouse Hepatocytes, but Not in Mice
Authors: GÖRGENS SVEN W. , RAICHUR SURYAPRAKSH , WOHLFART PAULUS , BRUNNER BODO , TENNAGELS NORBERT , BIELOHUBY MAXIMILIAN
Published: June 2018

Gene expression control by histone-deacetylases Class 2a (HDACs 4,5,7,9) has been demonstrated to be involved in the negative regulation of hepatic gluconeogenesis and consequently inhibition of HDACs was shown to result in improved glucose and pyruvate tolerance in rodent type 2 diabetes (T2D) models. However, pan-inhibition of HDACs in all tissues leads also to severe side effects in various tissues, excluding this approach for treatment of T2D. Here, we have investigated in vitro and in vivo safety and efficacy of a liver-selective HDAC knock-down via single or combinatorial siRNAs for HDAC 4, 5 or 7. In primary mouse and human hepatocytes, specific siRNAs directed against HDAC 4, 5 or 7, as well as their combination led to a selective knock-down of the respective target gene(s) by about 80-90%, which paralleled with a significant reduction of genes involved in the regulation of gluconeogenesis. These siRNAs were administered in a liver-specific lipid-particle formulation for treatment of healthy C57BL/6J mice. Mice received five intravenous injections with either PBS, a control siRNA, one of the respective silencing siRNAs (0.75mg/kg) or all possible dual or triple combinations for 25 days. Quantitative real-time PCR demonstrated that siRNA treatment led to a significant downregulation of the respective HDACs in the liver. However no reduction of gluconeogenic genes (PCK1 and G6PC) was detected. Consequently, also no improvement in intraperitoneal pyruvate tolerance test (PTT) could be observed. In addition, no significant differences in fasting blood glucose or plasma insulin were observed between treatment groups.

In summary, although active in cellular systems, liver-targeted siRNA knockdown of class 2a HDACs did not result in a clear inhibition of genes regulating gluconeogenesis in healthy mice suggesting no clear path forward for development of this approach for treatment of T2D.

Disclosure

S.W. Görgens: Employee; Self; Sanofi-Aventis Deutschland GmbH. S. Raichur: None. P. Wohlfart: Employee; Self; Sanofi-Aventis Deutschland GmbH. B. Brunner: Employee; Self; Sanofi. N. Tennagels: Employee; Self; Sanofi R&D. Stock/Shareholder; Self; Sanofi. M. Bielohuby: Employee; Self; Sanofi-Aventis Deutschland GmbH, Sanofi R&D.
Genetic Ablation of Liver CPT-1A Gene Protects Mice against High-Fat Diet–Induced Obesity and Insulin Resistance
Authors: WU DONGHAI
Published: June 2018

Carnitine palmitoyl transferase 1A (CPT-1A) catalyzes the transfer of the acyl group of long-chain fatty acid-CoA moiety onto carnitine, an essential step for the uptake of long-chain fatty acids and their subsequent beta-oxidation in the mitochondrion. It is the rate-limiting step in beta-oxidation and is highly expressed in the liver where it plays an important role in triglyceride metabolism. The physiological consequences of CPT-1A deficiency in liver, however, are yet to be reported. In this study, we have generated mouse model with liver specific deletion of CPT-1A gene. We found that deletion of CPT-1A in liver caused an increased triglyceride content in liver and decreased epididymal adipose tissue weight compared with control mice under high fat diet feeding conditions. RNA-seq analysis between livers of CPT-1A knockout vs. wild type mice revealed altered expression of many metabolic pathways such as steroid hormone biosynthesis and PPAR signaling pathways as well as some novel hepatokines. Further analysis of the significance of these changes is under way but the data so far suggest that a pharmacological agent that alters hepatic fatty acid oxidation (perhaps acting on CPT-1A) could provide a novel approach to target nonalcohol fatty liver disease (NAFLD).

Disclosure

D. Wu: None.
Loss of Insulin/IGF-1 Receptors in Muscle Coordinately Downregulates Mitochondrial Metabolism and Alters Mitophagy via Foxo Transcription Factors
Authors: BHARDWAJ GOURAV , PENNIMAN CHRISTIE M. , SUAREZ BELTRAN PABLO A. , FOSTER COLLIN M. , O'NEILL BRIAN T.
Published: June 2018

Diabetes is associated with decreased muscle strength and energy production. There is a strong association between muscle insulin resistance and mitochondrial abnormalities, but cause/effect remains hotly debated. Recently, we showed that insulin receptor (IR) and IGF-1 receptor (IGF1R) signaling controls muscle atrophy by FoxO1/3/4-dependent regulation of autophagy. To directly test the roles of IR, IGF1R, and FoxO signaling on muscle metabolism and mitochondrial autophagy (mitophagy) we measured mitochondrial function in mice lacking IR (M-IR-/-), both IR/IGF1R (MIGIRKO), or IR/IGF1R/FoxO1/3/4 (QKO) in muscle. RNA seq analysis revealed decreases in multiple mitochondrial pathways from M-IR-/- and MIGIRKO muscle, including OXPHOS. Respiratory capacity and ATP production were decreased in mitochondria and soleus fibers isolated from M-IR-/- and MIGIRKO mice using complex I substrates. These decreases in OXPHOS and mitochondrial function were normalized when FoxOs were deleted in QKO mice. MIGIRKO muscle showed decreased PINK1 (mitophagy protein) and increased LC3II/I ratio, indicating changes in respiration and mitophagy occurred together. To determine if this was cell autonomous, C2C12 myotubes were pre-treated with insulin and/or CCCP (an uncoupler and mitophagy-inducer), and assayed for respiration. Insulin increased respiration and PINK1 levels after 24 hours. Four hour CCCP pre-treatment reduced basal respiration and increased LC3II/I which were partially, but significantly, rescued with insulin in C2C12 myotubes. PINK1 was also decreased in primary myotubes with acute deletion of IR/IGF1R. Thus, loss of IR/IGFR signaling in skeletal muscle directly reduces mitochondrial respiratory capacity and alters mitophagy, which is regulated by FoxO transcription factors.

Disclosure

G. Bhardwaj: None. C.M. Penniman: None. P.A. Suarez Beltran: None. C.M. Foster: None. B.T. O'Neill: None.
A Role for Macrophage Adenosine 2A Receptor in Diet-Induced Nonalcoholic Fatty Liver Disease
Authors: ZHOU JING , LI HONGGUI , LUO XIANJUN , MA LINQIANG , WU CHAODONG
Published: June 2018

Recent evidence suggests that adenosine 2A receptor (A2AR) exerts a protective role in NAFLD. However, the role of the A2AR in macrophages in NAFLD pathophysiology remains unknown. In the present study, hepatic inflammation and fat deposition were examined in myeloid cell-specific A2AR-deficient mice and control mice fed a HFD for 12 weeks. In addition, bone marrow cells were isolated from global and/or myeloid cell-specific A2AR-deficient mice and differentiated into macrophages (BMDM) for inflammatory analysis and for macrophage-hepatocyte co-cultures. Upon HFD feeding, the activity of JNK p46 and NFκB p65 and the mRNA levels of proinflammatory cytokines such as TNFα, IL-1β, and IL-6 in livers of myeloid cell-specific A2AR-defcient mice were significantly higher than their respective levels in livers of control mice. These increases were accompanied with increased severity of diet-induced hepatic steatosis, indicated by the results from liver histology and by elevated levels of liver triglycerides. When BMDM were analyzed for inflammatory activation, the phosphorylation states of JNK p46 and NFκB p65 and the production of proinflammatory cytokines in A2AR-deficient BMDM were significantly higher than their respective levels in control BMDM. Moreover, in macrophage-hepatocyte co-culture systems, hepatocytes co-cultured with A2AR-deficient BMDM revealed significant increases in TNFα, IL-1β, and IL-6 mRNAs in relative to hepatocytes co-cultured with control BMDM. Additionally, hepatocytes co-cultured with A2AR-deficient BMDM accumulated much more fat upon palmitate stimulation than hepatocytes co-cultured with control BMDM. Taken together, these results suggest that the inflammatory status of macrophages can be altered by A2AR in a manner to critically determine the development of NAFLD. Moreover, targeting A2AR to suppress inflammation may be a viable preventive and/or therapeutic approach for inflammation-associated diseases including NAFLD.

Disclosure

J. Zhou: None. H. Li: None. X. Luo: None. L. Ma: None. C. Wu: None.
Integration of BCAA and Lipid Metabolism by the BCKDH Kinase and Phosphatase
Authors: WHITE PHILLIP J. , MCGARRAH ROBERT W. , GRIMSRUD PAUL A. , YANG WEN-HSUAN , ZHANG GUO-FANG , HALDEMAN JONATHAN M. , ARLOTTO MICHELLE , ILKAYEVA OLGA , WYNN RICHARD M. , CHUANG DAVID T. , NEWGARD CHRISTOPHER B.
Published: June 2018

Strong associations exist between branched chain amino acids (BCAA) and dysregulated glucose and lipid metabolism, but underlying mechanisms are not well understood. One factor contributing to elevated BCAA in obesity is inhibitory phosphorylation of the branched chain keto-acid dehydrogenase complex (BCKDH) in liver. We studied the impact of modulating the activity of the BCKDH kinase (BDK) and phosphatase (PPM1K) on amino acid, glucose and lipid metabolism in Zucker fatty rats (ZFR). Daily administration of a selective inhibitor of BDK, BT2 (20mg.kg-1, IP), for one week or expression of a recombinant adenovirus overexpressing PPM1K significantly lowered BCAA and branched chain keto acid (BCKA) levels in ZFR. Remarkably, these effects were accompanied by strong lowering of liver triacylglycerides and improved glucose tolerance in the absence of weight loss. Proteomics analysis of liver samples from these studies revealed that both inhibition of BDK with BT2 and adenoviral mediated overexpression of PPM1K results in reduced phosphorylation of the lipogenic enzyme ATP-citrate lyase (ACL) on its regulatory serine 454. Phosphorylation of ACL on this site is activating. Sequence analysis revealed the presence of a canonical BDK phosphorylation motif surrounding ser454 in ACL and incubation of ACL with purified BDK resulted in ACL phosphorylation in vitro. Furthermore, adenovirus-mediated overexpression of BDK increased ACL phosphorylation and activated de novo lipogenesis in liver of lean Wistar rats. Together these studies reveal a novel regulatory function for BDK and PPM1K in integration of BCAA and lipid metabolism. Moreover, we show that modulation of this node provides broad protection against metabolic abnormalities associated with obesity.

Disclosure

P.J. White: None. R.W. McGarrah: None. P.A. Grimsrud: None. W. Yang: None. G. Zhang: None. J.M. Haldeman: None. M. Arlotto: None. O. Ilkayeva: None. R.M. Wynn: None. D.T. Chuang: None. C.B. Newgard: Advisory Panel; Self; Eli Lilly and Company. Research Support; Self; Eli Lilly and Company.
Estrogen Improves Insulin Sensitivity and Suppresses Hepatic Glucose Production via the Transcription Factor Foxo1
Authors: YAN HUI , ZHOU FENGHUA , YANG WANBAO , PAN QUAN , SHEN ZHENG , HAN GUICHUN , ALLRED KIMBERLY , ALLRED CLINTON , XU YONG , GUO SHAODONG
Published: June 2018

Premenopausal women exhibit enhanced insulin sensitivity and reduced incidence of type 2 diabetes compared with age-matched men, but this advantage disappears after menopause with disrupted glucose homeostasis, in part, owing to a reduction in circulating 17β-estradiol (E2). Fasting hyperglycemia, a feature of diabetes mellitus, is largely derived from dysregulation of hepatic glucose production (HGP), in which hyper-activated Foxo1 induces transcription of genes coding gluconeogenic enzymes. We investigated the E2 action on the regulation of glucose hemostasis in male and ovariectomized (OVX) female of control and liver-specific Foxo1 knockout (L-F1KO) mice, and sought to understand the mechanism by which E2 regulates gluconeogenesis via the interaction with liver Foxo1. E2 subcutaneous implant led to decreased fasting blood glucose and improvement of insulin sensitivity in both male and OVX female mice in parallel with the suppression of gluconeogenesis and hepatic gluconeogenic gene expression, including glucose 6-phosphatase (G6pase) and phosphoenolpyruvate carboxykinase 1 (Pck1). Importantly, these effects of E2 were only observed in control mice but disrupted in L-F1KO mice. Moreover, suppression of HGP and gluconeogenesis by E2 only occurred in hepatocytes isolated from control mice and we confirmed that Foxo1 was required for the E2 action on gluconeogenesis. E2 suppressed Foxo1 via activation of ERα and phosphorylation of Akt, while inhibition of both ERα and Akt signaling abolished the effect of E2 on gluconeogenesis. Collectively we demonstrated that the activation of ERα-Akt-Foxo1 signaling serves as an important mechanism of estrogen in control of glucose homeostasis. These results may help explain the gender difference in the incidence of type 2 diabetes and suggest an approach to target ERα to modulate glucose metabolism in diabetic patients.

Disclosure

H. Yan: None. F. Zhou: None. W. Yang: None. Q. Pan: None. Z. Shen: None. G. Han: None. K. Allred: None. C. Allred: None. Y. Xu: None. S. Guo: None.
Serum Apolipoprotein C-III and Cardiovascular Events in Type 1 Diabetes
Authors: BASU ARPITA , BEBU IONUT , JENKINS ALICIA , STONER JULIE , ZHANG YING , KLEIN RICHARD L. , LOPES-VIRELLA MARIA F. , TIMOTHY GARVEY W. , BUDOFF MATTHEW , LYONS TIMOTHY ,   DCCT/EDIC RESEARCH GROUP
Published: June 2018

Background: Apolipoproteins Apo-B, Apo-C-III, and Apo-E are atherogenic and are associated with diabetic dyslipidemia and cardiovascular disease (CVD). Apolipoprotein-defined lipoprotein subclasses (ADLS) also show associations with CVD, but prospective associations of these detailed metrics with CVD in people with type 1 diabetes (T1DM) have not been explored.

Objective: Prospective associations of serum apolipoproteins and ADLS with CVD and major atherosclerotic cardiovascular events (MACE: composite of CVD death, nonfatal myocardial infraction or nonfatal stroke) in T1DM.

Methods: Serum apolipoproteins and ADLS (14 biomarkers in total) measured in sera (1997-2000) in a subset (n=465) of the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications cohort. Prospective associations of CVD and MACE over 5942 and 6180 patient-years follow-up, respectively, were investigated using Cox proportional hazards models unadjusted and adjusted for risk factors.

Results: During 15 years follow-up, 50 CVD and 24 MACE events occurred. Significant positive univariate associations for CVD were: Lp-B, Apo-B, Apo-C-III (total, heparin precipitate (HP), i.e., Apo-C-III in VLDL, and heparin soluble (HS), i.e., Apo-C-III in HDL) and Lp-B:C; for MACE: Apo-C-III sub fractions (total, Apo-C-III-HP, and Apo-C-III-HS). Adjusting for sex, age, HbA1c, systolic blood pressure, pulse rate, LDL-cholesterol, and triglycerides: for CVD, Apo-C-III-HS remained significant (HR (95% CI): 1.18 (1.03, 1.35); P=0.01). For MACE, total Apo-C-III and Apo-C-III-HS persisted when adjusted for age, HbA1c and LDL [1.11 (1.01, 1.22); P=0.02, and 1.22 (1.02, 1.45); P=0.02, respectively], but not for age, HbA1c and (log) triglycerides. No significant associations of other ADLS, Apo-A-I, A-II and Apo-E with CVD/MACE were observed.

Conclusions: Total serum Apo-C-III and Apo-C-III in HDL and Apo-B-containing lipoproteins may serve as predictive biomarkers for CVD events in T1DM adults.

Disclosure

A. Basu: None. I. Bebu: None. A. Jenkins: Research Support; Self; Medtronic, Mylan, Sanofi-Aventis. J. Stoner: None. Y. Zhang: None. R.L. Klein: None. M.F. Lopes-Virella: None. W. Garvey: Advisory Panel; Self; Novo Nordisk Inc., Merck & Co., Inc.. Research Support; Self; Sanofi, Pfizer Inc., Novo Nordisk Inc., AstraZeneca, Merck & Co., Inc., Elcelyx Therapeutics, Inc., Lexicon Pharmaceuticals, Inc., Eisai Inc. M. Budoff: Speaker's Bureau; Self; Janssen Pharmaceuticals, Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Amarin Corporation. T. Lyons: None.
Praliciguat, a Clinical-Stage sGC Stimulator, Improved Glucose Tolerance and Insulin Sensitivity and Lowered Triglycerides in a Mouse Diet-Induced Obesity Model
Authors: SCHWARTZKOPF CHAD D. , HADCOCK JOHN , JONES JULI E. , CURRIE MARK , MILNE GEORGE T. , MASFERRER JAIME
Published: June 2018

Background: Praliciguat (IW-1973) is a soluble guanylate cyclase (sGC) stimulator in clinical development. In animal models, praliciguat has broad tissue distribution and enhances nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signaling, which has been shown to elicit hemodynamic, anti-inflammatory, and anti-fibrotic effects. This study assessed the metabolic effects of praliciguat in a mouse DIO model.

Methods: Male C57BL/6J mice (12 weeks old at the start of treatment) were either maintained on regular chow (lean mice) or switched to a 60% high fat diet (HFD) starting at age 6 weeks (obese mice). At the start of treatment, obese mice received either praliciguat (6 mg/kg) or vehicle in the HFD. The lean mice remained on regular chow. On day 28, an oral glucose tolerance test (OGTT) was performed after a 3-h fast. On day 29, organs and terminal blood were collected for analysis after a 3-h fast.

Results: Praliciguat treatment lowered OGTT glucose area under the curve (-22%, P<0.05), fasting plasma insulin (-53%, P<0.01) and HOMA-IR, an index of insulin resistance (-53%, P<0.01) vs. vehicle treatment. Praliciguat-treated mice also had lower liver (-36%, P<0.05) and fasting circulating triglycerides (-30%, P<0.01) vs. vehicle-treated mice. Furthermore, several fasting biomarkers did not change in treated vs. vehicle including beta-hydroxybutyrate, free fatty acids, plasma glucose, cholesterol, leptin, and adiponectin.

Conclusions: Praliciguat improved insulin sensitivity, glucose tolerance, and yielded lower circulating and liver triglycerides in a mouse diet-induced obesity model.

Disclosure

C.D. Schwartzkopf: None. J. Hadcock: None. J.E. Jones: None. M. Currie: Employee; Self; Ironwood Pharmaceuticals, Inc. G.T. Milne: Employee; Self; Ironwood Pharmaceuticals, Inc.. Stock/Shareholder; Self; Ironwood Pharmaceuticals, Inc.. Employee; Spouse/Partner; Catabasis Pharmaceuticals. Stock/Shareholder; Spouse/Partner; Catabasis Pharmaceuticals. J. Masferrer: Employee; Self; Ironwood Pharmaceuticals, Inc..
Uric Acid and Cardiovascular Disease in Type 1 Diabetes
Authors: BASU ARPITA , BRAFFETT BARBARA , JENKINS ALICIA , BEBU IONUT , KLEIN RICHARD L. , LOPES-VIRELLA MARIA F. , DAGOGO-JACK SAMUEL , ORCHARD TREVOR J. , WALLIA AMISHA , LACHIN JOHN , TIMOTHY GARVEY W. , LYONS TIMOTHY ,   DCCT/EDIC RESEARCH GROUP
Published: June 2018

Background: Serum uric acid (UA) is associated with the pathogenesis of diabetes complications, including cardiovascular disease (CVD) and kidney injury. Current epidemiological evidence is limited regarding the association between UA and the development of CVD in type 1 diabetes mellitus (T1DM).

Objective: To examine associations of serum UA with CVD and major atherosclerotic cardiovascular events (MACE) in T1DM.

Methods: UA was measured in sera (1997-2000) from a subset of participants (n=973; males=540, females=433) in the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) study. Subsequent CVD events were adjudicated by a review committee masked to DCCT assignment and HbA1c levels, and defined as the time to the first of any of the following: non-fatal myocardial infarction (MI) or stroke, CVD death, silent MI on annual ECG, angina confirmed by ischemic changes with exercise tolerance testing, congestive heart failure, or revascularization; MACE were defined as a composite of CVD death, nonfatal MI, or stroke. Cox proportional hazards models were used to evaluate prospectively the effect of UA as a fixed covariate on CVD and MACE, separately by gender, and adjusted for age and HbA1c.

Results: Over 15 years follow-up, there were 201 adjudicated CVD events among 110 participants (HR=1.10 per 1 mg/dl change in UA, 95% CI 0.93-1.30); 61 males experienced 96 events (HR=1.15, 95% CI 0.90-1.46) and 49 females, 105 events (HR=1.17, 95% CI 0.88-1.56). There were 62 adjudicated MACE among 53 participants (HR=1.18, 95% CI 0.93-1.49); 29 males experienced 31 events (HR=1.10, 95% CI 0.77-1.58) and 24 females, 31 events (HR=1.47, 95% CI 1.01-2.14). No significant associations between quartiles of UA and either CVD or MACE were observed in either sex.

Conclusions: Our results show UA may not serve as a predictive biomarker for CVD events in T1DM adults. Sex-specific associations with MACE deserve further investigation.

Disclosure

A. Basu: None. B. Braffett: None. A. Jenkins: Research Support; Self; Medtronic, Mylan, Sanofi-Aventis. I. Bebu: None. R.L. Klein: None. M.F. Lopes-Virella: None. S. Dagogo-Jack: None. T.J. Orchard: None. A. Wallia: Research Support; Self; UnitedHealth Group. Consultant; Self; Glytec Systems, Lexicon Pharmaceuticals, Inc.. Research Support; Self; Eli Lilly and Company. J. Lachin: Board Member; Self; AbbVie Inc., Celgene Corporation. W. Garvey: Advisory Panel; Self; Novo Nordisk Inc., Merck & Co., Inc.. Research Support; Self; Sanofi, Pfizer Inc., Novo Nordisk Inc., AstraZeneca, Merck & Co., Inc., Elcelyx Therapeutics, Inc., Lexicon Pharmaceuticals, Inc., Eisai Inc.. T. Lyons: None.
The Effect of Nutritional Transition on the Differentiation of Bone Marrow Stem Cells and the Therapeutic Effect of GLP-1
Authors: XIA WENFANG , CHEN LULU , ZHANG QING
Published: June 2018

Background: Catch up growth is associated with glucose and lipid metabolism disorders, that can lead to osteoporosis and related fractures. Glucagon-like peptide-1 receptor agonists, such as liraglutide, exert an anabolic effect on bone in insulin resistant mice, but the mechanism for the action remains unclear. We established an animal model of catch-up growth to explore the actions of GLP-1 on bone mass and structure.

Methods: 30 male C57BL/6 mice were randomly divided into the normal diet (ND, n=10), the catch-up growth model (RH, n=10) and the catch-up growth model +GLP-1 (RH+GLP-1, n=10) groups. Liraglutide was subcutaneously administered (200ug/kg, twice a day, for four weeks). After killing, tibia and fibula were removed for bone histomorphometry, bilateral femoral bone marrow mesenchymal stem cells were collected to evaluate their differentiation ability.

Results: GLP-1 administration decreased weight of visceral fat tissue, body weight, ratio between them, and blood lipids of the RH group. GLP-1 decreased triglyceride levels, and fatty acids in the liver, visceral fat tissue, subcutaneous fat tissue and skeletal muscle tissue. Compared to the ND group, GLP-1 secretion decreased in the RH group, but noticeably increased after GLP-1 administration. The areas under curves of intraperitoneal glucose tolerance test and insulin tolerance test and HOMA-IR in RH+GLP-1 group significantly declined. GLP-1 reversed deteriorated tibial and fibular bone structure in RH mice and induced osteoblastic differentiation.

Conclusion: Decreased ability of osteoblastic/osteogenesis differentiation of MSCs in RH mice led to impaired bone formation and GLP-1 intervention reversed the damage and enhanced insulin sensitivity.

Disclosure

W. Xia: None. L. Chen: None. Q. Zhang: None.
Exercise Is Required to Improve Mitochondrial Respiratory Capacity in Skeletal Muscle of Older Obese Subjects during Calorie Restriction–Induced Weight Loss to Improve Insulin Sensitivity
Authors: DISTEFANO GIOVANNA , STANDLEY ROBERT , CARNERO ELVIS A. , COEN PAUL M. , VEGA RICK B. , GOODPASTER BRET H.
Published: June 2018

Calorie restriction has been shown to have “anti-aging” effects in animal models, and calorie restriction-induced weight loss (CRWL) and exercise can both improve insulin sensitivity.

The aim of this study was to investigate the effect of 6-month diet-induced weight loss intervention with or without the addition of exercise training on skeletal muscle mitochondrial respiratory capacity and IS.

Methods: Forty-four older adults with obesity were randomized to one of the following 6-month intervention: Health education (CON: n=14, age=70±4 years, BMI=34±5kg/m2), Diet induced weight-loss (DIWL: n=14, age=70±4 years, BMI=36±5kg kg/m2), or Weight-loss and exercise (WLEX: n=16, age=66±3 years, BMI=37±5kg/m2). CRWL and WLEX participants had a goal of 10% weight-loss through calorie restriction. Subjects in the WLEX group completed a supervised exercise program. Mitochondrial respiratory capacity of permeabilized myofibers from biopsies was evaluated by high-resolution respirometry. Complex I+II supported maximal oxidative phosphorylation (OXPHOS), and maximal uncoupled respiration were evaluated. IS was determined by hyperinsulinemic euglycemic clamps.

Results: IS improved in both CRWL (10.4%) and WLEX (50%) compared to CON (0.7%). However, while mitochondrial respiration was unchanged in the CON and CRWL groups after the 6-month intervention (p>0.05), subjects in the WLEX group presented an increased maximal OXPHOS (PRE: 50.7±10.1 vs. POST: 72.6±17.9 pmol/s*mg) and uncoupled respiration (PRE: 57.2±10.5 vs. POST: 79.6±19.3 pmol/s*mg) after the exercise training.

Conclusions: Despite weight loss and exercise both improving insulin sensitivity, exercise is required to improve mitochondrial respiratory capacity in skeletal muscle of older obese human subjects undergoing calorie restriction-induced weight loss.

Disclosure

G. Distefano: None. R. Standley: None. E.A. Carnero: None. P.M. Coen: None. R.B. Vega: None. B.H. Goodpaster: None.
Long-Term Insulin Treatment Alters Muscle-Related Protein in OLETF Rat Skeletal Muscle
Authors: KWON HYUK-SANG , HONG OAKKEE , KWON HEE SUN , KANG YANGGYO , LEE SEONG-SU , KIM SUNG RAE , YOO SOON JIB
Published: June 2018

Purpose: The objective of this study was to investigate molecular and physiological changes in response to long-term insulin glargine treatment in the skeletal muscle of Otsuka Long-Evans Tokushima Fatty (OLETF) rats.

Methods: Protein changes in the skeletal muscle of type 2 diabetic rats was detected by Western blot analysis, and muscle fiber type composition was assessed by real-time PCR.

Results: Insulin glargine (1 U/kg) treatment in OLETF rats (OLETF-G) for 24 weeks were changed in blood glucose levels in intraperitoneal glucose tolerance tests compared with age-matched, untreated OLETF rats (OLETF-C), and the area under the curve was significantly decreased for OLETF-G rats compared with OLETF-C rats (P < 0.05). The protein levels of myosin heavy chain (MHC) isoforms were altered in gastrocnemius muscle of OLETF rats, and the proportions of MHC type I and II fibers were lower and higher, respectively, in OLETF-G compared with OLETF-C rats. Activation of myokines (interleukin [IL]-6, IL-15, fibronectin type III domain containing 5 [FNDC5]/irisin, and myostatin) in gastrocnemius muscle was significantly inhibited in OLETF-G compared with OLETF-C rats (P < 0.05). Moreover, myogenin determination gene D and myogenin levels were decreased, while insulin-like growth factor-I levels were increased, in the skeletal muscle of OLETF-G rats (P < 0.05). Insulin glargine treatment significantly increased the phosphorylation levels of 5’-AMP-activated protein kinase, sirtuin 1, and peroxisome proliferator-activated receptor γ coactivator 1α.

Conclusions: Our results suggest that long-term insulin glargine treatment can effectively alter protein expression in the gastrocnemius muscle of OLETF rats.

Disclosure

H. Kwon: None. O. Hong: None. H. Kwon: None. Y. Kang: None. S. Lee: None. S. Kim: None. S. Yoo: None.
The Rates of Overtreatment and Deintensification of Antidiabetic and Antihypertensive Medications in Patients with Diabetes Mellitus
Authors: SONMEZ ALPER , TASCI ILKER , DEMIRCI IBRAHIM , HAYMANA CEM , BARCIN CEM , AYDIN HASAN , CETINKALP SEVKI , OZTURK FEYZA YENER , GUL KAMILE , SABUNCU TEVFIK , SATMAN ILHAN , BAYRAM FAHRI ,   TEMD STUDY GROUP
Published: June 2018

Background: Targeting better HbA1c and blood pressure (BP) goals may endanger older adults with type 2 diabetes mellitus (T2DM) for a number of risks. Overtreatment of T2DM and hypertension (HTN) is a trending issue but awareness and attitudes of physicians need to be explored.

Objective: To assess the rates and predictors of overtreatment and undertreatment of glycemia and blood pressure in older adults with T2DM. Treatment deintensification or intensification by the physicians were also investigated.

Method: Data from older adults (≥65 years) enrolled in a large nationwide T2DM survey in 2017 across Turkey were analyzed. Overtreatment was defined as HbA1c <6.5% plus use of ≥2 oral hypoglycemics or insulin, and systolic BP <120 mmHg or diastolic BP <65 mmHg plus use of ≥2 drugs). Undertreatment was defined as HbA1c >9% at all, and SBP >140 mmHg or DBP >90 mmHg plus use of <3 drugs. Deintensification or intensification rates were calculated according to treatment modification by the physicians.

Results: A total of 1276 patients were included. The overtreatment rates for glycemia and BP were 9.8% and 5.9%, whereas undertreatment rates were 14.2% and 17.7%, respectively. In the adjusted model, use of oral hypoglycemics only (OR:3.1, 95% CI:1.9-5.3) and follow-up at a private clinic (OR:2.2, 95% CI:1.2-3.9) were the predictors of glycemia overtreatment. Presence of microvascular complications (OR:2.0, 95% CI:1.1-3.5) was the only predictor of BP overtreatment. The deintensification and intensification rates for glycemia were 25% and 75.7% respectively, and for the BP 10.9% and 9.0% respectively.

Conclusion: The overtreatment rates of diabetes and BP in Turkish older adults with T2DM were consistent with the previous studies, while the undertreatment rates were much higher. Doctors seem to feel more comfortable to intensify glycemic management and largely ignore BP control. The results warrant enforced measures to improve care of older adults with T2DM.

Disclosure

A. Sonmez: None. I. Tasci: None. I. Demirci: None. C. Haymana: None. C. Barcin: None. H. Aydin: None. S. Cetinkalp: None. F. Yener Ozturk: None. K. Gul: None. T. Sabuncu: None. I. Satman: None. F. Bayram: None.
Exogenous Delta-Like 1 Homolog (DLK1)-Fc Treatment Attenuates Muscle Atrophy in Both Dexamethasone and High-Fat Diet–Induced Obesity Mice Models
Authors: LEE JI YOUNG , LEE MINYOUNG , JUNG HYE-IN , LEE JI-YEON , CHO YONGIN , BAE JAEHYUN , LEE YONG-HO , LEE BYUNG-WAN , KANG EUN SEOK , CHA BONG-SOO
Published: June 2018

Skeletal muscle degeneration is accelerated in elderly patients with diabetes and the imbalance between muscle growth and wasting can lead to metabolic dysfunction. DLK1 is a member of the EGF-like family and essential for skeletal muscle development and regeneration. To identify whether exogenous DLK1 treatment can prevent muscle wasting, we assessed muscle mass as well as muscle differentiation and atrophy markers in two different models: dexamethasone and high fat diet (HFD)-induced obesity mice models (n=7-8 per each group). In the dexamethasone model, mice were divided into (1) control, (2) dexamethasone treatment (1mg/Kg, 2 weeks), and (3) DLK1 (0.8mg/Kg, 2 weeks) and dexamethasone treatment groups. In the HFD model, mice were divided into (1) control, (2) HFD (60% calories from fat, 8 weeks), and (3) HFD with DLK1 treatment (0.8mg/Kg, 8 weeks) groups. The expressions of genes related to muscle differentiation and atrophy were determined using RT-PCR. We observed that dexamethasone reduced muscle mass and markedly increased atrophy makers including atrogin-1 and murf-1. DLK1 treatment attenuated these degenerative changes. Furthermore, the level of myostatin, which inhibits muscle cell growth, was reduced in DLK1 treatment group compared to dexamethasone group. Additionally, compared to HFD group, DLK1 inhibited muscle atrophy by increasing the differentiation marker: myoD, and decreasing the atrophy markers: mysotatin, atrogin-1, and murf-1. Taken together, these results suggest that DLK1 attenuates both dexamethasone and HFD-induced muscle atrophy in mice by suppressing the downstream signaling of myostatin/atrogin-1/murf-1 pathway. The increased expression of myoD indicates that DLK1 treatment can also improve the quality of muscle formation. Our study implies that DLK1 could be a promising candidate in the treatment of aging or diabetes-related sarcopenia, characterized by muscle atrophy and dysfunction.

Disclosure

J. Lee: None. M. Lee: None. H. Jung: None. J. Lee: None. Y. Cho: None. J. Bae: None. Y. Lee: None. B. Lee: None. E. Kang: None. B. Cha: None.
Skeletal Muscle Clock Is Essential for Nutrient-Sensing and Interorgan Metabolic Fuel Partitioning
Authors: MA KE , CHATTERJEE SOMIK , YIN HONGSHAN
Published: June 2018

The circadian clock exerts temporal control in metabolism and its disruption leads to the development of diabetes and obesity. Tissue-intrinsic clock circuits are integral components of global metabolic homeostasis, although how they sense nutrient signals to orchestrate metabolic flux is not clear. Skeletal muscle is major site for metabolic substrate oxidation, and its utilization of glucose and fatty acid depends on availability of nutrients accompanying feeding-fasting transitions. Interestingly, nearly 30% of rhythmic transcripts in skeletal muscle belongs to metabolism. By generating a mouse model with myocyte-selective ablation of the clock transcription activator Bmal1, here we show that cell-autonomous muscle clock plays an essential role in coordinating nutrient utilization with feeding-fasting induced cycles. Bmal1 in skeletal muscle was robustly induced by feeding, and its loss markedly impaired feeding-induced switch to glucose metabolism from fatty acid utilization. As a result, Bmal1-deficient muscle displays nearly 50% reduction of glucose oxidation whereas fatty acid oxidation was enhanced, resembling a constant fasting state. This metabolic shift was accompanied by muscle fiber type switching to an oxidative phenotype, and led to a remarkable resistance to hepatic lipid accumulation induced by prolonged fasting or high-fat diet feeding. In contrast, systemic fasting glucose levels in muscle Bmal1-deficient mice was elevated, which is due to a failure of suppressing hepatic glucose output as revealed by hyperinsulinemic-euglycemic glucose clamp study. Collectively, our results reveal a novel function of the Bmal1-driven muscle clock as a key metabolic sensor that coordinates metabolic fuel oxidation with oscillatory nutrient availability in fasting-feeding cycles. This temporal mechanism in orchestrating global nutrient flux may contribute to metabolic abnormalities induced by circadian misalignment.

Disclosure

K. Ma: None. S. Chatterjee: None. H. Yin: None.
Effects of Diet/Exercise and Pioglitazone on the Association between Fitness and Intramyocellular FFA Trafficking
Authors: SHADID SAMYAH ,   N ENDOCRINE RESEARCH UNIT, MAYO CLINIC, ROCHESTER, M, JENSEN MICHAEL D.
Published: June 2018

Background: Intramyocellular (im) triglycerides (TG) are increased in athletes, but also insulin resistant (IR) adults. To elucidate this discrepancy, we studied FFA storage in imTG and im oleyl-carnitine (OC) concentrations in IR humans pre and post 2 methods of insulin sensitization (IS) in the context of fitness (VO2max).

Methods: 39 adults (BMI 28-36 kg/m2) were given a diet/exercise program (DE; 11.7 ± 3.2 kg weight loss) or 30 mg/day pioglitazone (PI) for ∼19 weeks. Blood and muscle samples were taken before and after a 6 hour [U-13C]oleate infusion with either a euglycemic, hyperinsulinemic clamp (n=20) or saline infusion. Plasma oleate storage in imTG (nmol FFA/g muscle/hr) was calculated.

Results: Quantative IS was similar in DE and PI groups. VO2max increased post DE (44 ± 1 to 46 ± 2 ml/kgFFM/min†), but not PI (45 ± 1 ml/kgFFM/min pre and post PI). At baseline, oleate storage in imTG during clamps correlated with VO2max (r=0.51*), but the δVO2max with treatment did not correlate with the δoleate storage in imTG. Following IS, VO2max correlated with imOC (r=0.46*); the latter was true for men (m) (r=0.89*), but not women (r=0.32), and for DE (m: r=0.97*) but not PI (m: r=0.70, p=0.18). δ VO2max correlated with δimOC (m: r=0.92*, f: r=0.57†), both post WL (r=0.91*) and PI (r=0.96*).Summary: In IR adults, IS influences the associations between fitness and markers of imFFA oxidation and storage in imTG during hyperinsulinemia. These relationships are more apparent in men and exaggerated post-IS.

Conclusion: Fitness in men regulates imFFA trafficking towards storage and oxidation. *p<0.05; †p=NS.

Disclosure

S. Shadid: None. M.D. Jensen: Other Relationship; Self; Novo Nordisk A/S. Consultant; Self; Novo Nordisk A/S.
Localization of ChREBPa and ChREBPß in Pancreatic ß Cells
Authors: KATZ LIORA S. , LAXMAN ANIL K. , ZHANG PILI , SCOTT DONALD
Published: June 2018

Glucose is a key physiological driver of adaptive β-cell mass expansion, promoting β-cell proliferation both in vitro and in vivo. Carbohydrate response element binding protein (ChREBP) is required for glucose-stimulated β-cell proliferation in both rodent and human β-cells. ChREBP has two splice isoforms - α and β. Translation of the ChREBPβ mRNA generates a truncated protein, resulting in a potent, constitutively active isoform. To study the role of the 2 splice isoforms in β-cell biology, we performed numerous experiments including immunostaining and chromatin immunoprecipitation with antibodies that recognize only ChREBPα or both isoforms. We found that ChREBPα is only transiently expressed in the nucleus. By contrast, ChREBPβ, once expressed at high enough levels to detect, is constitutively nuclear. Our data indicate that mice fed on high fat diet and diabetic db/db mice have abundant ChREBPβ localized in the nucleus while control mice have mostly cytoplasmic ChREBPα. We generated Ins-1 cell lines where we can distinguish between the splice isoforms using CRISPR/Cas9: 1. The C-terminus of ChREBPα and ChREBPβ was labeled with eGFP; 2. The N-terminus of ChREBPα was labeled by mCherry; and 3. ChREBPα was labeled with mCherry and eGFP on the N- and C- terminus, respectively and ChREBPβ is labeled with eGFP on the C-terminus. We validated the three cell lines by sequencing, ChIP, qRT-PCR and also showed that the glucose response is not altered in these cells. We found that in low glucose, ChREBPβ is present at very low levels in the nucleus. By contrast, in high glucose, ChREBPβ is present at high levels in the nucleus. Our study suggests that in response to glucose, ChREBPα, which is mostly cytoplasmic, transiently enters the nucleus, binds to a carbohydrate response element (ChoRE), and induces ChREBPβ expression, which is mostly nuclear. We propose that while the induction of ChREBPβ drives adaptive beta cell expansion, ChREBPβ contributes to glucose toxicity after prolonged exposure to high glucose and in diabetes.

Disclosure

L.S. Katz: None. A.K. Laxman: None. P. Zhang: None. D. Scott: None.
Diabetes and High-Fat Diet Induce Different Pathologies in Mouse Skeletal Muscle Extracellular Matrix
Authors: MARTINEZ-HUENCHULLAN SERGIO F. , BAN LINDA A. , TAO ANH , SUTANTO SURYA S. , TAM CHARMAINE , MCLENNAN SUSAN , TWIGG STEPHEN M.
Published: June 2018

Whether obesity and diabetes (DM) similarly affect composition and function of skeletal muscle extracellular matrix (ECM) has not been resolved. This study examines the effects of long term high fat diet (HFD) and DM on rodent skeletal muscle ECM were addressed. Five week old C57BL/6 male mice were randomly allocated to standard chow (control; n=26) or HFD (45% kcal fat; n=25). After 15 weeks chow and HFD mice were rendered diabetic (by low dose STZ, 65mg/kgx3d, I.P.) (DM, n=14; HFD+DM, n=14); nondiabetic mice acted as controls. Mice were euthanased after 30 weeks and plasma and quadriceps muscle were collected for analysis. Body weight and plasma insulin expectedly decreased with DM induction and increased with HFD (all p<0.05). High glucose occurred only in DM mice (∼14 mM; p<0.05), whereas only HFD increased muscle triglyceride (p<0.05). Both conditions induced a decrease in quadriceps weight and in grip strength performance (p<0.05). Quadriceps of DM mice showed increased CTGF/CCN2 (1.5-fold; p<0.05), TIMP-1 (2-fold; p<0.05), collagen-I (3-fold; p<0.05), -IV (2-fold; p<0.05), and -VI (2-fold; p<0.05) protein. In contrast, HFD induced a downregulation of CTGF/CCN2 (0.6-fold; p<0.05), MMP-1 (0.6-fold; p<0.05), and TIMP-2 (0.4-fold; p<0.05). In DM mice quadriceps centralized nuclei (p<0.05) and caspase 3 (protein; 1.5-fold; p<0.05) were increased, whereas caspase 3 was decreased in HFD (0.5-fold; p<0.05). Moreover, HFD induced a downregulation of ubiquitin ligase atrogin-1 protein (0.5-fold; p<0.05) and mRNA levels of serine/threonine kinase p70S6k (0.75-fold; p<0.05). Notably, no greater dysregulation of the muscle changes observed with DM alone or with HFD alone, was seen in HFD+DM mice. These data suggest that DM and HFD differentially affect skeletal muscle ECM: DM promotes a pro-fibrotic profile with signs of muscle damage, and HFD impairs regulatory protein turnover pathways, each inducing muscle dysfunction.

Disclosure

S.F. Martinez-Huenchullan: None. L.A. Ban: None. A. Tao: None. S.S. Sutanto: None. C. Tam: None. S. McLennan: None. S.M. Twigg: Advisory Panel; Self; Abbott. Consultant; Self; Abbott. Advisory Panel; Self; Novo Nordisk Inc., Sanofi-Aventis, AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company. Speaker's Bureau; Self; AstraZeneca, Merck Sharp & Dohme Corp.. Other Relationship; Self; Abbott.
Skeletal Muscle Dysfunction and Metabolic Abnormalities in Individuals with Familial Partial Lipodystrophy Is Associated with Diminished Mitochondrial Fatty Acid Oxidation
Authors: SIMHA VINAYA , LANZA IAN R. , LEBRASSEUR NATHAN , KLAUS KATHERINE , PORT JOHN , LAURENTI MARCELLO C. , COBELLI CLAUDIO , SREEKUMARAN NAIR K.
Published: June 2018

Background: Familial Partial Lipodystrophy, Dunnigan variety (FPLD) is characterized by post-natal fat loss leading to severe insulin resistance. Affected individuals also display striking skeletal muscle hypertrophy, but there is limited information on muscle function and energy metabolism. We therefore systematically investigated muscle and mitochondrial function in 6 individuals with FPLD and 6 matched controls.

Methods: Body composition and intramyocellular lipid content (IMCL) were assessed by DEXA and magnetic resonance spectroscopy, respectively. Maximal voluntary strength and fatigue were assessed for both recumbent leg press and seated chest press. Vastus lateralis muscle biopsies were obtained prior to and following a high fat (40%) mixed-meal. Oxygen consumption in isolated mitochondria was measured using substrates supporting carbohydrate or lipid-based respiration. Insulin Sensitivity Index (SI) was calculated by minimal modelling.

Results: Individuals with FPLD had significantly lower SI, lower body fat content, and higher lean mass. Peak leg press and chest press force were not different, but FPLD individuals had earlier fatigue during chest press (number of repetitions: 15 ± 0.6 vs. 20 ± 0.3, p = 0.01). They also had a 50-60% decrease in state 3 respiration measured using substrates supporting lipid-based respiration (p = 0.04). Further, mitochondrial oxidative capacity showed a strong correlation with muscle fatigue (r = 0.76, p = 0.003), and inversely correlated with fasting and post-prandial lipemia and measures of insulin resistance.

Conclusion: Individuals with FPLD have higher lean body mass and lower insulin sensitivity. Despite preserved peak muscle strength, they demonstrated earlier muscle fatigue. Decreased mitochondrial fatty acid oxidative capacity, likely related to elevated IMCL, may contribute to muscular and metabolic abnormalities.

Disclosure

V. Simha: None. I.R. Lanza: None. N. LeBrasseur: None. K. Klaus: None. J. Port: None. M.C. Laurenti: Other Relationship; Self; GlySens Incorporated. C. Cobelli: Research Support; Self; Sanofi-Aventis. Advisory Panel; Self; Novo Nordisk Inc.. K. Nair: None.
Skeletal Muscle Lipid Composition Parallels Clinical Phenotype Extremes
Authors: CHOW LISA S. , MASHEK DOUGLAS G. , BOSCH TYLER , BANTLE ANNE , PETTERSON MAI , DENGEL DONALD R. , JENSEN MICHAEL D.
Published: June 2018

Intramyocellular lipid (IMCL) is generally associated with insulin resistance and lipotoxicity. However, the “athlete’s paradox” is well documented, where IMCL levels are similar between athletes and patients with type 2 diabetes despite discrepant levels of insulin sensitivity. Differences in muscle lipid metabolites between aerobically trained, lean, insulin-sensitive (LT) vs. sedentary, overweight/obese, insulin-resistant (OIR) individuals remain insufficiently characterized and may help to explain the “athlete’s paradox.” LT [n=14: 50% females, 28.5±1.6 years (Mean±SE)] and OIR [n=24: 79% females, 29.8±1.6 years] subjects each provided 2 muscle biopsies from the vastus lateralis (bilateral, 6 hours apart, ≥48 hours after last exercise, ≥8 fasting, overnight admission prior to biopsy) for mass spectrometry analysis. At a separate visit, insulin sensitivity was quantified by a hyperinsulinemic-euglycemic clamp (mg glucose/kg FFM/min). As the sequential biopsy results were not significantly different, the results were averaged for each subject. Compared with the OIR group, the LT group had lower BMI (22.3±0.5 vs. 36.2±1.2 kg/m2:p<0.01), higher fitness (VO2max:53.4±2.2 vs. 26.7±1.1 ml/kg/min: p<0.01), and higher insulin sensitivity (M-value: 17.1±0.7 vs. 8±0.5, p<0.01). Muscle triglycerides in the LT group were lower (nmol/mg muscle) in oleic acid (3.2±0.6 vs. 6.0±1.0, p=0.02) and palmitic acid (1.5±0.2 vs. 3.1±0.5, p<0.01) than the OIR group. Diacylglycerol (DAG) and palmitoylcarnitine concentrations were not significantly different. Only in the OIR group, palmitoylcarnitine correlated with the following: oleic acid (r=0.73, p<0.01), palmitic acid (r=0.73, p<0.01) and DAG (dipalmitate:r=0.53, p=0.03, palmitate+oleate: r=0.51, p=0.04). These findings suggest that either incomplete muscle lipid oxidation or increased acylcarnitine generation is present in the OIR group, thus warranting further research on training effects on muscle lipid turnover.

Disclosure

L.S. Chow: Research Support; Self; Eli Lilly and Company, National Institute of Diabetes and Digestive and Kidney Diseases. D.G. Mashek: None. T. Bosch: None. A. Bantle: None. M. Petterson: None. D.R. Dengel: None. M.D. Jensen: Other Relationship; Self; Novo Nordisk A/S. Consultant; Self; Novo Nordisk A/S.