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2.
J Dev Orig Health Dis ; 8(2): 188-195, 2017 Apr.
Article in English | MEDLINE | ID: mdl-28115035

ABSTRACT

Intrauterine exposure to the rainy season in the tropics may be accompanied by high rates of infection and nutritional deficiencies. It is unknown whether this exposure is related to the extrauterine timing of development. Our aim was to evaluate the relations of prenatal exposure to the rainy season and altitude of residence with age at menarche. The study included 15,370 girls 10 to <18 years old who participated in Colombia's 2010 National Nutrition Survey. Primary exposures included the number of days exposed to the rainy season during the 40 weeks preceding birth, and altitude of residence at the time of the survey. We estimated median menarcheal ages and hazard ratios with 95% confidence interval (CI) according to exposure categories using Kaplan-Meier cumulative probabilities and Cox proportional hazards models, respectively. All tests incorporated the complex survey design. Girls in the highest quintile of gestation days exposed to the rainy season had an earlier age at menarche compared with those in the lowest (adjusted hazard ratios (HR)=1.08; 95% CI 1.00-1.18, P-trend=0.03). Girls living at altitudes ⩾2000 m had a later age at menarche compared with those living <1000 m (adjusted HR=0.88; 95% CI 0.82-0.94, P-trend <0.001). The inverse association between gestation days during the rainy season and menarche was most apparent among girls living at altitudes ⩾2000 m (P, interaction=0.04). Gestation days exposed to the rainy season and altitude of residence were associated with the timing of sexual maturation among Colombian girls independent of socioeconomic status and ethnicity.


Subject(s)
Altitude , Menarche , Prenatal Exposure Delayed Effects , Seasons , Sexual Maturation , Adolescent , Adolescent Development , Age Factors , Child , Female , Humans , Pregnancy , Social Class , Tropical Climate
3.
J Public Health (Oxf) ; 36(2): 235-42, 2014 Jun.
Article in English | MEDLINE | ID: mdl-23995713

ABSTRACT

BACKGROUND: Climate change has contributed to increasing temperatures, earlier snowmelts and thinning ice packs in the Arctic, where crossing frozen bodies of water is essential for transportation and subsistence living. In some Arctic communities, anecdotal reports indicate a growing belief that falling-through-the-ice (FTI) are increasing. The objective of this study was to describe the morbidity and mortality associated with unintentional FTIs in Alaska. METHODS: We searched newspaper reports to identify FTI events from 1990 to 2010. We also used data from a trauma registry, occupational health and law enforcement registries and vital statistics to supplement the newspaper reports. Morbidity and mortality rates were calculated for Alaska Native (AN) people and all Alaskans. RESULTS: During the 21-year period, we identified 307 events, affecting at least 449 people. Events ranged from no morbidity to fatalities of five people. More than half of the events involved transportation by snow machine. Mortality rates were markedly higher for AN people than that for all Alaskans. CONCLUSIONS: We provide a numeric estimate of the importance of FTI events in Alaska. FTIs may represent an adverse health outcome related to climate changes in the Arctic, and may be particularly critical for vulnerable populations such as AN people.


Subject(s)
Accidental Falls/statistics & numerical data , Ice , Accidental Falls/mortality , Alaska/epidemiology , Climate Change , Drowning/epidemiology , Female , Humans , Male , Registries , Risk Factors , Wounds and Injuries/epidemiology
4.
J Epidemiol Community Health ; 64(1): 16-21, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19643766

ABSTRACT

BACKGROUND: The field of epidemiology struggles both with enhancing causal inference in observational studies and providing useful information for policy makers and public health workers focusing on interventions. Population intervention models, analogous to population attributable fractions, estimate the causal impact of interventions in a population, and are one option for understanding the relative importance of various risk factors. With population intervention parameters, risk factors are effectively standardised, allowing one to compare their values directly and determine which potential intervention may have the greatest impact on the outcome. METHODS: The difference between total effects and population intervention parameters was examined using naïve, G-computation and inverse probability of treatment weighting approaches. The differences between these parameters and the intuitions they provide were explored using data from a 2003 cross-sectional study in rural Mexico. RESULTS: The assumptions, specific analytic steps, limitations and interpretations of the total effects and population intervention parameters are discussed, and code is provided in Stata. CONCLUSION: Population intervention parameters are a valuable and straightforward approach in epidemiological studies for making causal inference from the data while also supplying information that is relevant for researchers, public health practitioners and policy makers.


Subject(s)
Causality , Depressive Disorder/epidemiology , Epidemiologic Methods , Cross-Sectional Studies , Humans , Latin America , Models, Statistical , Observation , Risk Factors , Social Support
5.
Gene Ther ; 16(3): 340-8, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19112449

ABSTRACT

The effectiveness of genetic engineering with lentivectors to protect transplanted cells from allogeneic rejection was examined using, as a model, type 1 diabetes treatment with beta-cell transplantation, whose widespread use has been limited by the requirement for sustained immunosuppressive treatment to prevent graft rejection. We examined whether lentivectors expressing select immunosuppressive proteins encoded by the adenoviral genome early region 3 (AdE3) would protect transplanted beta-cells from an alloimmune attack. The insulin-producing beta-cell line beta TC-tet (C3HeB/FeJ-derived) was transduced with lentiviruses encoding the AdE3 proteins gp19K and RID alpha/beta. The efficiency of lentiviral transduction of beta TC-tet cells exceeded 85%. Lentivector expression of gp19K decreased surface class I major histocompatibility complex expression by over 90%, whereas RID alpha/beta expression inhibited cytokine-induced Fas upregulation by over 75%. beta TC-tet cells transduced with gp19K and RID alpha/beta lentivectors, but not with a control lentivector, provided prolonged correction of hyperglycemia after transplantation into diabetic BALB/c severe combined immunodeficient mice reconstituted with allogeneic immune effector cells or into diabetic allogeneic BALB/c mice. Thus, genetic engineering of beta-cells using gp19K- and RID alpha/beta-expressing lentiviral vectors may provide an alternative that has the potential to eliminate or reduce treatment with the potent immunosuppressive agents necessary at present for prolonged engraftment with transplanted islets.


Subject(s)
Diabetes Mellitus, Experimental/therapy , Diabetes Mellitus, Type 1/therapy , Genetic Engineering/methods , Graft Rejection/prevention & control , Insulin-Secreting Cells/immunology , Islets of Langerhans Transplantation/methods , Adenovirus E3 Proteins/genetics , Adenovirus E3 Proteins/immunology , Adenovirus Early Proteins/genetics , Adenovirus Early Proteins/immunology , Animals , Diabetes Mellitus, Experimental/immunology , Diabetes Mellitus, Type 1/immunology , Disease Models, Animal , Female , Genetic Vectors , Graft Rejection/immunology , Immune Tolerance , Lentivirus/genetics , Mice , Mice, Inbred C3H , Mice, Inbred Strains , Reverse Transcriptase Polymerase Chain Reaction/methods , Transduction, Genetic
6.
J Epidemiol Community Health ; 62(9): 842-6, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18701738

ABSTRACT

BACKGROUND: Directed acyclic graphs, or DAGs, are a useful graphical tool in epidemiologic research that can help identify appropriate analytical strategies in addition to potential unintended consequences of commonly used methods such as conditioning on mediators. The use of DAGs can be particularly informative in the study of the causal effects of social factors on health. METHODS: The authors consider four specific scenarios in which DAGs may be useful to neighbourhood health effects researchers: (1) identifying variables that need to be adjusted for in estimating neighbourhood health effects, (2) identifying the unintended consequences of estimating "direct" effects by conditioning on a mediator, (3) using DAGs to understand possible sources and consequences of selection bias in neighbourhood health effects research, and (4) using DAGs to identify the consequences of adjustment for variables affected by prior exposure. CONCLUSIONS: The authors present simplified sample DAGs for each scenario and discuss the insights that can be gleaned from the DAGs in each case and the implications these have for analytical approaches.


Subject(s)
Health Status , Residence Characteristics , Social Environment , Causality , Confounding Factors, Epidemiologic , Data Interpretation, Statistical , Humans , Selection Bias , Socioeconomic Factors
7.
HIV Med ; 7(6): 389-96, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16903984

ABSTRACT

OBJECTIVES: To determine factors associated with diabetes, insulin resistance, and abnormal glucose tolerance in older men with or at risk of HIV infection. METHODS: Diabetes was assessed by self-report in 643 men >or=49 years old with or at risk of HIV infection. In a subset of 216 men without previously diagnosed diabetes [including 90 HIV-uninfected men, 28 HIV-infected, antiretroviral-naive men, 28 HIV-infected men taking non-protease inhibitor (PI)-containing highly active antiretroviral therapy (HAART), and 70 HIV-infected men taking PI-containing HAART], an oral glucose tolerance test with insulin levels was performed. HIV serology, CD4 cell count, weight, height and waist circumference were measured. Antiretroviral use, drug use, family history of diabetes, physical activity and sociodemographic data were obtained using standardized interviews. RESULTS: Of 643 participants, 116 (18%) had previously diagnosed diabetes. With the oral glucose tolerance test, 15 of 216 men (7%) were found to have undiagnosed diabetes and 40 (18%) impaired glucose tolerance. Factors independently associated with previously diagnosed diabetes included use of non-PI-containing HAART, methadone treatment, positive CAGE test for alcoholism, obesity and family history of diabetes. Factors independently associated with greater insulin resistance included waist circumference and heroin use. Factors independently associated with abnormal glucose tolerance (impaired glucose tolerance or diabetes) included age >or=55 years and Hispanic ethnicity. CONCLUSIONS: HIV-infected men with diabetes risk factors should undergo screening for diabetes regardless of HAART use. Interventions targeting modifiable risk factors, including overweight and physical inactivity, are warranted. The potential impact of opiate and alcohol abuse on glucose metabolism should be recognized in clinical care, and addressed in future research studies of HIV-infected persons.


Subject(s)
Diabetes Complications , Diabetes Mellitus/epidemiology , Glucose Intolerance/complications , HIV Infections/complications , Antiretroviral Therapy, Highly Active , Body Composition , Cohort Studies , Diabetes Mellitus/diagnosis , Glucose Intolerance/diagnosis , Glucose Intolerance/epidemiology , Glucose Tolerance Test , HIV Infections/drug therapy , HIV Infections/metabolism , HIV Protease Inhibitors/therapeutic use , Humans , Insulin Resistance , Interviews as Topic , Male , Middle Aged , Risk Factors , Substance-Related Disorders/complications
8.
Conf Proc IEEE Eng Med Biol Soc ; 2006: 2009-12, 2006.
Article in English | MEDLINE | ID: mdl-17945690

ABSTRACT

This paper demonstrates the first steps of an automation process to develop models of signal transduction pathways using discrete modelling languages. The whole approach consists of modelling, validation, animation, linking databases to simulation tools and also the qualitative analysis of the data. In this paper, we detail the modelling and simulation of the TLR4 pathway with a coloured petri net simulation tool and the validation of this model against the semantic and mechanistic map from a biological database. These graphical maps contain all necessary reactions as a figure. We start with an UML class diagram to understand the static structure of molecules involved in the TLR4 pathway. Afterwards we model and simulate each "pathway step reaction" - one after another - to get the behaviour of the final system. The result is a model of the pathway which can be used in simulations, derived solely from basic chemical reactions in the database. Also, it is a lesson on critical points where human decision-making is needed, because not all the required information is stored directly in the database.


Subject(s)
Color , Computer Graphics , Models, Biological , Signal Transduction/physiology , Toll-Like Receptor 4/metabolism , User-Computer Interface , Computer Simulation
9.
Cell Transplant ; 10(7): 645-50, 2001.
Article in English | MEDLINE | ID: mdl-11714200

ABSTRACT

Pancreatic beta cell lines may offer an abundant source of cells for beta-cell replacement in type I diabetes. Using regulatory elements of the bacterial tetracycline (tet) operon for conditional expression of SV40 T antigen oncoprotein in transgenic mouse beta cells, we have shown that reversible immortalization is an efficient approach for regulated beta-cell expansion, accompanied by enhanced cell differentiation upon growth arrest. The original system employed the tet-off approach, in which the cells proliferate in the absence of tet ligands and undergo growth arrest in their presence. The disadvantage of this system is the need for continuous treatment with the ligand in vivo for maintaining growth arrest. Here we utilized the tet-on regulatory system to generate beta cell lines in which proliferation is regulated in reverse: these cells divide in the presence of tet ligands, and undergo growth arrest in their absence, as judged by [3H]thymidine and BrdU incorporation assays. These cell lines were derived from insulinomas, which heritably developed in transgenic mice continuously treated with the tet derivative doxycycline (dox). The cells produce and secrete high amounts of insulin, and can restore and maintain euglycemia in syngeneic streptozotocin-induced diabetic mice in the absence of dox. Such a system is more suitable for transplantation, compared with cells regulated by the tet-off approach, because ligand treatment is limited to cell expansion in culture and is not required for long-term maintenance of growth arrest in vivo.


Subject(s)
Diabetes Mellitus, Type 1/therapy , Hyperglycemia/therapy , Islets of Langerhans Transplantation , Islets of Langerhans/cytology , Animals , Antigens, Polyomavirus Transforming/genetics , Cell Division , Cell Line, Transformed/transplantation , Insulin/metabolism , Insulin Secretion , Insulinoma , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Transgenic , Plasmids , Repressor Proteins
10.
Diabetes ; 50(2): 348-52, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11272146

ABSTRACT

The demonstration of leptin receptors on the pancreatic beta-cells suggests the possibility of direct actions of leptin on insulin secretion. In vitro studies on islets or perfused pancreas and beta-cell lines produced inconsistent results. We performed an in vivo study to distinctly examine whether leptin has an effect on glucose-stimulated insulin secretion. Young chronically catheterized Sprague-Dawley rats (n = 28) were subjected to a 4-h hyperglycemic clamp study (approximately 11 mmol/l). At minute 120 to 240, rats were assigned to receive either saline or leptin (0.1, 0.5, and 5 microg x kg(-1) x min) infusion. Leptin decreased plasma insulin levels abruptly, and an approximately twofold decrease in plasma insulin levels compared with saline control was sustained over the 2 h of the study (14.8 +/- 5.8 vs. 34.8 +/- 2.6 ng/ml with leptin and saline infusion, respectively, P < 0.001). Moreover, a dose-dependent decrease in plasma insulin levels was noted (r = -0.731, P < 0.01). Since milrinone, an inhibitor of cAMP phosphodiesterase (PDE) 3, did not reverse the effect of leptin on glucose-induced insulin secretion, its action may be independent of PDE3. These findings suggest that acute physiological increase in plasma leptin levels acutely and significantly inhibits glucose-stimulated insulin secretion in vivo. The site of leptin effects on insulin secretion remains to be determined.


Subject(s)
Insulin/metabolism , Leptin/blood , Animals , Dose-Response Relationship, Drug , Glucose Clamp Technique , Insulin/blood , Insulin Secretion , Male , Milrinone/pharmacology , Phosphodiesterase Inhibitors/pharmacology , Rats , Rats, Sprague-Dawley
11.
J Biol Chem ; 274(48): 34343-9, 1999 Nov 26.
Article in English | MEDLINE | ID: mdl-10567411

ABSTRACT

The role of the calcium-binding protein, calbindin-D(28k) in potassium/depolarization-stimulated increases in the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and insulin release was investigated in pancreatic islets from calbindin-D(28k) nullmutant mice (knockouts; KO) or wild type mice and beta cell lines stably transfected and overexpressing calbindin. Using single islets from KO mice and stimulation with 45 mM KCl, the peak of [Ca(2+)](i) was 3.5-fold greater in islets from KO mice compared with wild type islets (p < 0.01) and [Ca(2+)](i) remained higher during the plateau phase. In addition to the increase in [Ca(2+)](i) in response to KCl there was also a significant increase in insulin release in islets isolated from KO mice. Evidence for modulation by calbindin of [Ca(2+)](i) and insulin release was also noted using beta cell lines. Rat calbindin was stably expressed in betaTC-3 and betaHC-13 cells. In response to depolarizing concentrations of K(+), insulin release was decreased by 45-47% in calbindin expressing betaTC cells and was decreased by 70-80% in calbindin expressing betaHC cells compared with insulin release from vector transfected betaTC or betaHC cells (p < 0.01). In addition, the K(+)-stimulated intracellular calcium peak was markedly inhibited in calbindin expressing betaHC cells compared with vector transfected cells (225 nM versus 1,100 nM, respectively). Buffering of the depolarization-induced rise in [Ca(2+)](i) was also observed in calbindin expressing betaTC cells. In summary, our findings, using both isolated islets from calbindin-D(28k) KO mice and beta cell lines, establish a role for calbindin in the modulation of depolarization-stimulated insulin release and suggest that calbindin can control the rate of insulin release via regulation of [Ca(2+)](i).


Subject(s)
Calcium/metabolism , Insulin/metabolism , S100 Calcium Binding Protein G/physiology , 1-Methyl-3-isobutylxanthine/pharmacology , Animals , Calbindins , Immunohistochemistry , Insulin Secretion , Islets of Langerhans/cytology , Islets of Langerhans/drug effects , Islets of Langerhans/metabolism , Mice , Mice, Knockout , Mutation , Potassium Chloride/pharmacology , Rats , Recombinant Fusion Proteins/drug effects , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , S100 Calcium Binding Protein G/genetics , S100 Calcium Binding Protein G/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Tissue Distribution , Tumor Cells, Cultured
12.
J Biol Chem ; 274(32): 22337-44, 1999 Aug 06.
Article in English | MEDLINE | ID: mdl-10428803

ABSTRACT

To understand the role cAMP phosphodiesterases (PDEs) play in the regulation of insulin secretion, we analyzed cyclic nucleotide PDEs of a pancreatic beta-cell line and used family and isozyme-specific PDE inhibitors to identify the PDEs that counteract glucose-stimulated insulin secretion. We demonstrate the presence of soluble PDE1C, PDE4A and 4D, a cGMP-specific PDE, and of particulate PDE3, activities in betaTC3 insulinoma cells. Selective inhibition of PDE1C, but not of PDE4, augmented glucose-stimulated insulin secretion in a dose-dependent fashion thus demonstrating that PDE1C is the major PDE counteracting glucose-dependent insulin secretion from betaTC3 cells. In pancreatic islets, inhibition of both PDE1C and PDE3 augmented glucose-dependent insulin secretion. The PDE1C of betaTC3 cells is a novel isozyme possessing a K(m) of 0.47 microM for cAMP and 0.25 microM for cGMP. The PDE1C isozyme of betaTC3 cells is sensitive to 8-methoxymethyl isobutylmethylxanthine and zaprinast (IC(50) = 7.5 and 4.5 microM, respectively) and resistant to vinpocetine (IC(50) > 100 microM). Increased responsiveness of PDE1C activity to calcium/calmodulin is evident upon exposure of cells to glucose. Enhanced cAMP degradation by PDE1C, due to increases in its responsiveness to calcium/calmodulin and in intracellular calcium, constitutes a glucose-dependent feedback mechanism for the control of insulin secretion.


Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases/metabolism , 3',5'-Cyclic-GMP Phosphodiesterases/metabolism , Glucose/pharmacology , Insulin/metabolism , Islets of Langerhans/enzymology , Phosphoric Diester Hydrolases , 3',5'-Cyclic-AMP Phosphodiesterases/antagonists & inhibitors , 3',5'-Cyclic-GMP Phosphodiesterases/antagonists & inhibitors , Animals , Calcium/pharmacology , Calmodulin/pharmacology , Cyclic AMP/analysis , Cyclic Nucleotide Phosphodiesterases, Type 1 , Down-Regulation , Feedback , Insulin Secretion , Male , Mice , Models, Biological , Nucleotides, Cyclic/metabolism , Phosphodiesterase Inhibitors/pharmacology , Substrate Specificity , Tumor Cells, Cultured
13.
Diabetes ; 47(9): 1419-25, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9726230

ABSTRACT

Development of beta-cell lines for cell therapy of diabetes is hindered by functional deviations of the replicating cells from the normal beta-cell phenotype. In a recently developed cell line, denoted betaTC-tet, derived from transgenic mice expressing the SV40 T antigen (Tag) under control of the tetracycline (Tc) gene regulatory system, growth arrest can be induced by shutting off Tag expression in the presence of Tc. Here, we compared differentiated cell functions in dividing and growth-arrested betaTC-tet cells, both in culture and in vivo. Proliferating cells stably maintained normal glucose responsiveness for >60 passages in culture. Growth-arrested cells survived for months in culture and in vivo and maintained normal insulin production and secretion. After growth arrest, the cells gradually increased their insulin content three- to fourfold. This occurred without significant changes in insulin biosynthetic rates. At high passage numbers, proliferating betaTC-tet cells exhibited an abnormal increase in hexokinase expression. However, the upregulation of hexokinase was reversible upon growth arrest. Growth-arrested cells transplanted intraperitoneally into syngeneic recipients responded to hyperglycemia by a significant increase in insulin secretion. These findings demonstrate that transformed beta-cells maintain function during long periods of growth arrest, suggesting that conditional transformation of beta-cells may be a useful approach for developing cell therapy for diabetes.


Subject(s)
Diabetes Mellitus, Experimental/surgery , Insulin/biosynthesis , Islets of Langerhans Transplantation/physiology , Islets of Langerhans/physiology , Animals , Antigens, Polyomavirus Transforming/biosynthesis , Antigens, Polyomavirus Transforming/genetics , Blood Glucose/metabolism , Cell Division , Cell Line, Transformed , Diabetes Mellitus, Experimental/blood , Glucokinase/metabolism , Glucose/metabolism , Glucose/pharmacology , Glucose Clamp Technique , Hexokinase/metabolism , Insulin/metabolism , Insulin Secretion , Islets of Langerhans/cytology , Islets of Langerhans/drug effects , Kinetics , Mice , Mice, Inbred C3H , Mice, Transgenic , Phosphorylation , Proinsulin/biosynthesis , Protein Biosynthesis , Regulatory Sequences, Nucleic Acid , Simian virus 40/genetics , Tetracycline Resistance/genetics
18.
Diabetes ; 45(10): 1412-8, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8826979

ABSTRACT

Two voltage-dependent calcium channels (VDCCs) have been reported in pancreatic islets: the beta-cell/endocrine-brain and cardiac subtypes. The cardiac-type alpha 1 subunit was isolated from cultured beta TC3 cells, a murine pancreatic beta-cell line, by immunoprecipitation with a specific polyclonal antibody. We have examined the effects of 1-isobutyl-3-methylxanthine (IBMX) and forskolin, agonists that elevate cAMP in these cells, on the phosphorylation of this subunit in intact beta TC3 cells using a sensitive back-phosphorylation technique. This technique allows quantitative detection of protein phosphorylation that is specifically stimulated by cAMP. The stimulation of intact beta TC3 cells with forskolin or IBMX resulted in the phosphorylation of the cardiac-type alpha 1 subunit as evidenced by a 40-60% decrease in the ability of the 257-kDa form to serve as a substrate in the in vitro back-phosphorylation reaction with [gamma-32P]ATP and the catalytic subunit of cAMP-dependent protein kinase (PKA). The effects of forskolin were time- and concentration-dependent. The concentration-dependency of forskolin-induced phosphorylation of the cardiac-type alpha 1 subunit and the potentiation of glucose-induced insulin secretion were highly correlated, a finding that is consistent with a role for such phosphorylation in mediating at least some of the effects of cAMP on secretion.


Subject(s)
Calcium Channels/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP/metabolism , Islets of Langerhans/metabolism , Myocardium/metabolism , 1-Methyl-3-isobutylxanthine/pharmacology , Animals , Calcium Channels/isolation & purification , Calcium Channels/physiology , Calcium Channels, L-Type , Cell Line , Colforsin/pharmacology , Electrophoresis, Polyacrylamide Gel , Gallopamil/pharmacology , Insulin/metabolism , Insulin Secretion , Kinetics , Macromolecular Substances , Mice , Phosphoproteins/analysis , Phosphoproteins/metabolism , Phosphorylation
19.
J Biol Chem ; 270(37): 21464-7, 1995 Sep 15.
Article in English | MEDLINE | ID: mdl-7665557

ABSTRACT

Glucokinase catalyzes a rate-limiting step in glucose metabolism in hepatocytes and pancreatic beta cells and is considered the "glucose sensor" for regulation of insulin secretion. Patients with maturity-onset diabetes of the young (MODY) have heterozygous point mutations in the glucokinase gene that result in reduced enzymatic activity and decreased insulin secretion. However, it remains unclear whether abnormal liver glucose metabolism contributes to the MODY disease. Here we show that disruption of the glucokinase gene results in a phenotype similar to MODY in heterozygous mice. Reduced islet glucokinase activity causes mildly elevated fasting blood glucose levels. Hyperglycemic clamp studies reveal decreased glucose tolerance and abnormal liver glucose metabolism. These findings demonstrate a key role for glucokinase in glucose homeostasis and implicate both islets and liver in the MODY disease.


Subject(s)
Diabetes Mellitus, Type 2/genetics , Glucokinase/genetics , Islets of Langerhans/enzymology , Liver/enzymology , Point Mutation , Animals , Base Sequence , Blood Glucose/metabolism , Cloning, Molecular , DNA Primers , Diabetes Mellitus, Type 2/enzymology , Diabetes Mellitus, Type 2/metabolism , Disease Models, Animal , Glucose/metabolism , Glucose Clamp Technique , Heterozygote , Humans , Insulin/metabolism , Insulin Secretion , Islets of Langerhans/metabolism , Mice , Mice, Mutant Strains , Molecular Sequence Data , Mutagenesis, Site-Directed , Polymerase Chain Reaction , Restriction Mapping
20.
Endocrinology ; 136(6): 2521-30, 1995 Jun.
Article in English | MEDLINE | ID: mdl-7750474

ABSTRACT

Protein carboxylmethylation is a reversible posttranslational modification that regulates protein function. We examined the carboxylmethylation of small GTP-binding proteins in a pancreatic beta-cell line (beta TC cells). In vitro assays showed that carboxylmethylation of a membrane 23-kDa protein was induced by guanine nucleotides, best demonstrated by the nonhydrolyzable GTP analog, guanosine 5'-(3-O-thio)triphosphate (GTP gamma S). GTP gamma S also induced translocation of this 23-kilodalton (kDa) protein from cytosol to particulate fractions. Immunoblotting with antiserum sc-65 raised against Rap1 identified the carboxyl-methylated 23-kDa protein as Rap1. 1) The 23-kDa carboxyl-methylated protein separated by two-dimensional electrophoresis overlapped with the 23-kDa protein detected by immunoblotting. 2) GTP gamma S, in the presence of cytosol, increased the amount of detectable membrane-associated Rap1. Studies in intact beta TC cells demonstrated the carboxylmethylation of the 23-kDa protein in response to glucose and depolarizing concentrations of potassium, an effect that was abolished by the calcium channel inhibitor, D600. Similarly, N-acetyl-S-trans,trans-farnesyl-L-cysteine, an inhibitor of in vivo carboxylmethylation at COOH-terminal S-farnesylcysteine by methyltransferase, inhibited carboxylmethylation of the 23-kDa protein in intact cells and reduced insulin secretion in response to glucose and potassium. These data establish a correlation between insulin secretion and carboxylmethylation of a 23-kDa protein that comigrates with Rap1.


Subject(s)
GTP-Binding Proteins/metabolism , Insulin/metabolism , Acetylcysteine/analogs & derivatives , Acetylcysteine/pharmacology , Animals , Cell Line , GTP-Binding Proteins/chemistry , Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology , Insulin Secretion , Islets of Langerhans/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Methylation , Molecular Weight , Subcellular Fractions/enzymology , rac GTP-Binding Proteins , rap GTP-Binding Proteins
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