Recent Publications of BIIC Members

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NCBI: db=pubmed; Term=Gray JP OR Heart E OR Zarrouki B OR Corkey BE OR Bonner-Weir S OR Urano F OR Cline GW OR Sharp GW OR Holz GG OR Weir GC OR Kulkarni RN OR Tornheim K OR Kibbey RG OR Fonseca SG OR Straub SG OR Jetton TL OR Poitout V OR Prentki M
Updated: 5 hours 44 min ago

Neuregulin-activated ERBB4 induces the SREBP-2 cholesterol biosynthetic pathway and increases low-density lipoprotein uptake.

Fri, 11/06/2015 - 15:03
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Neuregulin-activated ERBB4 induces the SREBP-2 cholesterol biosynthetic pathway and increases low-density lipoprotein uptake.

Sci Signal. 2015;8(401):ra111

Authors: Haskins JW, Zhang S, Means RE, Kelleher JK, Cline GW, Canfrán-Duque A, Suárez Y, Stern DF

Abstract
Cholesterol is a lipid that is critical for steroid hormone production and the integrity of cellular membranes, and, as such, it is essential for cell growth. The epidermal growth factor receptor (EGFR) family member ERBB4, which forms signaling complexes with other EGFR family members, can undergo ligand-induced proteolytic cleavage to release a soluble intracellular domain (ICD) that enters the nucleus to modify transcription. We found that ERBB4 activates sterol regulatory element binding protein-2 (SREBP-2) to enhance low-density lipoprotein (LDL) uptake and cholesterol biosynthesis. Expression of the ERBB4 ICD in mammary epithelial cells or activation of ERBB4 with the ligand neuregulin 1 (NRG1) induced the expression of SREBP target genes involved in cholesterol biosynthesis, including HMGCR and HMGCS1, and lipid uptake, LDLR, which encodes the LDL receptor. Addition of NRG1 increased the abundance of the cleaved, mature form of SREBP-2 through a pathway that was blocked by addition of inhibitors of PI3K (phosphatidylinositol 3-kinase) or dual inhibition of mammalian target of rapamycin complex 1 (mTORC1) and mTORC2, but not by inhibition of AKT or mTORC1. Pharmacological inhibition of the activity of SREBP site 1 protease or of all EGFR family members (with lapatinib), but not EGFR alone (with erlotinib), impaired NRG1-induced expression of cholesterol biosynthesis genes. Collectively, our findings indicated that activation of ERBB4 promotes SREBP-2-regulated cholesterol metabolism. The connections of EGFR and ERBB4 signaling with SREBP-2-regulated cholesterol metabolism are likely to be important in ERBB-regulated developmental processes and may contribute to metabolic remodeling in ERBB-driven cancers.

PMID: 26535009 [PubMed - in process]

Neuronal control of bone and muscle.

Tue, 10/13/2015 - 02:39
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Neuronal control of bone and muscle.

Bone. 2015 Nov;80:95-100

Authors: Houweling P, Kulkarni RN, Baldock PA

Abstract
The functional interplay between bone and muscle that enables locomotion is a fundamental aspect of daily life. However, other interactions between bone and muscle continue to attract attention as our understanding of the breath and importance of this inter-relationship continues to expand. Of particular interest is the regulatory connection between bone and muscle, which adds a new insight to the coordination of the bone/muscle unit. We have appreciated the importance of neuronal signaling to the control of bone turnover and muscle contraction, but recent data indicate that neuronal inputs control a far wider range of bone and muscle physiology than previously appreciated. This review outlines the role of the sympathetic nervous system and neuronal/neuropeptide inputs upon the regulation of bone and muscle tissue, and the potential for co-regulatory actions, particularly involving the sympathetic nervous system. This article is part of a Special Issue entitled "Muscle Bone Interactions".

PMID: 26453499 [PubMed - in process]

KCl -Permeabilized Pancreatic Islets: An Experimental Model to Explore the Messenger Role of ATP in the Mechanism of Insulin Secretion.

Sat, 10/10/2015 - 00:49
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KCl -Permeabilized Pancreatic Islets: An Experimental Model to Explore the Messenger Role of ATP in the Mechanism of Insulin Secretion.

PLoS One. 2015;10(10):e0140096

Authors: Pizarro-Delgado J, Deeney JT, Martín-Del-Río R, Corkey BE, Tamarit-Rodriguez J

Abstract
Our previous work has demonstrated that islet depolarization with KCl opens connexin36 hemichannels in β-cells of mouse pancreatic islets allowing the exchange of small metabolites with the extracellular medium. In this study, the opening of these hemichannels has been further characterized in rat islets and INS-1 cells. Taking advantage of hemicannels'opening, the uptake of extracellular ATP and its effect on insulin release were investigated. 70 mM KCl stimulated light emission by luciferin in dispersed rat islets cells transduced with the fire-fly luciferase gene: it was suppressed by 20 mM glucose and 50 μM mefloquine, a specific connexin36 inhibitor. Extracellular ATP was taken up or released by islets depolarized with 70 mM KCl at 5 mM glucose, depending on the external ATP concentration. 1 mM ATP restored the loss of ATP induced by the depolarization itself. ATP concentrations above 5 mM increased islet ATP content and the ATP/ADP ratio. No ATP uptake occurred in non-depolarized or KCl-depolarized islets simultaneously incubated with 50 μM mefloquine or 20 mM glucose. Extracellular ATP potentiated the secretory response induced by 70 mM KCl at 5 mM glucose in perifused rat islets: 5 mM ATP triggered a second phase of insulin release after the initial peak triggered by KCl-depolarization itself; at 10 mM, it increased both the initial, KCl-dependent, peak and stimulated a greater second phase of secretion than at 5 mM. These stimulatory effects of extracellular ATP were almost completely suppressed by 50 μM mefloquine. The magnitude of the second phase of insulin release due to 5 mM extracellular ATP was decreased by addition of 5 mM ADP (extracellular ATP/ADP ratio = 1). ATP acts independently of KATP channels closure and its intracellular concentration and its ATP/ADP ratio seems to regulate the magnitude of both the first (triggering) and second (amplifying) phases of glucose-induced insulin secretion.

PMID: 26444014 [PubMed - in process]

Simplified assays of lipolysis enzymes for drug discovery and specificity assessment of of known inhibitors.

Sun, 10/04/2015 - 00:01
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Simplified assays of lipolysis enzymes for drug discovery and specificity assessment of of known inhibitors.

J Lipid Res. 2015 Sep 30;

Authors: Iglesias J, Lamontagne J, Erb H, Gezzar S, Zhao S, Joly E, Truong VL, Skorey K, Crane S, Madiraju SR, Prentki M

Abstract
Lipids are used as cellular building blocks and condensed energy stores and also act as signaling molecules. Glycerolipid/ fatty acid cycle, encompassing lipolysis and lipogenesis, generates many lipid signals. Reliable procedures are not available for measuring activities of several lipolytic enzymes for the purposes of drug screening and this resulted in questionable selectivity of various known lipase inhibitors. We now describe simple assays for lipolytic enzymes, including adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), sn1-diacylglycerol lipase (DAGL), monoacylglycerol lipase, α/β-hydrolase domain-6 as well as carboxylesterase-1 using recombinant human and mouse enzymes either in cell extracts or using purified enzymes. We observed that many of the reported inhibitors lack specificity. Thus, Cay10499 (HSL inhibitor) and RHC20867 (DAGL inhibitor) inhibit other lipases also. Marked differences in the inhibitor sensitivities of human ATGL and HSL compared to the corresponding mouse enzymes, was noticed. Thus, ATGListatin inhibited mouse ATGL but not human ATGL and the HSL inhibitors WWL11 and Compound13f were effective against mouse enzyme but much less potent against human enzyme. Many of these lipase inhibitors also inhibited human carboxylesterase-1. Results describe reliable assays for measuring lipase activities that are amenable for drug screening and also caution about the specificity of the many earlier described lipase inhibitors.

PMID: 26423520 [PubMed - as supplied by publisher]

Dissecting diabetes/metabolic disease mechanisms using pluripotent stem cells and genome editing tools.

Wed, 09/30/2015 - 22:50
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Dissecting diabetes/metabolic disease mechanisms using pluripotent stem cells and genome editing tools.

Mol Metab. 2015 Sep;4(9):593-604

Authors: Teo AK, Gupta MK, Doria A, Kulkarni RN

Abstract
BACKGROUND: Diabetes and metabolic syndromes are chronic, devastating diseases with increasing prevalence. Human pluripotent stem cells are gaining popularity in their usage for human in vitro disease modeling. With recent rapid advances in genome editing tools, these cells can now be genetically manipulated with relative ease to study how genes and gene variants contribute to diabetes and metabolic syndromes.
SCOPE OF REVIEW: We highlight the diabetes and metabolic genes and gene variants, which could potentially be studied, using two powerful technologies - human pluripotent stem cells (hPSCs) and genome editing tools - to aid the elucidation of yet elusive mechanisms underlying these complex diseases.
MAJOR CONCLUSIONS: hPSCs and the advancing genome editing tools appear to be a timely and potent combination for probing molecular mechanism(s) underlying diseases such as diabetes and metabolic syndromes. The knowledge gained from these hiPSC-based disease modeling studies can potentially be translated into the clinics by guiding clinicians on the appropriate type of medication to use for each condition based on the mechanism of action of the disease.

PMID: 26413465 [PubMed]

Integrated, Step-Wise, Mass-Isotopomeric Flux Analysis of the TCA Cycle.

Wed, 09/30/2015 - 22:50
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Integrated, Step-Wise, Mass-Isotopomeric Flux Analysis of the TCA Cycle.

Cell Metab. 2015 Sep 22;

Authors: Alves TC, Pongratz RL, Zhao X, Yarborough O, Sereda S, Shirihai O, Cline GW, Mason G, Kibbey RG

Abstract
Mass isotopomer multi-ordinate spectral analysis (MIMOSA) is a step-wise flux analysis platform to measure discrete glycolytic and mitochondrial metabolic rates. Importantly, direct citrate synthesis rates were obtained by deconvolving the mass spectra generated from [U-(13)C6]-D-glucose labeling for position-specific enrichments of mitochondrial acetyl-CoA, oxaloacetate, and citrate. Comprehensive steady-state and dynamic analyses of key metabolic rates (pyruvate dehydrogenase, β-oxidation, pyruvate carboxylase, isocitrate dehydrogenase, and PEP/pyruvate cycling) were calculated from the position-specific transfer of (13)C from sequential precursors to their products. Important limitations of previous techniques were identified. In INS-1 cells, citrate synthase rates correlated with both insulin secretion and oxygen consumption. Pyruvate carboxylase rates were substantially lower than previously reported but showed the highest fold change in response to glucose stimulation. In conclusion, MIMOSA measures key metabolic rates from the precursor/product position-specific transfer of (13)C-label between metabolites and has broad applicability to any glucose-oxidizing cell.

PMID: 26411341 [PubMed - as supplied by publisher]

Peretinoin after curative therapy of hepatitis C-related hepatocellular carcinoma: a randomized double-blind placebo-controlled study.

Wed, 09/30/2015 - 22:50
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Peretinoin after curative therapy of hepatitis C-related hepatocellular carcinoma: a randomized double-blind placebo-controlled study.

J Gastroenterol. 2015 Feb;50(2):191-202

Authors: Okita K, Izumi N, Matsui O, Tanaka K, Kaneko S, Moriwaki H, Ikeda K, Osaki Y, Numata K, Nakachi K, Kokudo N, Imanaka K, Nishiguchi S, Okusaka T, Nishigaki Y, Shiomi S, Kudo M, Ido K, Karino Y, Hayashi N, Ohashi Y, Makuuchi M, Kumada H, Peretinoin Study Group

Abstract
BACKGROUND: Effective prophylactic therapies have not been established for hepatocellular carcinoma recurrence. Peretinoin represents one novel option for patients with hepatitis C virus-related hepatocellular carcinoma (HCV-HCC), and it was tested in a multicenter, randomized, double-blind, placebo-controlled study.
METHODS: Patients with curative therapy were assigned to one of the following regimens: peretinoin 600, 300 mg/day, or placebo for up to 96 weeks. The primary outcome was recurrence-free survival (RFS).
RESULTS: Of the 401 patients initially enrolled, 377 patients were analyzed for efficacy. The RFS rates in the 600-mg group, the 300-mg group, and the placebo group were 71.9, 63.6, and 66.0 % at 1 year, and 43.7, 24.9, and 29.3 % at 3 years, respectively. The primary comparison of peretinoin (300 and 600-mg) with placebo was not significant (P = 0.434). The dose-response relationship based on the hypothesis that "efficacy begins to increase at 600 mg/day" was significant (P = 0.023, multiplicity-adjusted P = 0.048). The hazard ratios for RFS in the 600-mg group vs. the placebo group were 0.73 [95 % confidence interval (CI) 0.51-1.03] for the entire study period and 0.27 (95 % CI 0.07-0.96) after 2 years of the randomization. Common adverse events included ascites, increased blood pressure, headache, presence of urine albumin, and increased transaminases.
CONCLUSIONS: Although the superiority of peretinoin to placebo could not be validated, 600 mg/day was shown to be the optimal dose, and treatment may possibly reduce the recurrence of HCV-HCC, particularly after 2 years. The efficacy and safety of peretinoin 600 mg/day should continue to be evaluated in further studies.

PMID: 24728665 [PubMed - indexed for MEDLINE]

Response to Comments on Nolan et al. Insulin Resistance as a Physiological Defense Against Metabolic Stress: Implications for the Management of Subsets of Type 2 Diabetes. Diabetes 2015;64:673-686.

Sun, 09/27/2015 - 22:15
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Response to Comments on Nolan et al. Insulin Resistance as a Physiological Defense Against Metabolic Stress: Implications for the Management of Subsets of Type 2 Diabetes. Diabetes 2015;64:673-686.

Diabetes. 2015 Oct;64(10):e38-9

Authors: Nolan CJ, Ruderman NB, Kahn SE, Pedersen O, Prentki M

PMID: 26405280 [PubMed - in process]

Isocitrate-to-SENP1 signaling amplifies insulin secretion and rescues dysfunctional β cells.

Thu, 09/24/2015 - 21:52
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Isocitrate-to-SENP1 signaling amplifies insulin secretion and rescues dysfunctional β cells.

J Clin Invest. 2015 Sep 21;

Authors: Ferdaoussi M, Dai X, Jensen MV, Wang R, Peterson BS, Huang C, Ilkayeva O, Smith N, Miller N, Hajmrle C, Spigelman AF, Wright RC, Plummer G, Suzuki K, Mackay JP, van de Bunt M, Gloyn AL, Ryan TE, Norquay LD, Brosnan MJ, Trimmer JK, Rolph TP, Kibbey RG, Manning Fox JE, Colmers WF, Shirihai OS, Neufer PD, Yeh ET, Newgard CB, MacDonald PE

Abstract
Insulin secretion from β cells of the pancreatic islets of Langerhans controls metabolic homeostasis and is impaired in individuals with type 2 diabetes (T2D). Increases in blood glucose trigger insulin release by closing ATP-sensitive K+ channels, depolarizing β cells, and opening voltage-dependent Ca2+ channels to elicit insulin exocytosis. However, one or more additional pathway(s) amplify the secretory response, likely at the distal exocytotic site. The mitochondrial export of isocitrate and engagement with cytosolic isocitrate dehydrogenase (ICDc) may be one key pathway, but the mechanism linking this to insulin secretion and its role in T2D have not been defined. Here, we show that the ICDc-dependent generation of NADPH and subsequent glutathione (GSH) reduction contribute to the amplification of insulin exocytosis via sentrin/SUMO-specific protease-1 (SENP1). In human T2D and an in vitro model of human islet dysfunction, the glucose-dependent amplification of exocytosis was impaired and could be rescued by introduction of signaling intermediates from this pathway. Moreover, islet-specific Senp1 deletion in mice caused impaired glucose tolerance by reducing the amplification of insulin exocytosis. Together, our results identify a pathway that links glucose metabolism to the amplification of insulin secretion and demonstrate that restoration of this axis rescues β cell function in T2D.

PMID: 26389676 [PubMed - as supplied by publisher]

Beta cells transfer vesicles containing insulin to phagocytes for presentation to T cells.

Thu, 09/03/2015 - 09:25
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Beta cells transfer vesicles containing insulin to phagocytes for presentation to T cells.

Proc Natl Acad Sci U S A. 2015 Aug 31;

Authors: Vomund AN, Zinselmeyer BH, Hughes J, Calderon B, Valderrama C, Ferris ST, Wan X, Kanekura K, Carrero JA, Urano F, Unanue ER

Abstract
Beta cells from nondiabetic mice transfer secretory vesicles to phagocytic cells. The passage was shown in culture studies where the transfer was probed with CD4 T cells reactive to insulin peptides. Two sets of vesicles were transferred, one containing insulin and another containing catabolites of insulin. The passage required live beta cells in a close cell contact interaction with the phagocytes. It was increased by high glucose concentration and required mobilization of intracellular Ca(2+). Live images of beta cell-phagocyte interactions documented the intimacy of the membrane contact and the passage of the granules. The passage was found in beta cells isolated from islets of young nonobese diabetic (NOD) mice and nondiabetic mice as well as from nondiabetic humans. Ultrastructural analysis showed intraislet phagocytes containing vesicles having the distinct morphology of dense-core granules. These findings document a process whereby the contents of secretory granules become available to the immune system.

PMID: 26324934 [PubMed - as supplied by publisher]

Effect of aging on muscle mitochondrial substrate utilization in humans.

Fri, 08/28/2015 - 07:06
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Effect of aging on muscle mitochondrial substrate utilization in humans.

Proc Natl Acad Sci U S A. 2015 Aug 24;

Authors: Petersen KF, Morino K, Alves TC, Kibbey RG, Dufour S, Sono S, Yoo PS, Cline GW, Shulman GI

Abstract
Previous studies have implicated age-associated reductions in mitochondrial oxidative phosphorylation activity in skeletal muscle as a predisposing factor for intramyocellular lipid (IMCL) accumulation and muscle insulin resistance (IR) in the elderly. To further investigate potential alterations in muscle mitochondrial function associated with aging, we assessed basal and insulin-stimulated rates of muscle pyruvate dehydrogenase (VPDH) flux relative to citrate synthase flux (VCS) in healthy lean, elderly subjects and healthy young body mass index- and activity-matched subjects. VPDH/VCS flux was assessed from the (13)C incorporation from of infused [1-(13)C] glucose into glutamate [4-(13)C] relative to alanine [3-(13)C] assessed by LC-tandem MS in muscle biopsies. Insulin-stimulated rates of muscle glucose uptake were reduced by 25% (P < 0.01) in the elderly subjects and were associated with ∼70% (P < 0.04) increase in IMCL, assessed by (1)H magnetic resonance spectroscopy. Basal VPDH/VCS fluxes were similar between the groups (young: 0.20 ± 0.03; elderly: 0.14 ± 0.03) and increased approximately threefold in the young subjects following insulin stimulation. However, this increase was severely blunted in the elderly subjects (young: 0.55 ± 0.04; elderly: 0.18 ± 0.02, P = 0.0002) and was associated with an ∼40% (P = 0.004) reduction in insulin activation of Akt. These results provide new insights into acquired mitochondrial abnormalities associated with aging and demonstrate that age-associated reductions in muscle mitochondrial function and increased IMCL are associated with a marked inability of mitochondria to switch from lipid to glucose oxidation during insulin stimulation.

PMID: 26305973 [PubMed - as supplied by publisher]

Deficiency of FcϵR1 increases body weight gain but improves glucose tolerance in diet-induced obese mice.

Tue, 08/25/2015 - 04:58
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Deficiency of FcϵR1 increases body weight gain but improves glucose tolerance in diet-induced obese mice.

Endocrinology. 2015 Aug 21;:en20151184

Authors: Lee YJ, Liu C, Liao M, Sukhova GK, Shirakawa J, Abdennour M, Iamarene K, Andre S, Inouye K, Clement K, Kulkarni RN, Banks AS, Libby P, Shi GP

Abstract
Prior studies demonstrated increased plasma immunoglobulin E (IgE) in diabetic patients, but the direct participation of IgE in diabetes or obesity remains unknown. This study found that plasma IgE levels correlated inversely with body weight, body mass index, and body fat mass among a population of randomly selected obese women. IgE receptor FcϵR1-deficient (Fcer1a(-/-)) mice and diet-induced obesity (DIO) mice demonstrated that FcϵR1 deficiency in DIO mice increased food intake, reduced energy expenditure, and increased body weight gain, but improved glucose tolerance and glucose-induced insulin secretion. White adipose tissue (WAT) from Fcer1a(-/-) mice showed increased expression of phospho-AKT, C/EBPα, PPARγ, Glut4, and Bcl-2, but reduced UCP1 and phospho-JNK expression, tissue macrophage accumulation, and apoptosis, suggesting that IgE reduces adipogenesis and glucose uptake, but induces energy expenditure, adipocyte apoptosis, and WAT inflammation. In 3T3-L1 cells, IgE inhibited the expression of C/EBPα and PPARγ, and preadipocyte adipogenesis, and induced adipocyte apoptosis. IgE reduced 3T3-L1 cell expression of Glut4, phospho-AKT, and glucose uptake, which concurred with improved glucose tolerance in Fcer1a(-/-) mice. This study established two novel pathways of IgE in reducing body weight gain in DIO mice by suppressing adipogenesis and inducing adipocyte apoptosis, while worsening glucose tolerance by reducing Glut4 expression, glucose uptake, and insulin secretion.

PMID: 26295369 [PubMed - as supplied by publisher]

The ΔF508 Mutation in the Cystic Fibrosis Transmembrane Conductance Regulator is Associated with Progressive Insulin Resistance and Decreased Functional Beta-Cell Mass in Mice.

Sat, 08/22/2015 - 04:06
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The ΔF508 Mutation in the Cystic Fibrosis Transmembrane Conductance Regulator is Associated with Progressive Insulin Resistance and Decreased Functional Beta-Cell Mass in Mice.

Diabetes. 2015 Aug 17;

Authors: Fontés G, Ghislain J, Benterki I, Zarrouki B, Trudel D, Berthiaume Y, Poitout V

Abstract
Cystic Fibrosis (CF) is due to mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Cystic Fibrosis-Related Diabetes affects 50% of adult CF patients. How CFTR deficiency predisposes to diabetes is unknown. Herein we examined the impact of the most frequent cftr mutation in humans, ΔF508, on glucose homeostasis in mice. We compared ΔF508 mutant mice to wild-type (WT) littermates. Twelve-week old male ΔF508 mutants had lower body weight, improved oral glucose tolerance and a trend towards higher insulin tolerance. Glucose-induced insulin secretion was slightly diminished in ΔF508 mutant islets, due to reduced insulin content, but ΔF508 mutant islets were not more sensitive to proinflammatory cytokines than WT islets. Hyperglycemic clamps confirmed an increase in insulin sensitivity with normal beta-cell function in 12- and 18-week old ΔF508 mutants. In contrast, 24-week old ΔF508 mutants exhibited insulin resistance and reduced beta-cell function. Beta-cell mass was unaffected at 11 weeks of age but was significantly lower in ΔF508 mutants vs. controls at 24 weeks. This was not associated with gross pancreatic pathology. We conclude that the ΔF508 CFTR mutation does not lead to an intrinsic beta-cell secretory defect but is associated with insulin resistance and a beta-cell mass deficit in aging mutants.

PMID: 26283735 [PubMed - as supplied by publisher]

Curcumin, Hesperidin, and Rutin Selectively Interfere with Apoptosis Signaling and Attenuate Streptozotocin-Induced Oxidative Stress-Mediated Hyperglycemia.

Sun, 08/16/2015 - 00:49
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Curcumin, Hesperidin, and Rutin Selectively Interfere with Apoptosis Signaling and Attenuate Streptozotocin-Induced Oxidative Stress-Mediated Hyperglycemia.

Curr Neurovasc Res. 2015 Aug 12;

Authors: Parmar MS, Syed I, Gray JP, Ray SD

Abstract
Type I Diabetes is characterized by the presence of hyperglycemia due to insulin deficiency and consequent impaired hepatic glucose metabolism. During diabetes, the liver becomes the most important tissue for the regulation of serum glucose. However, elevated glucose causes continuous oxidative damage to the liver, reducing its capacity to ameliorate hyperglycemia, which contributes to macrovascular complications [1] . Numerous epidemiological studies have demonstrated that excess human consumption of diets rich in specific bioflavonoid phytochemicals attenuates the effects of diabetes. Thus, this study was designed to investigate whether a bioflavonoid mixture could : i) attenuate streptozotocin (STZ)-induced hyperglycemia, ii) potentiate antioxidant signaling in the liver, and iii) ameliorate the apoptotic signaling cascade in the liver of STZ-induced hyperglycemic mice. In order to examine our hypothesis, three well-investigated antioxidant phytochemicals, curcumin, hesperidin and rutin, were combined into a mixture (CHR) for this study. Diabetes was induced in 6-month-old female ICR mice by STZ (100 mg/kg, i.p.) administration, and CHR or vehicle control was orally administered (200 mg/kg per body weight of each ingredient) to the hyperglycemic mice (blood glucose levels > 250 mg/dl) for a period of 14 days. Administration of CHR to the hyperglycemic mice significantly reduced blood glucose levels, attenuated STZ-induced lipid peroxidation and total nitrate/nitrite levels, and significantly augmented the expression of superoxide dismutase and glutathione in the liver. STZ-induced hyperglycemia resulted in downregulation of antiapoptotic proteins Bcl-2 by 66% and Bcl-XL by 51%, and upregulation of the pro-apoptotic Bad (69%) with an increase in the ratio of cytosolic/mitochondrial cytochrome c by 81% in hepatic tissue. Administration of CHR significantly ameliorated apoptotic signaling in STZ-induced diabetic mice, significantly increasing Bad/Bcl-2 and Bad/Bcl-XL ratios to 410% and 244% respectively in the hyperglycemic group. This study demonstrated that a bioflavonoid mixture of curcumin, hesperidin and rutin (CHR) ameliorates hepatic oxidative stress caused by STZ-induced hyperglycemia, resulting in improved hepatic function and glucose regulation.

PMID: 26265154 [PubMed - as supplied by publisher]

Enhanced Peptide Stability Against Protease Digestion Induced by Intrinsic Factor Binding of a Vitamin B12 Conjugate of Exendin-4.

Wed, 08/12/2015 - 22:46
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Enhanced Peptide Stability Against Protease Digestion Induced by Intrinsic Factor Binding of a Vitamin B12 Conjugate of Exendin-4.

Mol Pharm. 2015 Aug 11;

Authors: Bonaccorso RL, Chepurny OG, Becker-Pauly C, Holz GG, Doyle RP

Abstract
Peptide digestion from proteases is a significant limitation in peptide therapeutic development. It has been hypothesized that the dietary pathway of vitamin B12 (B12) may be exploited in this area, but an open question is whether B12-peptide conjugates bound to the B12 gastric uptake protein intrinsic factor (IF) can provide any stability against proteases. Herein, we describe a new conjugate of B12 with the incretin peptide exendin 4 that demonstrates picomolar agonism of the glugacon-like peptide-1 receptor (GLP1-R). Stability studies reveal that Ex-4 is digested by pancreatic proteases trypsin and chymotrypsin and by the kidney endopeptidase meprin β. Prebinding the B12 conjugate to IF, however, resulted in up to a 4-fold greater activity of the B12-Ex-4 conjugate relative to Ex-4, when the IF-B12-Ex-4 complex was exposed to 22 μg/mL of trypsin, 2.3-fold greater activity when exposed to 1.25 μg/mL of chymotrypsin, and there was no decrease in function at up to 5 μg/mL of meprin β.

PMID: 26260673 [PubMed - as supplied by publisher]

Islet Oxygen Consumption Rate (OCR) Dose Predicts Insulin Independence in Clinical Islet Autotransplantation.

Wed, 08/12/2015 - 22:46
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Islet Oxygen Consumption Rate (OCR) Dose Predicts Insulin Independence in Clinical Islet Autotransplantation.

PLoS One. 2015;10(8):e0134428

Authors: Papas KK, Bellin MD, Sutherland DE, Suszynski TM, Kitzmann JP, Avgoustiniatos ES, Gruessner AC, Mueller KR, Beilman GJ, Balamurugan AN, Loganathan G, Colton CK, Koulmanda M, Weir GC, Wilhelm JJ, Qian D, Niland JC, Hering BJ

Abstract
BACKGROUND: Reliable in vitro islet quality assessment assays that can be performed routinely, prospectively, and are able to predict clinical transplant outcomes are needed. In this paper we present data on the utility of an assay based on cellular oxygen consumption rate (OCR) in predicting clinical islet autotransplant (IAT) insulin independence (II). IAT is an attractive model for evaluating characterization assays regarding their utility in predicting II due to an absence of confounding factors such as immune rejection and immunosuppressant toxicity.
METHODS: Membrane integrity staining (FDA/PI), OCR normalized to DNA (OCR/DNA), islet equivalent (IE) and OCR (viable IE) normalized to recipient body weight (IE dose and OCR dose), and OCR/DNA normalized to islet size index (ISI) were used to characterize autoislet preparations (n = 35). Correlation between pre-IAT islet product characteristics and II was determined using receiver operating characteristic analysis.
RESULTS: Preparations that resulted in II had significantly higher OCR dose and IE dose (p<0.001). These islet characterization methods were highly correlated with II at 6-12 months post-IAT (area-under-the-curve (AUC) = 0.94 for IE dose and 0.96 for OCR dose). FDA/PI (AUC = 0.49) and OCR/DNA (AUC = 0.58) did not correlate with II. OCR/DNA/ISI may have some utility in predicting outcome (AUC = 0.72).
CONCLUSIONS: Commonly used assays to determine whether a clinical islet preparation is of high quality prior to transplantation are greatly lacking in sensitivity and specificity. While IE dose is highly predictive, it does not take into account islet cell quality. OCR dose, which takes into consideration both islet cell quality and quantity, may enable a more accurate and prospective evaluation of clinical islet preparations.

PMID: 26258815 [PubMed - in process]

Excessive Cellular Proliferation Negatively Impacts Reprogramming Efficiency of Human Fibroblasts.

Sun, 08/09/2015 - 22:19
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Excessive Cellular Proliferation Negatively Impacts Reprogramming Efficiency of Human Fibroblasts.

Stem Cells Transl Med. 2015 Aug 7;

Authors: Gupta MK, Teo AK, Rao TN, Bhatt S, Kleinridders A, Shirakawa J, Takatani T, Hu J, De Jesus DF, Windmueller R, Wagers AJ, Kulkarni RN

Abstract
: The impact of somatic cell proliferation rate on induction of pluripotent stem cells remains controversial. Herein, we report that rapid proliferation of human somatic fibroblasts is detrimental to reprogramming efficiency when reprogrammed using a lentiviral vector expressing OCT4, SOX2, KLF4, and cMYC in insulin-rich defined medium. Human fibroblasts grown in this medium showed higher proliferation, enhanced expression of insulin signaling and cell cycle genes, and a switch from glycolytic to oxidative phosphorylation metabolism, but they displayed poor reprogramming efficiency compared with cells grown in normal medium. Thus, in contrast to previous studies, our work reveals an inverse correlation between the proliferation rate of somatic cells and reprogramming efficiency, and also suggests that upregulation of proteins in the growth factor signaling pathway limits the ability to induce pluripotency in human somatic fibroblasts.
SIGNIFICANCE: The efficiency with which human cells can be reprogrammed is of interest to stem cell biology. In this study, human fibroblasts cultured in media containing different concentrations of growth factors such as insulin and insulin-like growth factor-1 exhibited variable abilities to proliferate, with consequences on pluripotency. This occurred in part because of changes in the expression of proteins involved in the growth factor signaling pathway, glycolysis, and oxidative phosphorylation. These findings have implications for efficient reprogramming of human cells.

PMID: 26253715 [PubMed - as supplied by publisher]

Preserved DNA Damage Checkpoint Pathway Protects against Complications in Long-Standing Type 1 Diabetes.

Thu, 08/06/2015 - 18:51
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Preserved DNA Damage Checkpoint Pathway Protects against Complications in Long-Standing Type 1 Diabetes.

Cell Metab. 2015 Aug 4;22(2):239-252

Authors: Bhatt S, Gupta MK, Khamaisi M, Martinez R, Gritsenko MA, Wagner BK, Guye P, Busskamp V, Shirakawa J, Wu G, Liew CW, Clauss TR, Valdez I, El Ouaamari A, Dirice E, Takatani T, Keenan HA, Smith RD, Church G, Weiss R, Wagers AJ, Qian WJ, King GL, Kulkarni RN

Abstract
The mechanisms underlying the development of complications in type 1 diabetes (T1D) are poorly understood. Disease modeling of induced pluripotent stem cells (iPSCs) from patients with longstanding T1D (disease duration ≥ 50 years) with severe (Medalist +C) or absent to mild complications (Medalist -C) revealed impaired growth, reprogramming, and differentiation in Medalist +C. Genomics and proteomics analyses suggested differential regulation of DNA damage checkpoint proteins favoring protection from cellular apoptosis in Medalist -C. In silico analyses showed altered expression patterns of DNA damage checkpoint factors among the Medalist groups to be targets of miR200, whose expression was significantly elevated in Medalist +C serum. Notably, neurons differentiated from Medalist +C iPSCs exhibited enhanced susceptibility to genotoxic stress that worsened upon miR200 overexpression. Furthermore, knockdown of miR200 in Medalist +C fibroblasts and iPSCs rescued checkpoint protein expression and reduced DNA damage. We propose miR200-regulated DNA damage checkpoint pathway as a potential therapeutic target for treating complications of diabetes.

PMID: 26244933 [PubMed - as supplied by publisher]

MAFA and T3 drive maturation of both fetal human islets and insulin-producing cells differentiated from hESC.

Sat, 07/25/2015 - 13:58

MAFA and T3 drive maturation of both fetal human islets and insulin-producing cells differentiated from hESC.

J Clin Endocrinol Metab. 2015 Jul 24;:jc20152632

Authors: Aguayo-Mazzucato C, Dilenno A, Hollister-Lock J, Cahill C, Sharma A, Weir G, Colton C, Bonner-Weir S

Abstract
CONTEXT: Human embryonic stem cells (hESC) differentiated towards beta cells and fetal human pancreatic islet cells resemble each other transcriptionally and are characterized by immaturity with a lack of glucose responsiveness, low levels of insulin content and impaired pro-insulin-to-insulin processing. However, their response to stimuli that promote functionality have not been compared.
OBJECTIVE: To evaluate the effects of our previous strategies for functional maturation developed in rodents in these two human models of beta-cell immaturity and compare their responses. Design, settings, participants and interventions: In proof-of-principle experiments using either adenoviral-mediated overexpression of MAFA or the physiologically driven path via thyroid hormone (T3), human fetal islet-like clusters (ICC) functional maturity was evaluated. Then, the effects of T3 were evaluated upon the functional maturation of hESC differentiated toward beta cells.
MAIN OUTCOME MEASURES: Functional maturation was evaluated by the following parameters: glucose responsiveness, insulin content, expression of the mature beta-cell transcription factor MAFA and proinsulin-to-insulin processing.
RESULTS: ICCs responded positively to MAFA overexpression and T3 treatment as assessed by two different maturation parameters: increased insulin secretion at 16.8 mM glucose and increased proinsulin-to-insulin processing. In hESC differentiated toward beta cells, T3 enhanced MAFA expression, increased insulin content (probably mediated by the increased MAFA) and increased insulin secretion at 16.8 mM glucose.
CONCLUSION: T3 is a useful in vitro stimulus to promote human beta-cell maturation as shown in both human fetal ICCs and differentiated hESCs. The degree of maturation induced varied in the two models, possibly due to the different developmental status at the beginning of the study.

PMID: 26207953 [PubMed - as supplied by publisher]

β-Cell differentiation of human pancreatic duct-derived cells after in vitro expansion.

Sun, 07/19/2015 - 09:07
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β-Cell differentiation of human pancreatic duct-derived cells after in vitro expansion.

Cell Reprogram. 2014 Dec;16(6):456-66

Authors: Corritore E, Dugnani E, Pasquale V, Misawa R, Witkowski P, Lei J, Markmann J, Piemonti L, Sokal EM, Bonner-Weir S, Lysy PA

Abstract
β-Cell replacement therapy is a promising field of research that is currently evaluating new sources of cells for clinical use. Pancreatic epithelial cells are potent candidates for β-cell engineering, but their large-scale expansion has not been evidenced yet. Here we describe the efficient expansion and β-cell differentiation of purified human pancreatic duct cells (DCs). When cultured in endothelial growth-promoting media, purified CA19-9(+) cells proliferated extensively and achieved up to 22 population doublings over nine passages. While proliferating, human pancreatic duct-derived cells (HDDCs) downregulated most DC markers, but they retained low CK19 and SOX9 gene expression. HDDCs acquired mesenchymal features but differed from fibroblasts or pancreatic stromal cells. Coexpression of duct and mesenchymal markers suggested that HDDCs were derived from DCs via a partial epithelial-to-mesenchymal transition (EMT). This was supported by the blockade of HDDC appearance in CA19-9(+) cell cultures after incubation with the EMT inhibitor A83-01. After a differentiation protocol mimicking pancreatic development, HDDC populations contained about 2% of immature insulin-producing cells and showed glucose-unresponsive insulin secretion. Downregulation of the mesenchymal phenotype improved β-cell gene expression profile of differentiated HDDCs without affecting insulin protein expression and secretion. We show that pancreatic ducts represent a new source for engineering large amounts of β-like-cells with potential for treating diabetes.

PMID: 25437872 [PubMed - indexed for MEDLINE]

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