Adipose tissue is required for the antidiabetic, but not for the hypolipidemic, effect of thiazolidinediones.

There is uncertainty about the site(s) of action of the antidiabetic thiazolidinediones (TZDs). These drugs are agonist ligands of the transcription factor PPAR gamma, which is abundant in adipose tissue but is normally present at very low levels in liver and muscle. We have studied the effects of TZDs in A-ZIP/F-1 mice, which lack white adipose tissue. The A-ZIP/F-1 phenotype strikingly resembles that of humans with severe lipoatrophic diabetes, including the lack of fat, marked insulin resistance and hyperglycemia, hyperlipidemia, and fatty liver. Rosiglitazone or troglitazone treatment did not reduce glucose or insulin levels, suggesting that white adipose tissue is required for the antidiabetic effects of TZDs. However, TZD treatment was effective in lowering circulating triglycerides and increasing whole body fatty acid oxidation in the A-ZIP/F-1 mice, indicating that this effect occurs via targets other than white adipose tissue. A-ZIP/F-1 mice have markedly increased liver PPAR gamma mRNA levels, which may be a general property of fatty livers. Rosiglitazone treatment increased the triglyceride content of the steatotic livers of A-ZIP/F-1 and ob/ob mice, but not the "lean" livers of fat-transplanted A-ZIP/F-1 mice. In light of this evidence that rosiglitazone acts differently in steatotic livers, the effects of rosiglitazone, particularly on hepatic triglyceride levels, should be examined in humans with hepatic steatosis.

Torpor in mice is induced by both leptin-dependent and -independent mechanisms.

We tested the effect of chronic leptin treatment on fasting-induced torpor in leptin-deficient A-ZIP/F-1 and ob/ob mice. A-ZIP/F-1 mice have virtually no white adipose tissue and low leptin levels, whereas ob/ob mice have an abundance of fat but no leptin. These two models allowed us to examine the roles of adipose tissue and leptin in the regulation of entry into torpor. Torpor is a short-term hibernation-like state that allows conservation of metabolic fuels. We first characterized the A-ZIP/F-1 animals, which have a 10-fold reduction in total body triglyceride stores. Upon fasting, A-ZIP/F-1 mice develop a lower metabolic rate and decreased plasma glucose, insulin, and triglyceride levels, with no increase in free fatty acids or beta-hydroxybutyrate. Unlike control mice, by 24 hr of fasting, they have nearly exhausted their triglycerides and are catabolizing protein. To conserve energy supplies during fasting, A-ZIP/F-1 (but not control) mice entered deep torpor, with a minimum core body temperature of 24 degrees C, 2 degrees C above ambient. In ob/ob mice, fasting-induced torpor was completely reversed by leptin treatment. In contrast, neither leptin nor thyroid hormone prevented torpor in A-ZIP/F-1 mice. These data suggest that there are at least two signals for entry into torpor in mice, a low leptin level and another signal that is independent of leptin and thyroid hormone levels. Studying rodent torpor provides insight into human torpor-like states such as near drowning in cold water and induced hypothermia for surgery.

Identification of putative sites of interaction between the human formyl peptide receptor and G protein.

Wild-type and 35 mutant formyl peptide receptors (FPRs) were stably expressed in Chinese hamster ovary cells. All cell surface-expressed mutant receptors bound N-formyl peptide with similar affinities as wild-type FPR, suggesting that the mutations did not affect the ligand-binding site. G protein coupling was examined by quantitative analysis of N-formyl-methionyl-leucyl-phenylalanine-induced increase in binding of (35)S-labeled guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) to membranes. The most prominent uncoupled FPR mutants were located in the N-terminal part of the second transmembrane domain (S63W and D71A) and the C-terminal interface of the third transmembrane domain (R123A and C124S/C126S). In addition, less pronounced uncoupling was detected with deletion mutations in the third cytoplasmic loop and in the cytoplasmic tail. Further analysis of some of the mutants that were judged to be uncoupled based on the [(35)S]GTPgammaS membrane-binding assay were found to transduce a signal, as evidenced by intracellular calcium mobilization and activation of p42/44 MAPK. Thus, these single point mutations in FPR did not completely abolish the interaction with G protein, emphasizing that the coupling site is coordinated by several different regions of the receptor. Mutations located in the putative fifth and sixth transmembrane domains near the N- and C-terminal parts of the third cytoplasmic loop did not result in uncoupling. These regions have previously been shown to be critical for G protein coupling to many other G protein-coupled receptors. Thus, FPR appears to have a G protein-interacting site distinct from the adrenergic receptors, the muscarinic receptors, and the angiotensin receptors.

Transgenic mice lacking white fat: models for understanding human lipoatrophic diabetes.

The human disease lipoatrophic (or lipodystrophic) diabetes is a rare syndrome in which a deficiency of adipose tissue is associated with Type 2 diabetes. This disease is an interesting contrast to the usual situation in which diabetes is associated with obesity, an excess of fat. Aside from obesity, patients with lipodystrophic diabetes have the other features associated with Metabolic Syndrome X, including hypertension and dyslipidemia. The contrast between diabetes with a lack of fat and diabetes with an excess of fat provides an opportunity to study the mechanisms causing Type 2 diabetes and its complications. Recently, three laboratories have produced transgenic mice that are deficient in white adipose tissue. These mice have insulin resistance and other features of lipoatrophic diabetes, and are a faithful model for the human disease. Here we review the different murine models of fat ablation and compare the murine and human diseases, addressing the questions: Is the lack of fat causative of the diabetes, and if so by what mechanism? How could the other clinical features be explained mechanistically? And finally, what can be gleaned about insight into treatment options?

Life without white fat: a transgenic mouse.

We have generated a transgenic mouse with no white fat tissue throughout life. These mice express a dominant-negative protein, termed A-ZIP/F, under the control of the adipose-specific aP2 enhancer/promoter. This protein prevents the DNA binding of B-ZIP transcription factors of both the C/EBP and Jun families. The transgenic mice (named A-ZIP/F-1) have no white adipose tissue and dramatically reduced amounts of brown adipose tissue, which is inactive. They are initially growth delayed, but by week 12, surpass their littermates in weight. The mice eat, drink, and urinate copiously, have decreased fecundity, premature death, and frequently die after anesthesia. The physiological consequences of having no white fat tissue are profound. The liver is engorged with lipid, and the internal organs are enlarged. The mice are diabetic, with reduced leptin (20-fold) and elevated serum glucose (3-fold), insulin (50- to 400-fold), free fatty acids (2-fold), and triglycerides (3- to 5-fold). The A-ZIP/F-1 phenotype suggests a mouse model for the human disease lipoatrophic diabetes (Seip-Berardinelli syndrome), indicating that the lack of fat can cause diabetes. The myriad of consequences of having no fat throughout development can be addressed with this model.

Regulation of leptin promoter function by Sp1, C/EBP, and a novel factor.

Leptin is a hormone produced in adipose cells that regulates energy expenditure, food intake, and adiposity. To understand leptin's transcriptional regulation, we are studying its promoter. Four conserved and functional regions were identified. Mutations in the C/EBP and TATA motifs each caused an approximately 10-fold decrease in promoter activity. The C/EBP motif bound recombinant C/EBP alpha and mediated trans-activation by C/EBP alpha, -beta, and -delta. Mutation of a consensus Sp1 site reduced promoter activity 2.5-fold and abolished binding of Sp1. Mutation of a fourth factor-binding site, denoted LP1, abolished protein binding and reduced promoter activity 2-fold. Factor binding to the LP1 motif was observed with adipocyte, but not with nonadipocyte extracts. Adipocytes from fa/fa Zucker rats transcribed the reporter plasmids more efficiently than did control adipocytes. No effect on the transient expression of leptin was noted upon treatment with a thiazolidinedione, BRL49653, or upon cotransfection with peroxisome proliferator-activated receptor-gamma/retinoid X receptor-alpha or sterol response element-binding protein-1. Mutations of the Sp1, LP1, and C/EBP sites in pairwise combinations diminished promoter activity to the extent predicted assuming these motifs contribute independently to leptin promoter function. Our identification of motifs regulating leptin transcription is an important step in the elucidation of the mechanisms underlying hormonal and metabolic regulation of this gene.

Identification of a placental enhancer for the human leptin gene.

Leptin is a hormone that regulates metabolic efficiency, energy expenditure, and food intake. Leptin is produced chiefly in adipose cells, but in humans, mRNA encoding leptin is also present in the placenta. Here we elucidate the basis for placental leptin production. The same promoter is used for adipose and placental transcription. An upstream enhancer functions in the JEG-3 and JAR choriocarcinoma cell lines but not in adipocytes or HeLa cells. The minimal positive acting region is 60 base pairs in length. This region is within a MER11 repetitive element, suggesting that human placental expression of leptin is the result of insertion of this element. Binding analyses demonstrated three protein binding sites, designated placental leptin enhancer elements (PLE)1, PLE2, and PLE3. PLE2 binds Sp1. Enhancer activity was reduced by mutation of the PLE1 or PLE3 sites but was unaffected by alteration of PLE2. Proteins binding to PLE3 were present in JEG-3 and human placental nuclear extracts but not in extracts from non-placental sources. Upon triplication, the PLE3 element was a strong enhancer in choriocarcinoma cells but not in HeLa cells. The protein binding to the PLE3 motif appears to be a novel, placenta-specific transcription factor.

Identification of functional elements of the chicken epsilon-globin promoter involved in stage-specific interaction with the beta/epsilon enhancer.

Expression of the chicken globin genes is regulated in part by competition between the betaA-globin and epsilon-globin promoters for the enhancer found between the genes. To understand the determinants of the enhancer-promoter interaction in stage-specific regulation, the functional elements of the embryonic chicken epsilon-globin promoter were characterized. In vitro assays demonstrated that: (a) the TATA motif at -30 bound GATA-1, (b) Sp1 bound to an element centered at -54, and (c) both Sp1 and another factor, designated CACCC (which appears related to erythroid Krüppel-like factor, EKLF) bound in the -120 to -128 region. The functions of these motifs were tested using transient expression in embryonic erythroid cells. In the absence of the enhancer, promoter point mutants showed that the TATA, Sp1, and CCAAT motifs (but not the CACCC motif) contributed to promoter activity. In contrast, in the presence of the enhancer, all four motifs (including the CACCC motif) contributed to transcription. Developmental regulation of the enhancer activity was observed, with enhancement decreasing sharply from 185-fold at 4 days (cells expressing epsilon-globin) to 16-fold at 10 days (when epsilon-globin is no longer expressed). Taken together, the data suggest that multiple transcription factors contribute to promoter-enhancer interaction and the developmental regulation of epsilon-globin expression, with EKLF-like factors having an especially important role. Regulation of stage specificity occurs at the level of enhancer/epsilon-promoter interaction, even in the absence of competition, and is not simply a property of the enhancer or promoter in isolation.

Expression of the chicken beta-globin gene cluster in mice: correct developmental expression and distributed control.

To investigate the regulation of gene clusters, we introduced the entire chicken beta-globin cluster into mice. This 35-kb region includes the four globin genes (rho-beta H-beta A-epsilon), the four upstream hypersensitive sites, and the intergenic beta A/epsilon enhancer. The chicken globins are not arranged in order of developmental expression, which is unlike the case for the human beta-globin cluster, in which gene order plays a role in the regulation of globin expression. Mice carrying the chicken cluster expressed the transgenes with the same developmental patterns as seen in the chicken. Therefore, stage-specific erythroid transcriptional milieus existed before the divergence of birds and mammals and have been conserved since then. Mice bearing the complete cluster except for a deletion removing the beta A/epsilon enhancer displayed markedly reduced expression of the beta H, beta A, and epsilon genes with efficient (but variable) rho expression. Mice carrying the four genes and beta A/epsilon enhancer but without the upstream hypersensitive sites showed reduced expression of rho, beta H, and beta A, with variable expression of epsilon. We conclude that (i) all of the genes (except possibly rho) are under the control of both the upstream hypersensitive sites and the enhancer, (ii) the influence of the control elements can extend beyond the nearest active gene, (iii) a single element (the enhancer) can influence more than one gene in a single developmental stage, (iv) the enhancer can work bidirectionally, and (v) neither the upstream sites (as a group) nor the enhancer showed developmental stage specificity. Thus, the regulation of this cluster is achieved by interaction of two distinct control regions with each of the globin genes.

The neuromuscular effects of ORG9426 in patients receiving balanced anesthesia.

In searching for a nondepolarizing muscle relaxant with intermediate duration but more rapid onset of action than the presently available compounds, the neuromuscular and circulatory effects of ORG9426 were investigated in two studies in humans receiving fentanyl, droperidol, thiopental, and nitrous oxide-oxygen anesthesia. Eighty patients, randomly assigned to one of four groups of 20 each, received 0.12, 0.16, 0.20, or 0.24 mg/kg ORG9426. In the first study, the doses (in milligrams per kilogram) of ORG9426 that caused 50% (ED50), 90% (ED90), or 95% (ED95) neuromuscular block were determined by the individual dose-response method; they were 0.170, 0.268, and 0.305 mg/kg, respectively. In the second study, after induction of anesthesia, patients received 0.6 mg/kg (about 2 x ED95) of ORG9426, either in a single bolus (group 1) or in two unequal (0.1 and 0.5 mg/kg) increments 4 min apart (group 2). After the administration of 0.6 mg/kg ORG9426, maximal neuromuscular block developed in 1.5 +/- 0.12 min in group 1 and in 1.2 +/- 0.14 min in group 2. Patients tracheas were intubated after development of the maximal neuromuscular effect of the intubating dose and after the recording of heart rate and systolic and diastolic blood pressure. There was no difference in the clinical duration of the intubating doses, which were 40.0 +/- 3.2 (15-73) min in group 1 and 39.3 +/- 2.4 (19-57) min in group 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the inverted duplication in the mitochondrial DNA of Candida albicans.

The mitochondrial DNA (mtDNA) of Candida albicans contains a large inverted duplication. As is the case with most chloroplast DNAs and one other mtDNA, the nonduplicated regions of the molecule occur in two orientations with respect to each other, indicating that internal recombination occurs. Like some other mtDNAs, the C. albicans mtDNA contains a single SalI restriction site located near one end of the large rRNA gene. In contrast to other cases, however, the inverted duplication does not appear to contain any sequences coding for rRNA.

Molecular probe for identification of medically important Candida species and Torulopsis glabrata.

A cloned DNA fragment from Candida albicans containing the gene for the protein actin was used to probe the molecular structure of the actin gene of several medically important yeasts (C. albicans, Candida stellatoidea, Candida tropicalis, Candida pseudotropicalis, Candida krusei, Candida parapsilosis, Candida guilliermondii, and Torulopsis glabrata). Whole-cell DNA from each species was digested with restriction endonucleases, electrophoresed on agarose gels, and transferred to nitrocellulose. Radioactively labeled C. albicans actin gene was hybridized to the DNA fragments on the nitrocellulose. The C. albicans probe produced a strong signal with all of the Candida DNAs tested, indicating considerable conservation of this gene. In addition, the actin genes of all of the species tested were found to have no internal EcoRI or SalI restriction sites. With the exception of C. guilliermondii, all of the species tested had a single internal HindIII recognition site. However, the location of flanking restriction sites was found to be species specific. For all of the enzymes tested, the locations of the flanking restriction sites in C. albicans and C. stellatoidea were identical; all of the other strains yielded fragments clearly distinct from one another. These differences provide a molecular tool for the differentiation of medically important Candida species.

A new and superior adrenal scanning agent, NP-59.

The first synthesis of 131I-19-iodocholesterol had a 10-25% radiochemical impurity that was not iodide ion. This impurity has been identified as 6beta-131I-iodomethyl-19-nor cholest 5(10)-en-3beta-ol (NP-59) and has been synthesized. Tissue distribution studies with 131I-NP-59 in rats and dogs revealed a higher adrenal uptake and adrenal-to-tissue ratios compared to 131I-19-iodocholesterol, probably less in vivo deiodination, and superior adrenal images. A high uptake was seen in the adrenal medulla in addition to that in the cortex. Iodine-131-NP-59 is being evaluated for the early detection of adrenal-cortical disorders and as a potential scanning agent for detecting structural abnormalities of the adrenal medulla.

Toxicology of the prostaglandins.

PIP: Available published material of adverse reactions to prostaglandins (PGs) at various dosages routes and levels is reviewed. Animal studies are of 2 kinds: studies of pharmacological effects and studies of toxicological reactions (i.e., acute dose levels). The pharmacology of PGs show the drugs have 3 areas of action: 1) smooth muscle effects, either contraction or relaxation (cardiovascular effects and reproductive tract; relaxation of vascular smooth muscles by some PGs and contraction by others); 2) nervous system effects; and 3) lipid and carbohydrate metabolism. In humans, PGE1 was administered intravenously and it was found that adverse effects were related to rate of administration rather than to total dose; side effects included flushing, headache, visual disturbances, and restlessness, factors which might suggest a correlation with central nervous system effects (as found in animal studies). PGE1 administered at acute doses orally, by inhalation, and intradermally show effects attributable to gastrointestinal disturbances, respiratory problems, and erythematous responses. In general, studies in humans of other PGs have found similar adverse reactions, all of which are explained by known mechanisms of action of PGs. Dose levels constituting acutity are dependent on route (i.e., oral doses are much higher than intravenous doses), with rapid intravenous infusion causing the most adverse reactions.^ieng