Susceptibility to apoptosis in insulin-like growth factor-I receptor-deficient brown adipocytes

Fetal brown adipocytes are insulin-like growth factor-I (IGF-I) target cells. To assess the importance of the IGF-I receptor (IGF-IR) in brown adipocytes during fetal life, we have generated immortalized brown adipocyte cell lines from the IGF-IR(-/-) mice. Using this experimental model, we demonstrate that the lack of IGF-IR in fetal brown adipocytes increased the susceptibility to apoptosis induced by serum withdrawal. Culture of cells in the absence of serum and growth factors produced rapid DNA fragmentation (4 h) in IGF-IR(-/-) brown adipocytes, compared with the wild type (16 h). Consequently, cell viability was decreased more rapidly in fetal brown adipocytes in the absence of IGF-IR. Furthermore, caspase-3 activity was induced much earlier in cells lacking IGF-IR. At the molecular level, IGF-IR deficiency in fetal brown adipocytes altered the balance of the expression of several proapoptotic (Bcl-xS and Bim) and antiapoptotic (Bcl-2 and Bcl-xL) members of the Bcl-2 family. This imbalance was irreversible even though in IGF-IR-reconstituted cells. Likewise, cytosolic cytochrome c levels increased rapidly in IGF-IR-deficient cells compared with the wild type. A rapid entry of Foxo1 into the nucleus accompanied by a rapid exit from the cytosol and an earlier activation of caspase-8 were observed in brown adipocytes lacking IGF-IR upon serum deprivation. Activation of caspase-8 was inhibited by 50% in both cell types by neutralizing anti-Fas-ligand antibody. Adenoviral infection of wild-type brown adipocytes with constitutively active Foxol (ADA) increased the expression of antiapoptotic genes, decreased Bcl-xL and induced caspase-8 and -3 activities, with the final outcome of DNA fragmentation. Up-regulation of uncoupling protein-1 (UCP-1) expression in IGF-IR-deficient cells by transduction with PGC-1alpha or UCP-1 ameliorated caspase-3 activation, thereby retarding apoptosis. Finally, insulin treatment prevented apoptosis in both cell types. However, the survival effect of insulin on IGF-IR(-/-) brown adipocytes was elicited even in the absence of phosphatidylinositol 3-kinase/Akt signaling. Thus, our results demonstrate for the first time the unique role of IGF-IR in maintaining the balance of death and survival in fetal brown adipocytes.     

Cellular and humoral responses to collagen-polyvinylpyrrolidone administered during short and long periods in humans

Collagen, particularly type I, and its related derivatives have been extensively employed in many areas of pharmacology. The present study was performed to determine the safety of collagen-polyvinylpyrrolidone (collagen-PVP) by in vitro and in vivo studies. Sera and peripheral blood cells from healthy donors without treatment and patients treated with collagen-PVP were evaluated. We observed that the biodrug does not stimulate lymphoproliferation or DNA damage in vitro, nor does it induce human anti-porcine type I collagen or anti-collagen-PVP antibodies in vivo. Furthermore, no hepatic or renal metabolic dysfunctions were observed when collagen-PVP was administered by intradermal or intramuscular routes in short- or long-term treatments. In conclusion, the present work shows that no cellular damage or immunological adverse effects (cellular and humoral) occurred during collagen-PVP treatment, even after more than 400 weeks of consecutive administrations.     

Maxi K+ channel mediates regulatory volume decrease response in a human bronchial epithelial cell line

The cell regulatory volume decrease (RVD) response triggered by hypotonic solutions is mainly achieved by the coordinated activity of Cl- and K+ channels. We now describe the molecular nature of the K(+) channels involved in the RVD response of the human bronchial epithelial (HBE) cell line 16HBE14o-. These cells, under isotonic conditions, present a K+ current consistent with the activity of maxi K+ channels, confirmed by RT-PCR and Western blot. Single-channel and whole cell maxi K+ currents were readily and reversibly activated following the exposure of HBE cells to a 28% hypotonic solution. Both maxi K+ current activation and RVD response showed calcium dependency, inhibition by TEA, Ba2+, iberiotoxin, and the cationic channel blocker Gd3+ but were insensitive to clofilium, clotrimazole, and apamin. The presence of the recently cloned swelling-activated, Gd3+-sensitive cation channels (TRPV4, also known as OTRPC4, TRP12, or VR-OAC) was detected by RT-PCR in HBE cells. This channel, TRPV4, which senses changes in volume, might provide the pathway for Ca2+ influx under hypotonic solutions and, consequently, for the activation of maxi K+ channels.     

Association of insulin receptor substrate 1 (IRS-1) y895 with Grb-2 mediates the insulin signaling involved in IRS-1-deficient brown adipocyte mitogenesis

We have recently generated immortalized fetal brown adipocyte cell lines from insulin receptor substrate 1 (IRS-1) knockout mice and demonstrated an impairment in insulin-induced lipid synthesis as compared to wild-type cell lines. In this study, we investigated the consequences of IRS-1 deficiency on mitogenesis in response to insulin. The lack of IRS-1 resulted in the inability of insulin-stimulated IRS-1-deficient brown adipocytes to increase DNA synthesis and enter into S/G2/M phases of the cell cycle. These cells showed a severe impairment in activating mitogen-activated protein kinase kinase (MEK1/2) and p42-p44 mitogen-activated protein kinase (MAPK) upon insulin stimulation. IRS-1-deficient cells also lacked tyrosine phosphorylation of SHC and showed no SHC-Grb-2 association in response to insulin. The mitogenic response to insulin could be partially restored by enhancing IRS-2 tyrosine phosphorylation and its association with Grb-2 by inhibition of phosphatidylinositol 3-kinase activity through a feedback mechanism. Reconstitution of IRS-1-deficient brown adipocytes with wild-type IRS-1 restored insulin-induced IRS-1 and SHC tyrosine phosphorylation and IRS-1-Grb-2, IRS-1-SHC, and SHC-Grb-2 associations, leading to the activation of MAPK and enhancement of DNA synthesis. Reconstitution of IRS-1-deficient brown adipocytes with the IRS-1 mutant Tyr895Phe, which lacks IRS-1-Grb-2 binding, restored SHC-IRS-1 association and SHC-Grb-2 association. However, the lack of IRS-1-Grb-2 association impaired MAPK activation and DNA synthesis in insulin-stimulated mutant cells. These data provide strong evidence for an essential role of IRS-1 and its direct association with Grb-2 in the insulin signaling pathway leading to MAPK activation and mitogenesis in brown adipocytes.     

Influence of heat treatment of casein in presence of reducing sugars on Zn solubility and Zn uptake by Caco-2 cells after in vitro digestion

The effect of the heat treatment of casein in presence of reducing sugars on some aspects of Zn availability was investigated. Samples were prepared by mixing casein with glucose-fructose, and were used unprocessed (C) or heated (HC). Changes in Zn speciation after the in vitro digestion of the samples, both as part of a diet and in isolation, were studied. The uptake of soluble Zn from the digested samples was investigated in Caco-2 cells. After in vitro digestion, the percentage of precipitated Zn was significantly higher with the HC sample, both when digested alone and as a part of the diet. In assays with Caco-2 cells, a significant decrease in Zn uptake was observed when the uptake buffer contained the sample C digest, by comparison with the control buffer, without casein digest. When the digested heated mixture was added, Zn uptake by the cells was significantly lower than in either of the two other cases. It may be concluded that the heat treatment of casein in the presence of glucose-fructose has a negative effect on Zn availability because, after in vitro digestion, Zn insolubilization was enhanced and Zn uptake by the enterocyte was impaired, compared with the unheated mixture. In addition, the usefulness of Caco-2 cells in this kind of research has been shown.     

Kinetic characterization of outer-ring deiodinase activity (ORD) in the liver, gill and retina of the killifish Fundulus heteroclitus

Conversion of T4 to T3 is the first step in TH action and deiodinases are the major determinants of TH tissue availability and disposal. We here report the kinetic characterization of the outer-ring deiodinating (ORD) enzymes in the liver, gill and retina of sea water-adapted killifish, by using both rT3 and T4 as substrates. In liver, by using rT3, we detected a high Km (84 nM) and a low Km (1.3 nM) component with kinetic characteristics similar to mammalian deiodinases DI and DII. In contrast, T4-ORD only generated a low Km (0.5 nM) component. As judged by its Vmax (920 fmol 125I/mg per h) this DII enzyme is very abundant, approximately five and 20 times higher than that found in trout liver and hypothyroid rat, respectively. Kinetic analysis in killifish gill showed only one enzymatic component, with a high rT3 Km (430 nM) and a relatively low Vmax (4.3 pmol 125I/mg per h). Our results in killifish retina show the expression of a T4-low Km (0.6 nM) deiodinase with high cofactor requirements akin to the mammalian DII. The Vmax value for this enzyme is 182 fmol 125I/mg per h, five times lower than the one found in killifish liver, but comparable to that in hypothyroid rat pituitary. The biochemical similarities between fish and mammalian deiodinases could reflect their high conservation during vertebrate evolution and thus their importance in the regulation of thyroid hormone action.     

Chamaejasmin,a biflavanone from wood of Diphysa robinioides

The biflavanone chamaejasmin has been isolated from the wood of Diphysa robinioides and its structure established by the spectral data of the biflavanone and its hexaacetyl derivative.