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1.
J Cell Biol ; 109(6 Pt 1): 2761-9, 1989 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-2592405

RESUMO

The prespore vesicle (PSV) is an organelle which secretes spore coat proteins and gal/galNAc polysaccharides from prespore cells of Dictyostelium. By combining the techniques of protein A-gold immunocytochemistry and ricin-gold affinity cytochemistry we have demonstrated colocalization of the lysosomal enzyme alpha-mannosidase with gal/galNAc polysaccharides in prespore vesicles and the spore coat. To determine the origin of prespore vesicles a series of pulse-chase experiments were performed. Cells were labeled with [35S]methionine or [35S]sulfate at different times during development and allowed to differentiate in the presence of unlabeled methionine or sulfate for various periods of time. The cells were homogenized and intracellular organelles were separated using Percoll density gradient centrifugation. The distribution of [35S]methionine-labeled alpha-mannosidase and [35S]sulfate-labeled glycoproteins in the Percoll gradients was determined. It was found that prespore vesicles contained protein which was previously found in lysosomes. Newly labeled protein also entered these vesicles. The data suggest that developing Dictyostelium cells either restructure preexisting lysosomes into prespore vesicles or transport protein between these two organelles. We propose that secretory granules and lysosomes may have a common biosynthetic origin and may be evolutionarily related.


Assuntos
Grânulos Citoplasmáticos/enzimologia , Dictyostelium/enzimologia , Manosidases/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Dictyostelium/crescimento & desenvolvimento , Dictyostelium/ultraestrutura , Glicoproteínas/biossíntese , Lisossomos/enzimologia , Lisossomos/ultraestrutura , Manosidases/biossíntese , Microscopia Eletrônica , Peso Molecular , alfa-Manosidase
2.
Diabetes ; 46(3): 444-50, 1997 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-9032101

RESUMO

Ras associated with diabetes (Rad), a new ras-related GTPase, was recently identified by subtractive cloning as an mRNA in skeletal muscle that is overexpressed in NIDDM. To better understand its metabolic significance, we measured skeletal muscle Rad expression in well-characterized insulin sensitive (IS) and insulin resistant (IR) subjects with normal glucose tolerance and in untreated NIDDM patients. We found no differences in expression of Rad mRNA levels among IS, IR, and NIDDM groups using a ribonuclease protection assay (0.22 +/- 0.06, 0.13 +/- 0.01, and 0.16 +/- 0.02 relative units, respectively; NS) and no differences in Rad protein expression using a specific anti-peptide Rad antibody (1.05 +/- 0.18, 1.14 +/- 0.08, and 1.08 +/- 0.21 units/mg protein, respectively; NS). However, Rad protein levels were positively correlated with BMI (r = 0.43, P = 0.03) and percentage body fat (r = 0.55, P < 0.005), two independent measures of obesity, and negatively correlated with resting metabolic rate (r = 0.49, P = 0.01). In multiple regression analyses, percentage body fat and resting metabolic rate independently accounted for 30 and 10% of individual variability in muscle Rad protein expression. In conclusion, Rad expression in skeletal muscle is not altered as a function of insulin resistance or NIDDM in humans. However, these data, for the first time, implicate a role for Rad in regulating body composition and energy expenditure and provide a framework for studies designed to elucidate Rad's cellular functions.


Assuntos
Composição Corporal , Diabetes Mellitus Tipo 2/metabolismo , Metabolismo Energético , GTP Fosfo-Hidrolases/biossíntese , Proteínas de Ligação ao GTP/biossíntese , Resistência à Insulina , Insulina/farmacologia , Músculo Esquelético/metabolismo , Proteínas ras , Absorciometria de Fóton , Tecido Adiposo/anatomia & histologia , Adulto , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Índice de Massa Corporal , Regulação da Temperatura Corporal , Feminino , GTP Fosfo-Hidrolases/metabolismo , Teste de Tolerância a Glucose , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/biossíntese , Análise de Regressão , Transcrição Gênica
3.
Diabetes ; 48(7): 1415-24, 1999 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10389847

RESUMO

The discovery that peroxisome proliferator-activated receptor (PPAR)-gamma was the molecular target of the thiazolidinedione class of antidiabetic agents suggested a key role for PPAR-gamma in the regulation of carbohydrate and lipid metabolism. Through the use of high-throughput biochemical assays, GW1929, a novel N-aryl tyrosine activator of human PPAR-gamma, was identified. Chronic oral administration of GW1929 or troglitazone to Zucker diabetic fatty (ZDF) rats resulted in dose-dependent decreases in daily glucose, free fatty acid, and triglyceride exposure compared with pretreatment values, as well as significant decreases in glycosylated hemoglobin. Whole body insulin sensitivity, as determined by the euglycemic-hyperinsulinemic clamp technique, was significantly increased in treated animals. Comparison of the magnitude of glucose lowering as a function of serum drug concentrations showed that GW1929 was 2 orders of magnitude more potent than troglitazone in vivo. These data were consistent with the relative in vitro potencies of GW1929 and troglitazone. Isolated perfused pancreas studies performed at the end of the study confirmed that pancreata from vehicle-treated rats showed no increase in insulin secretion in response to a step change in glucose from 3 to 10 mmol/l. In contrast, pancreata from animals treated with GW1929 showed a first- and second-phase insulin secretion pattern. Consistent with the functional data from the perfusion experiments, animals treated with the PPAR-gamma agonist had more normal islet architecture with preserved insulin staining compared with vehicle-treated ZDF rats. This is the first demonstration of in vivo efficacy of a novel nonthiazolidinedione identified as a high-affinity ligand for human PPAR-gamma. The increased potency of GW1929 compared with troglitazone both in vitro and in vivo may translate into improved clinical efficacy when used as monotherapy in type 2 diabetic patients. In addition, the significant improvement in daily meal tolerance may impact cardiovascular risk factor management in these patients.


Assuntos
Benzofenonas/farmacologia , Diabetes Mellitus Experimental/fisiopatologia , Obesidade/fisiopatologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Tiazolidinedionas , Fatores de Transcrição/metabolismo , Tirosina/análogos & derivados , Animais , Cromanos/uso terapêutico , Células Clonais , Diabetes Mellitus Experimental/genética , Técnica Clamp de Glucose , Humanos , Hipoglicemiantes/uso terapêutico , Imuno-Histoquímica , Modelos Logísticos , Obesidade/genética , Fenótipo , Ratos , Ratos Zucker , Receptores Citoplasmáticos e Nucleares/agonistas , Tiazóis/uso terapêutico , Fatores de Transcrição/agonistas , Troglitazona , Tirosina/farmacologia
4.
Arterioscler Thromb Vasc Biol ; 20(12): 2625-9, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11116063

RESUMO

Hyperlipidemia may complicate the use of HIV protease inhibitors (PIs) in AIDS therapy. To determine the cause of hyperlipidemia, the effect of PIs on lipid metabolism was examined with HepG2 liver cells and AKR/J mice. In HepG2 cells, the PIs ABT-378, nelfinavir, ritonavir, and saquinavir stimulated triglyceride synthesis; ritonavir increased cholesterol synthesis; and amprenavir and indinavir had no effect. Moreover, nelfinavir increased mRNA expression of diacylglycerol acyltransferase and fatty acid synthase. The retinoid X receptor agonist LG100268, but not the antagonist LG100754, further increased PI-stimulated triglyceride synthesis and mRNA expression of fatty acid synthase in vitro. In fed mice, nelfinavir or ritonavir did not affect serum glucose and cholesterol, whereas triglyceride and fatty acids increased 57% to 108%. In fasted mice, ritonavir increased serum glucose by 29%, cholesterol by 40%, and triglyceride by 99%, whereas nelfinavir had no effect, suggesting these PIs have different effects on metabolism. Consistent with the in vitro results, nelfinavir and ritonavir increased triglyceride 2- to 3-fold in fasted mice injected with Triton WR-1339, an inhibitor of triglyceride clearance. We propose that PI-associated hyperlipidemia is due to increased hepatic triglyceride synthesis and suggest that retinoids or meal restriction influences the effects of select PIs on lipid metabolism.


Assuntos
Inibidores da Protease de HIV/farmacologia , Hiperlipidemias/induzido quimicamente , Fígado/efeitos dos fármacos , Triglicerídeos/biossíntese , Aciltransferases/biossíntese , Animais , Glicemia/análise , Radioisótopos de Carbono , Colesterol/biossíntese , Colesterol/sangue , Diacilglicerol O-Aciltransferase , Jejum , Ácido Graxo Sintases/biossíntese , Ácidos Graxos/sangue , Inibidores da Protease de HIV/toxicidade , Hiperlipidemias/sangue , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos AKR , Nelfinavir/farmacologia , Ácidos Nicotínicos/farmacologia , Ácidos Nicotínicos/toxicidade , Período Pós-Prandial , RNA Mensageiro/biossíntese , Receptores do Ácido Retinoico/agonistas , Receptores X de Retinoides , Ritonavir/farmacologia , Saquinavir/farmacologia , Tetra-Hidronaftalenos/farmacologia , Tetra-Hidronaftalenos/toxicidade , Fatores de Transcrição/agonistas , Triglicerídeos/sangue , Células Tumorais Cultivadas
5.
J Med Chem ; 41(25): 5037-54, 1998 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-9836621

RESUMO

We previously reported the identification of (2S)-((2-benzoylphenyl)amino)-3-¿4-[2-(5-methyl-2-phenyloxazol-4-y l)e thoxy]phenyl¿propanoic acid (2) (PPARgamma pKi = 8.94, PPARgamma pEC50 = 9.47) as a potent and selective PPARgamma agonist. We now report the expanded structure-activity relationship around the phenyl alkyl ether moiety by pursuing both a classical medicinal chemistry approach and a solid-phase chemistry approach for analogue synthesis. The solution-phase strategy focused on evaluating the effects of oxazole and phenyl ring replacements of the 2-(5-methyl-2-phenyloxazol-4-yl)ethyl side chain of 2 with several replacements providing potent and selective PPARgamma agonists with improved aqueous solubility. Specifically, replacement of the phenyl ring of the phenyloxazole moiety with a 4-pyridyl group to give 2(S)-((2-benzoylphenyl)amino)-3-¿4-[2-(5-methyl-2-pyridin-4-yloxazol+ ++- 4-yl)ethoxy]phenyl¿propionic acid (16) (PPARgamma pKi = 8.85, PPARgamma pEC50 = 8.74) or a 4-methylpiperazine to give 2(S)-((2-benzoylphenyl)amino)-3-(4-¿2-[5-methyl-2-(4-methylpiperazin+ ++- 1-yl)thiazol-4-yl]ethoxy¿phenyl)propionic acid (24) (PPARgamma pKi = 8.66, PPARgamma pEC50 = 8.89) provided two potent and selective PPARgamma agonists with increased solubility in pH 7.4 phosphate buffer and simulated gastric fluid as compared to 2. The second strategy took advantage of the speed and ease of parallel solid-phase analogue synthesis to generate a more diverse set of phenyl alkyl ethers which led to the identification of a number of novel, high-affinity PPARgamma ligands (PPARgamma pKi's 6.98-8.03). The combined structure-activity data derived from the two strategies provide valuable insight on the requirements for PPARgamma binding, functional activity, selectivity, and aqueous solubility.


Assuntos
Proteínas de Ligação a DNA/agonistas , Hipoglicemiantes/síntese química , Hipolipemiantes/síntese química , Oxazóis/síntese química , Propionatos/síntese química , Receptores Citoplasmáticos e Nucleares/agonistas , Tiazóis/síntese química , Fatores de Transcrição/agonistas , Tirosina/análogos & derivados , Tirosina/síntese química , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Hipolipemiantes/química , Hipolipemiantes/farmacologia , Ligantes , Lipídeos/biossíntese , Camundongos , Oxazóis/química , Oxazóis/farmacologia , Propionatos/química , Propionatos/farmacologia , Ensaio Radioligante , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Recombinantes de Fusão/agonistas , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade , Relação Estrutura-Atividade , Tiazóis/química , Tiazóis/farmacologia , Fatores de Transcrição/metabolismo , Transfecção , Tirosina/química , Tirosina/farmacologia
6.
J Med Chem ; 41(25): 5055-69, 1998 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-9836622

RESUMO

3-¿4-[2-(Benzoxazol-2-ylmethylamino)ethoxy]phenyl¿-(2S)-((2- benzoylph enyl)amino)propionic acid (1) and (2S)-((2-benzoylphenyl)amino)-3-¿4-[2-(5-methyl-2-phenyloxazol-4-y l)e thoxy]phenyl¿propionic acid (2) are peroxisome proliferator-activated receptor gamma (PPARgamma) agonists and have antidiabetic activity in rodent models of type 2 diabetes. As part of an effort to develop the SAR of the N-2-benzoylphenyl moiety of 1 and 2, a series of novel carboxylic acid analogues, 23-66, modified only in the N-2-benzoylphenyl moiety were synthesized from L-tyrosine and evaluated as PPARgamma agonists. In general, only modest changes in the N-2-benzoylphenyl moiety of 1 and 2 are tolerated. More specifically, the best changes involve bioisosteric replacement of one of the two phenyl rings of this moiety. Addition of substituents to this moiety generally produced compounds that are less active in the cell-based functional assays of PPARgamma activity although binding affinity to PPARgamma may be maintained. A particularly promising set of analogues is the anthranilic acid esters 63-66 in which the phenyl ring in the 2-benzoyl group of 1 and 2 has been replaced by an alkoxy group. In particular, (S)-2-(1-carboxy-2-¿4-[2-(5-methyl-2-phenyloxazol-4-yl)ethoxy]phen yl¿ ethylamino)benzoic acid methyl ester (63) has a pKi of 8.43 in the binding assay using human PPARgamma ligand binding domain and a pEC50 of 9.21 in the in vitro murine lipogenesis functional assay of PPARgamma activity. Finally, 63 was found to normalize glycemia when dosed at 3 mg/kg bid po in the Zucker diabetic fatty rat model of type 2 diabetes.


Assuntos
Benzoatos/síntese química , Proteínas de Ligação a DNA/agonistas , Hipoglicemiantes/síntese química , Hipolipemiantes/síntese química , Oxazóis/síntese química , Receptores Citoplasmáticos e Nucleares/agonistas , Fatores de Transcrição/agonistas , Tirosina/análogos & derivados , Tirosina/síntese química , Administração Oral , Animais , Benzoatos/química , Benzoatos/farmacologia , Glicemia/metabolismo , Linhagem Celular , Diabetes Mellitus Experimental/sangue , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Hipolipemiantes/química , Hipolipemiantes/farmacologia , Ligantes , Lipídeos/biossíntese , Masculino , Camundongos , Oxazóis/química , Oxazóis/farmacologia , Ensaio Radioligante , Ratos , Receptores Citoplasmáticos e Nucleares/metabolismo , Solubilidade , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismo , Tirosina/química , Tirosina/farmacologia , ortoaminobenzoatos
7.
J Med Chem ; 41(25): 5020-36, 1998 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-9836620

RESUMO

We have identified a novel series of antidiabetic N-(2-benzoylphenyl)-L-tyrosine derivatives which are potent, selective PPARgamma agonists. Through the use of in vitro PPARgamma binding and functional assays (2S)-3-(4-(benzyloxy)phenyl)-2-((1-methyl-3-oxo-3-phenylpropenyl)+ ++amin o)propionic acid (2) was identified as a structurally novel PPARgamma agonist. Structure-activity relationships identified the 2-aminobenzophenone moiety as a suitable isostere for the chemically labile enaminone moiety in compound 2, affording 2-((2-benzoylphenyl)amino)-3-(4-(benzyloxy)phenyl)propionic acid (9). Replacement of the benzyl group in 9 with substituents known to confer in vivo potency in the thiazolidinedione (TZD) class of antidiabetic agents provided a dramatic increase in the in vitro functional potency and affinity at PPARgamma, affording a series of potent and selective PPARgamma agonists exemplified by (2S)-((2-benzoylphenyl)amino)-3-¿4-[2-(methylpyridin-2-ylamino+ ++)ethoxy ]phenyl¿propionic acid (18), 3-¿4-[2-(benzoxazol-2-ylmethylamino)ethoxy]phenyl¿-(2S)-((2- benzoylph enyl)amino)propanoic acid (19), and (2S)-((2-benzoylphenyl)amino)-3-¿4-[2-(5-methyl-2-phenyloxazol-4-y l)e thoxy]phenyl¿propanoic acid (20). Compounds 18 and 20 show potent antihyperglycemic and antihyperlipidemic activity when given orally in two rodent models of type 2 diabetes. In addition, these analogues are readily prepared in chiral nonracemic fashion from L-tyrosine and do not show a propensity to undergo racemization in vitro. The increased potency of these PPARgamma agonists relative to troglitazone may translate into superior clinical efficacy for the treatment of type 2 diabetes.


Assuntos
Aminopiridinas/síntese química , Proteínas de Ligação a DNA/agonistas , Hipoglicemiantes/síntese química , Hipolipemiantes/síntese química , Oxazóis/síntese química , Propionatos/síntese química , Receptores Citoplasmáticos e Nucleares/agonistas , Fatores de Transcrição/agonistas , Tirosina/análogos & derivados , Tirosina/síntese química , Administração Oral , Aminopiridinas/química , Aminopiridinas/farmacologia , Animais , Glicemia/metabolismo , Linhagem Celular , Diabetes Mellitus Experimental/sangue , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Hipolipemiantes/química , Hipolipemiantes/farmacologia , Ligantes , Lipídeos/biossíntese , Masculino , Camundongos , Oxazóis/química , Oxazóis/farmacologia , Propionatos/química , Propionatos/farmacologia , Ensaio Radioligante , Ratos , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Recombinantes de Fusão/agonistas , Proteínas Recombinantes de Fusão/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismo , Transfecção , Tirosina/química , Tirosina/farmacologia
8.
Biochem Pharmacol ; 59(9): 1063-8, 2000 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-10704935

RESUMO

HIV protease inhibitors (PIs) are effective drugs for the treatment of AIDS. However, PI therapy is sometimes associated with side-effects including increased plasma lipids and altered body fat distribution, although fat redistribution may occur in some patients not treated with PIs. Overdosage with vitamin A(1) acid (all-trans-retinoic acid, ATRA) or its metabolites may cause similar changes in lipid metabolism. Moreover, the PI indinavir and retinoids have been associated with nail, skin, and hair defects, suggesting that indinavir and retinoids may exert their effects through similar molecular mechanisms. This hypothesis was tested by examining the effects of PIs on retinoid signaling in vitro. Mesenchymal stem cells (C3H10T1/2) were cultured in the presence of various PIs (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) and synthetic retinoids, and the metabolic response was assessed by measuring the activity of a retinoid-regulated protein, alkaline phosphatase (ALP). Of the PIs tested, only indinavir stimulated ATRA-dependent ALP activity and altered stem cell morphology; the effects of indinavir occurred in the presence of ATRA, but not in its absence. Moreover, indinavir increased the effects of ATRA on lipid accumulation during fat cell differentiation. AGN 193109 (4-[[5,6-dihydro-5, 5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl]ethynyl]-benzoic acid), a retinoic acid receptor (RAR) antagonist, inhibited the synergistic effects of indinavir and ATRA, indicating that indinavir increases RAR signaling. However, indinavir did not potentiate ALP activity in the presence of the RAR agonist CH55 (3,5-di-tert-butylchalcone 4'-carboxylic acid). Unlike ATRA, CH55 does not bind to cytosolic retinoic acid binding protein (CRABP), suggesting that CRABP may regulate the effects of indinavir on RAR signaling. These observations support the proposal that altered retinoid signaling promotes some of the adverse reactions associated with indinavir therapy, such as altered lipid metabolism.


Assuntos
Inibidores da Protease de HIV/farmacologia , Indinavir/farmacologia , Transdução de Sinais/efeitos dos fármacos , Vitamina A/metabolismo , Animais , Antineoplásicos/farmacologia , Células Cultivadas , Inibidores da Protease de HIV/efeitos adversos , Indinavir/efeitos adversos , Camundongos , Tretinoína/farmacologia
9.
Biochem Pharmacol ; 54(7): 801-8, 1997 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-9353134

RESUMO

Troglitazone and metformin are antidiabetic agents that belong to the thiazolidinedione and biguanide classes of drugs, respectively. To evaluate how these drugs influence fuel utilization, we compared their effects on several pathways regulating carbohydrate and lipid metabolism in vitro. Both drugs stimulated glucose transport and utilization in C3H10T1/2 cells, a cell line capable of differentiating into adipocytes when treated with thiazolidinediones. However, we observed that these drugs had a number of different in vitro effects. Unlike metformin, troglitazone stimulated beta3-adrenergic receptor-mediated lipolysis, lipogenesis, and transcriptional activity of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma). Further, by using a mitochondrial-specific fluorescent dye, we found troglitazone to be more effective than metformin at increasing mitochondrial mass. In contrast to troglitazone, metformin was more effective at increasing mitochondrial fatty acid beta-oxidation, peroxisomal fatty acid beta-oxidation, and anaerobic respiration (i.e. lactate production). Additionally, metformin stimulated and troglitazone inhibited both aerobic respiration and basal lipolysis. Insulin enhanced the effects of troglitazone, but not those of metformin, on these cells. Taken together, the data show that troglitazone and metformin affect two distinct metabolic pathways: one that is anabolic (i.e. troglitazone) and the other that is catabolic (i.e. metformin). Further, these observations suggest that the metabolic activity of mitochondria may be lower in cells treated with troglitazone than with metformin.


Assuntos
Cromanos/farmacologia , Glucose/metabolismo , Hipoglicemiantes/farmacologia , Metabolismo dos Lipídeos , Metformina/farmacologia , Tiazóis/farmacologia , Tiazolidinedionas , Animais , Transporte Biológico/efeitos dos fármacos , Ácido Láctico/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Mitocôndrias/efeitos dos fármacos , Palmitoil Coenzima A/metabolismo , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Fatores de Transcrição/efeitos dos fármacos , Troglitazona
10.
Antiviral Res ; 51(3): 151-77, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11448728

RESUMO

Mortality rates in the HIV-infected patient population have decreased with the advent of highly active antiretroviral therapy (HAART) for the treatment of AIDS. Due to the chronic nature of HAART, long-term metabolic complications are associated with therapy, such as hyperlipidemia, fat redistribution and diabetes mellitus. Currently, all of these symptoms are classified as the lipodystrophy (LD) syndrome(s). However, hyperlipidemia and fat redistribution occur independently, indicating there may be multiple syndromes associated with HAART. Although fat gain/loss and dyslipidemia occur in protease inhibitor (PI) naïve patients treated with nucleoside reverse transcriptase inhibitors (NRTIs), combination therapies (PI and NRTI) accelerate the syndrome. Recent clinical trials, cell culture and animal studies indicate that these effects are not drug class specific and select PIs, NRTIs and non-nucleoside reverse transcriptase inhibitors (NNRTIs) can be associated with metabolic complications. Moreover, the effects can vary between various members of the same class of antiretroviral agents (i.e. not all PIs cause the same adverse reactions) and may be influenced by duration of infection, genetics and environmental factors. Although HAART increases the risk of metabolic complications, this does not outweigh the benefits of survival. In this review, we summarize the latest clinical and scientific information on these metabolic complications, examine current hypotheses explaining the syndromes and comment on the existing methods available to manage these metabolic side effects.


Assuntos
Terapia Antirretroviral de Alta Atividade/efeitos adversos , Infecções por HIV/tratamento farmacológico , Lipodistrofia/induzido quimicamente , Diabetes Mellitus/induzido quimicamente , Glucose/metabolismo , Inibidores da Protease de HIV/efeitos adversos , Humanos , Hiperlipidemias/induzido quimicamente , Resistência à Insulina , Inibidores da Transcriptase Reversa/efeitos adversos , Fatores de Risco , Síndrome
11.
Antiviral Res ; 47(2): 121-9, 2000 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10996400

RESUMO

AIDS therapies employing HIV protease inhibitors (PIs) are associated with changes in fat metabolism. However, the cellular mechanisms affected by PIs are not clear. Thus, the affects of PIs on adipocyte differentiation were examined in vitro using C3H10T1/2 stem cells. In these cells the PIs, nelfinavir, saquinavir, and ritonavir, reduced triglyceride accumulation, lipogenesis, and expression of the adipose markers, aP2 and LPL. Histological analysis revealed nelfinavir, saquinavir and ritonavir treatment decreased oil red O-staining of cytoplasmic fat droplets. Inhibition occurred in the presence of the RXR agonist LGD1069, indicating the inhibitory effects were not due to an absence of RXR ligand. Moreover, these three PIs increased acute lipolysis in adipocytes. In contrast, two HIV PIs, amprenavir and indinavir, had little effect on lipolysis, lipogenesis, or expression of aP2 and LPL. Although, saquinavir, inhibited ligand-binding to PPARgamma with an IC(50) of 12.7+/-3.2 microM, none of the other PIs bound to the nuclear receptors RXRalpha or PPARgamma, (IC(50)s>20 microM), suggesting that inhibition of adipogenesis is not due to antagonism of ligand binding to RXRalpha or PPARgamma. Taken together, the results suggest that some, but not all, PIs block adipogenesis and stimulate fat catabolism in vitro and this may contribute to the effects of PIs on metabolism in the clinic.


Assuntos
Adipócitos/metabolismo , Inibidores da Protease de HIV/farmacologia , Lipólise/efeitos dos fármacos , Proteínas de Neoplasias , Tiazolidinedionas , Triglicerídeos/metabolismo , Adipócitos/citologia , Animais , Compostos Azo/farmacologia , Bexaroteno , Carbamatos , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Corantes/farmacologia , Proteínas de Ligação a Ácido Graxo , Furanos , Indinavir/farmacologia , Insulina/farmacologia , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Nelfinavir/farmacologia , RNA Mensageiro/análise , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Ácido Retinoico/agonistas , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides , Ritonavir/farmacologia , Rosiglitazona , Saquinavir/farmacologia , Células-Tronco , Sulfonamidas/farmacologia , Tetra-Hidronaftalenos/farmacologia , Tiazóis/farmacologia , Fatores de Transcrição/agonistas , Fatores de Transcrição/metabolismo
15.
Recept Channels ; 7(4): 249-58, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11697231

RESUMO

Type 2 diabetes is associated with insulin resistance in peripheral tissues, such as muscle and fat. Novel therapies that improve insulin action include ligands that bind and activate the nuclear receptors peroxisome proliferator activating receptor gamma (PPAR gamma) and retinoid X receptor (RXR). PPAR gamma/RXR form heterodimers that regulate transcription of genes involved in insulin action, adipocyte differentiation, lipid metabolism and inflammation. PPAR gamma activators include prostanoids, fatty acids, thiazolidinediones and N-(2-benzoylphenyl)tyrosine analogues. RXR ligands include naturally occurring retinoic acid and synthetic rexinoids. Selective ligands for these receptors improve metabolic abnormalities associated with type 2 diabetes, such as hyperglycemia, hyperlipidemia, insulin resistance and other cardiovascular risk factors. Although adipose tissue mediates some of the effects of PPAR gamma/RXR ligands, other tissues also regulate the effects of these receptors. The activity of the PPAR gamma/RXR heterodimer is influenced by posttranslational modifications, receptor turnover, polymorphisms, splice variants, coactivators and corepressors. This article reviews recent developments in research on these receptors, with particular emphasis on metabolic effects, ligand selectivity, structure and regulation of the PPAR gamma/RXR heterodimer.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Ácido Retinoico/metabolismo , Fatores de Transcrição/metabolismo , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 2/terapia , Dimerização , Humanos , Insulina , Ligantes , Conformação Proteica , Receptores Citoplasmáticos e Nucleares/química , Receptores do Ácido Retinoico/química , Proteínas Repressoras/metabolismo , Receptores X de Retinoides , Transativadores/metabolismo , Fatores de Transcrição/química , Transcrição Gênica
16.
Cell Tissue Res ; 290(1): 79-87, 1997 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9377645

RESUMO

Although there are a number of cell lines committed to differentiate into brown adipocytes, the stem-cell origin of brown fat remains unclear. To address this problem, we explored the effects of various pharmacological agents on differentiation of C3H10T1/2 cells, a pluripotent stem-cell line of mesodermal origin. Histochemical and biochemical analysis revealed that, when these cells were treated with retinoic acid, they expressed the osteoblastic marker alkaline phosphatase. Upon addition of thiazolidinediones and insulin, these cells accumulated lipid and expressed the adipocyte marker aP2, indicating differentiation into adipocytes. Treatment during the growth phase with thiazolidinediones resulted in maximal lipogenesis indicating a need for clonal expansion for efficient adipogenic differentiation. Further analysis revealed that addition of thiazolidinediones to the cells increased (1) the lipolytic response of the cells to beta3-agonists, (2) the expression of uncoupling protein (UCP), (3) the expression of mRNA for type II iodothyronine 5'-deiodinase (5'D-II), and (4) mitochondrial staining. These results suggest the anti-diabetic effects of thiazolidinediones may, in part, involve increased brown adipocyte differentiation. Moreover, this is the first direct evidence indicating that brown adipocytes and osteoblasts may arise from the same stem cell.


Assuntos
Fosfatase Alcalina/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Iodeto Peroxidase/metabolismo , Proteínas de Membrana/metabolismo , Células-Tronco/enzimologia , Tiazóis/farmacologia , Adipócitos/citologia , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/enzimologia , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Biomarcadores , Proteínas de Transporte/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Fibroblastos/química , Fibroblastos/citologia , Fibroblastos/enzimologia , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Canais Iônicos , Ceratolíticos/farmacologia , Proteínas de Membrana/genética , Mesoderma/citologia , Camundongos , Camundongos Endogâmicos C3H , Mitocôndrias/química , Mitocôndrias/enzimologia , Proteínas Mitocondriais , Osteoblastos/citologia , Fenótipo , RNA Mensageiro/análise , Receptores Adrenérgicos beta/biossíntese , Coloração e Rotulagem , Células-Tronco/química , Células-Tronco/citologia , Tretinoína/farmacologia , Proteína Desacopladora 1
17.
Arch Biochem Biophys ; 312(2): 474-9, 1994 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-8037460

RESUMO

An in vitro assay that measures endosome fusion was used to characterize the role of guanosine triphosphate (GTP)-binding proteins in endocytosis. Guanosine 5',3-(thio)triphosphate (GTP gamma S), a nonhydrolyzable analog of GTP, stimulates the binding of cytosolic factors to the endosomal membrane (priming). GTP gamma S also enhances vesicle aggregation, resulting in the formation of an intermediate that is resistant to dilution. In this report we demonstrate that priming precedes the appearance of a dilution-resistant intermediate. Thus, GTP-binding proteins are involved in multiple sequential events preceding endosome fusion. Both heterotrimeric G proteins (G proteins) and ADP-ribosylation factors (ARFs) are GTP-binding proteins that regulate undefined steps involved in endocytosis. The addition of G beta gamma subunits of G proteins to the in vitro fusion assay resulted in inhibition of priming. In contrast, addition of ARF to the assay enhanced priming. Thus, heterotrimeric G proteins and ARF may regulate endocytosis by mediating the binding of cytosolic factor(s) required for fusion to the endosomal membrane. Taken together, the results show that multiple GTP-binding proteins regulate a series of distinct biochemical events required for endosome fusion.


Assuntos
Endocitose/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Membranas Intracelulares/metabolismo , Fusão de Membrana/fisiologia , Fatores de Ribosilação do ADP , Animais , Transporte Biológico , Linhagem Celular , Endocitose/efeitos dos fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Fusão de Membrana/efeitos dos fármacos , Camundongos , Fragmentos de Peptídeos/farmacologia
18.
Biochem Biophys Res Commun ; 174(1): 197-203, 1991 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-1899190

RESUMO

Biochemical and morphological techniques were used to investigate the GTP-binding proteins of clathrin-coated vesicles. Binding of [3H]GTP to clathrin coats was demonstrated by electron microscopic autoradiography. Purified coated vesicles bound 5.2 pmol [35S]GTP gamma S/mg protein. Addition of GTP or GTP gamma S, but not ATP nor GMP, inhibited binding of [35S]GTP gamma S to intact coated vesicles and Triton X-100-extracted coats. A series of 23-24 kDa GTP-binding proteins with isoelectric points between pH 5-8 were detected in coated vesicles. We suggest that the low molecular weight ras-like GTP-binding protein(s) play a role in regulating vesicle-mediated protein transport or signal transduction within intracellular organelles.


Assuntos
Clatrina/química , Proteínas de Ligação ao GTP/química , Organelas/metabolismo , Placenta/ultraestrutura , Autorradiografia , Membrana Celular/ultraestrutura , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Humanos , Ponto Isoelétrico , Peso Molecular , Octoxinol , Polietilenoglicóis , Transdução de Sinais
19.
J Biol Chem ; 267(3): 1896-903, 1992 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-1730725

RESUMO

An in vitro endosome fusion assay using Dictyostelium discoideum is described. The method requires endocytosis of anti-dinitrophenol (DNP) IgG or DNP-derivitized beta-glucuronidase into two sets of cells. After homogenizing the cells, the vesicles were mixed, and fusion was measured by quantitating immune complex formation between DNP-beta-glucuronidase and anti-DNP IgG. Fusion was dependent upon ATP, temperature, pH, ionic strength, and cytosol and sensitive to detergent, dilution, trypsin, N-ethylmaleimide, and guanosine 5'-3-O-(thio)triphosphate. Although weak bases, ionophores, hadacidin, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, and caffeine inhibit endocytosis in vivo, these reagents had no affect on in vitro endosome fusion. Comparison of Dictyostelium with mammalian cells showed differences in the temperature, pH, and salt requirements for fusion, possibly reflecting differences in the life-styles of various cell types. Like mammalian cells, Dictyostelium required GTP-binding protein(s) and an N-ethylmaleimide-sensitive factor for endosome fusion. Thus, the mechanism driving endosome fusion may have been conserved throughout evolution. Electron microscopic studies confirmed in vitro endosome fusion and revealed endosomes were being engulfed by other endosomes, resulting in formation of multivesicular elements (i.e. autophagic vesicles). This system may be useful for characterizing mutations, evolution, and developmental regulation along the endocytic pathway.


Assuntos
Dictyostelium/fisiologia , Endocitose , Fusão de Membrana , Organelas/fisiologia , Trifosfato de Adenosina/farmacologia , Animais , Fracionamento Celular , Sistema Livre de Células , Centrifugação com Gradiente de Concentração , Detergentes/farmacologia , Dictyostelium/ultraestrutura , Glucuronidase/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Cinética , Fusão de Membrana/efeitos dos fármacos , Microscopia Eletrônica , Octoxinol , Organelas/efeitos dos fármacos , Organelas/ultraestrutura , Polietilenoglicóis/farmacologia , Cloreto de Potássio/farmacologia
20.
J Biol Chem ; 267(18): 13047-52, 1992 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-1618802

RESUMO

We have used an in vitro endosome fusion assay, recombinant ARF, synthetic peptides, and guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) to study the role of ARF during endocytosis. Previous work has shown that GTP gamma S stimulates in vitro endosome fusion in dilute cytosol (less than 0.5 mg/ml) but inhibits fusion in concentrated cytosol (greater than 1.0 mg/ml). Two peptides corresponding to the NH2-terminal 16 amino acids of human ARF1 and ARF4 blocked GTP gamma S stimulation of fusion in dilute cytosol and reversed GTP gamma S inhibition of fusion in concentrated cytosol. The addition of recombinant human ARF1 to endosomes in dilute or concentrated cytosol resulted in GTP gamma S-dependent inhibition of fusion. Only the myristoylated form of ARF inhibited fusion. The NH2-terminal ARF1 peptide reversed inhibition by recombinant ARF1. Preincubation experiments showed that endosomes could form an ARF-resistant intermediate during the fusion process. Western blot analysis revealed clathrin-coated vesicles extracted with detergent retained ARF. The results suggest that ARF is involved in both the stimulatory and inhibitory effects of GTP gamma S in dilute and concentrated cytosol, respectively. Furthermore, myristoylation, the NH2-terminal domain, and binding to GTP appear to be critical for ARF activity during an early prefusion step required for endocytosis.


Assuntos
Proteínas de Ligação ao GTP/fisiologia , Membranas Intracelulares/metabolismo , Organelas/metabolismo , Fator 1 de Ribosilação do ADP , Fatores de Ribosilação do ADP , Sequência de Aminoácidos , Animais , Transporte Biológico , Linhagem Celular , Invaginações Revestidas da Membrana Celular/metabolismo , Proteínas de Ligação ao GTP/química , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Cinética , Fusão de Membrana , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes/metabolismo
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