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.

Design of a leucine zipper coiled coil stabilized 1.4 kcal mol-1 by phosphorylation of a serine in the e position.

Using a dimeric bZIP protein, we have designed a leucine zipper that becomes more stable after a serine in the e position is phosphorylated by protein kinase A (delta delta GP = -1.4 kcal mol-1 dimer-1 or -0.7 kcal mol-1 residue-1). Mutagenesis studies indicate that three arginines form a network of inter-helical (i,i' + 5; i, i' + 2) and intra-helical (i, i + 4) attractive interactions with the phosphorylated serine. When the arginines are replaced with lysines, the stabilizing effect of serine phosphorylation is reduced (delta delta GP = -0.5 kcal mol-1 dimer-1). The hydrophobic interface of the leucine zipper needs a glycine in the d position to obtain an increase in stability after phosphorylation. The phosphorylated protein binds DNA with a 15-fold higher affinity. Using a transient transfection assay, we document a PKA dependent four-fold activation of a reporter gene. Phosphorylation of a threonine in the same e position decreases the stability by delta delta GP = +1.2 kcal mol-1 dimer-1. We present circular dichroism (CD) thermal denaturations of 15 bZIP proteins before and after phosphorylation. These data provide insights into the structural determinants that result in stabilization of a coiled coil by phosphorylation.

Cytoskeletal rearrangement and caspase activation in sphingosine 1-phosphate-induced lung capillary tube formation.

Angiogenesis is a multistep process involving the endothelial cell (EC) cytoskeleton in migration, proliferation, and barrier stabilization. Although precise intracellular pathways by which angiogenic tube formation occurs remain poorly understood, we speculated that interactions between the cytoskeleton and apoptosis are involved and explored cytoskeletal rearrangement and caspase activation in human lung microvascular EC capillary-like tube formation induced by sphingosine 1-phosphate (Sph 1-P) and vascular endothelial growth factor (VEGF). Sph 1-P and VEGF enhance tube formation quantified by a Tube Immaturity Index (TII) generated from the ratio of cell number to tube length, with concomitant morphologic and actomyosin network changes. Angiogenesis was temporally grouped into three stages with early changes characterized by cortical actin localization, whereas midstage tube development demonstrated elongated EC with peripheral actin labeling with transcellular stress fibers. Late tube formation was characterized by broad actin distribution and presence of caspase-positive EC. Phosphorylated MLC immunoreactivity was present at all stages, suggesting that coordinate Rho kinase and MLCK involvement is important to Sph 1-P-induced cell motility; however, chemical inhibition of either MLCK or Rho kinase failed to alter early tube formation. To address whether gaps created by apoptosis expand the lumen, Sph 1-P-induced tubes were differentiated in the presence of caspase inhibitor z-Val-Ala-Asp-fluoromethylketone (zVAD-FMK). Capillary-like tube maturation, but not length, was decreased by zVAD-FMK treatment. These studies suggest that Sph 1-P may induce EC tube formation by regulating early cytoskeletal rearrangement, whereas EC apoptosis within capillary-like tubes is necessary for late stage Sph 1-P-induced tube maturation and lumen formation.

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?

Opioid receptor labeling with the chloromethyl ketone derivative of [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol (DAMGO) II: Covalent labeling of mu opioid binding site by 3H-Tyr-D-Ala-Gly-(Me)Phe chloromethyl ketone.

Opioid receptors of rat brain membranes were prelabeled with 3H-Tyr-D-Ala2-(Phe4)-Gly-CH2Cl, a chloromethyl ketone derivative of enkephalin, and solubilized in 1% digitonin. Hydrodynamic parameters of the receptor detergent complex derived from gel filtration and sucrose density gradient ultracentrifugation were found to be 51 A and 8.7 S, respectively, and the size was estimated to be about 200 kDa. Sodium dodecyl sulfate gel electrophoresis followed by fluorography revealed specific alkylation of a major protein at 58 kDa.

Determination of lead, zinc, potassium, calcium, copper and sodium in human cataract lenses.

Concentrations of Pb, Zn, K, Ca, Cu and Na were determined in several human cataract and clear lenses, obtained from patients from two contrasting environmental regions in India, and the values were compared. When compared with the results obtained for samples of clear lenses, the mean concentration values of cataract lenses showed significant changes on the basis of the Mann-Whitney test. In cataract lenses, Pb and Zn showed an inverse correlation of r = -0.83; y = -1.64x + 457. Ca and K also showed an inverse correlation of r = -0.71; y = -0.34x + 3.6. No significant correlations were found between any other elements. The distribution of Pb, Zn, Ca and K were studied and their importance in ocular tissues is discussed. The molecules present in the eye lens lattice have undergone some conformational alterations due to intrusion of Pb and Ca ions and extrusion of Zn and K ions through the process of ion-exchange, thereby influencing the transparency of the lens.

Biological significance of [14C]phenol accumulation in different organs of a murrel, Channa punctatus, and the common carp, Cyprinus carpio.

Phenol, a ubiquitous component of industrial effluents, is a common pollutant of water resources and a serious threat to fish. The present work demonstrates that a significant amount of phenol is retained by various tissues of the common carp, Cyprinus carpio, and the snake-headed murrel, Channa punctatus. The rate of [14C]phenol accumulation was higher carp than in the murrel. It is suggested that retention of phenol in the brain and ovary may seriously affect the reproductive potential of the fish.

Thermodynamic parameters of frog brain kappa-opioid receptors.

The thermodynamic parameters for [3H]-ethylketocyclazocine binding in frog (Rana esculenta) brain membranes have been examined. Computer-based nonlinear regression analysis of the untransformed equilibrium displacement data showed that this ligand bound to two sites with different affinities and capacities in this tissue. KA values derived from equilibrium displacement curves have been used for calculating the changes in the standard Gibbs energy, enthalpy, and entropy during the binding process. Van't Hoff plots are bipartite, with transitions occurring at 18 degrees C for both the high- and the low-affinity sites. For the high-affinity site, the reaction appears to be associated with a decrease in enthalpy below the transition temperature and a significant gain in entropy above this temperature. The reverse appears to be true for the low-affinity site. We conclude that this profile fairly approximates the mixed agonist-antagonist nature of this ligand and surmise that thermodynamic analysis could be a very useful tool for characterization of the nature of cloned opioid receptors in vitro.

Phosphorylation destabilizes alpha-helices.

Phosphorylation of threonine destabilizes the leucine zipper of a bZIP protein by 4.6 kcal mol-1 dimer-1, which reduces DNA binding 100-fold. This decrease in stability reflects the low alpha-helix forming propensity of a phosphorylated threonine.

Leucine is the most stabilizing aliphatic amino acid in the d position of a dimeric leucine zipper coiled coil.

The energetic contribution of seven amino acids in the d position of a dimeric leucine zipper coiled coil structure was measured by determining the thermal stability. The d position contains the conserved leucines found in the leucine zipper. We used a natural bZIP protein as our host-guest system that remains dimeric when a single d position is mutated. We have determined the thermal stability, monitored by circular dichroism, of 14 proteins which indicate that alanine is 4.6 kcal mol-1 per residue less stabilizing than leucine. The similarly sized amino acid isoleucine is 2.9 kcal mol-1 per residue less stabilizing than leucine, suggesting that leucine is well-packed. Model building indicates that the beta-branched amino acids isoleucine and valine in the d position produced interhelical clashes between the Cgamma2 methyl groups when placed in the favored rotamer conformation. The stabilization by leucine in different d positions is context-dependent; it varies by over 2 kcal mol-1 in the two positions examined. The order of stabilization is L, M, I, V, C, A, and S. Cysteine in the d position can form a disulfide bond which stabilizes the coiled coil.

Identification of a nuclear factor-I family protein-binding site in the silencer region of the cartilage matrix protein gene.

Cartilage matrix protein (CMP) is synthesized by chondrocytes in a developmentally regulated manner. Here we have dissected promoter upstream elements involved in its transcriptional regulation. We show that although the 79-base pair CMP minimal promoter is promiscuous, 1137 base pairs of 5'-flanking region are capable of directing tissue- and developmental stage-specific transcription when fused to a reporter gene. This results from two positive control regions which, in proliferating chondrocytes, relieve the repression mediated by two non-tissue-specific negative control regions. Characterization of the promoter proximal silencer by DNase I footprinting and gel shifts revealed the presence of two elements, SI and SII, which bound mesenchymal cell proteins. Methylation interference analysis indicated a gapped palindromic binding site similar to nuclear factor I (NF-I) family proteins within SI, but only a half-site within SII. Gel shift assays with specific NF-I and mutated SI competitors, binding of recombinant NF-I, as well as supershift analysis with NF-I-specific antiserum verified the binding of NF-I family proteins to the SI element. Double-stranded SI and SII oligonucleotides inserted in single copy in either orientation were found to repress both homologous and heterologous promoters upon transfection into mesenchymal cells. Transcriptional repression also occurred when a consensus NF-I site itself was fused to the CMP minimal promoter. We conclude that NF-I-related protein(s) can mediate transcriptional repression in cells of mesenchymal origin.

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.