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We demonstrate here the catalytic activity and subcellular localization of the Nm23-H4 protein, product of nm23-H4, a new member of the human nm23/nucleoside diphosphate (NDP) kinase gene family (Milon, L., Rousseau-Merck, M., Munier, A., Erent, M., Lascu, I., Capeau, J., and Lacombe, M. L. (1997) Hum. Genet. 99, 550-557). Nm3-H4 was synthesized in escherichia coli as the full-length protein and as a truncated form missing the N-terminal extension characteristic of mitochondrial targeting. The truncated form possesses NDP kinase activity, whereas the full-length protein is inactive, suggesting that the extension prevents enzyme folding and/or activity. X-ray crystallographic analysis was performed on active truncated Nm23-H4. Like other eukaryotic NDP kinases, it is a hexamer. Nm23-H4 naturally possesses a serine residue at position 129, equivalent to the K-pn mutation of the Drosophila NDP kinase. The x-ray structure shows that the presence of Ser(129) has local structural effects that weaken subunit interactions. Site-directed mutagenesis shows that the serine is responsible for the lability of Nm23-H4 to heat and urea treatment, because the S129P mutant is greatly stabilized. Examination of human embryonic kidney 293 cells transfected with green fluorescent protein fusions by confocal microscopy shows a specific mitochondrial localization of Nm23-H4 that was also demonstrated by Western blot analysis of subcellular fractions of these cells. Import into mitochondria is accompanied by cleavage of the N-terminal extension that results in NDP kinase activity. Submitochondrial fractionation indicates that Nm23-H4 is associated with mitochondrial membranes, possibly to the contact sites between the outer and inner membranes.

We recently showed that the antiapoptotic function of insulin requires nuclear factor kappaB (NF-kappaB) activation (Bertrand, F., Atfi, A., Cadoret, A., L'Allemain, G., Robin, H., Lascols, O., Capeau, J., and Cherqui, G. (1998) J. Biol. Chem. 273, 2931-2938). Here we sought to identify the NF-kappaB-dependent survival genes that are activated by insulin to mediate this function. Insulin increased the expression of tumor necrosis factor receptor-associated factor 2 (TRAF2) mRNA and protein in Chinese hamster ovary cells overexpressing insulin receptors (IRs). This effect required (i) IR activation since it was abrogated by IR mutation at tyrosines 1162 and 1163 and (ii) NF-kappaB activation since it was abolished by overexpression of dominant-negative IkappaB-alpha(A32/36) and mimicked by overexpression of the NF-kappaB c-Rel subunit. TRAF2 contributed to insulin protection against serum withdrawal-induced apoptosis since TRAF2 overexpression mimicked insulin protection, whereas overexpression of dominant-negative TRAF2-(87-501) reduced this process. Along with its protective effect, overexpressed TRAF2 increased basal and insulin-stimulated NF-kappaB activities. All effects were inhibited by IkappaB-alpha(A32/36), suggesting that an amplification loop involving TRAF2 activation of NF-kappaB is implicated in insulin antiapoptotic signaling. We also show that insulin increased manganese-superoxide dismutase (Mn-SOD) mRNA expression through NF-kappaB activation and that Mn-SOD contributed to insulin antiapoptotic signaling since expression of antisense Mn-SOD RNA decreased this process. This study provides the first evidence that insulin activates the NF-kappaB-dependent survival genes encoding TRAF2 and Mn-SOD and thereby clarifies the role of NF-kappaB in the antiapoptotic function of insulin.

In comparison to skin fibroblasts from normal subjects, those from patients with cystic fibrosis (CF): (1) bound [20-3H] phorbol 12,13-dibutyrate (PDBu) with a higher affinity (Kd=25.8 vs 12.8 nM respectively) but expressed a similar number of total phorbol ester binding sites (about 2.5 pmol PDBu bound/mg of protein); (2) exhibited a faster and higher response to 4?-phorbol 12?-myristate 13?-acetate (PMA) for the stimulation of [35S]-labelled glycoconjutate release, but were equally sensitive to the synergistic effect of A23187 on this process; and (3) secreted glycoconjugates with similar [35S]-sulfate and [14C]-leucine to [14C]-glucosamine labelling ratios. Taken together, these results provide further evidence for abnormal protein kinase C (PKC) regulation of macromolecule secretion in CF disease.

Lectins specific for D-mannose (concanavalin A), N-acetyl-D-glucosamine (wheat-germ agglutinin) or D-galactose (Ricinus communis agglutinin I) inhibited insulin binding and activated glucose transport in rat adipocytes [Cherqui, Caron, Capeau & Picard (1982) Mol. Cell. Endocrinol. 28, 627-643]. In the present investigation, the intracellular activities of insulin and lectins on lipogenesis and protein synthesis were studied under conditions where neither agent had an effect on membrane transport processes. (1) When glucose transport was rate-limiting (0.5 mM-glucose), insulin (0.8 ng/ml) and lectins (20 micrograms/ml) increased lipogenesis by 2.4-3-fold. (2) When passive diffusion of glucose was amplified (10 mM-glucose), insulin (0.8 ng/ml) and lectins (20 micrograms/ml) increased lipogenesis by 1.6-1.8-fold even in the presence of 50 microM-cytochalasin B, which completely blocked glucose transport. (3) Insulin (6 ng/ml), concanavalin A and wheat-germ agglutinin (40 micrograms/ml) stimulated the incorporation of L-[U-14C]leucine into fat-cell protein 1.5-fold but did not modify alpha-aminoisobutyric acid uptake or 14C-labelled protein degradation. (4) Peanut and soya-bean agglutinins (specific for O-glycosidically-linked oligosaccharides), known not to alter insulin binding, were ineffective. (5) Lectin effects were dose-dependent and were markedly inhibited by specific monosaccharides (50 mM). (6) Insulin and lectin maximal effects were not additive and were completely abolished by neuraminidase treatment of fat-cells (0.05 unit/ml). These data indicate involvement of surface sialylated glycoproteins of the complex N-linked type in the insulin stimulation of glucose and amino acid intracellular metabolic processes. They suggest, together with our previous results, that the transmission of the insulin signal for both membrane and intracellular effects occurs via glycosylated effector entities of, or closely linked to, the insulin-receptor complex.

The general aim is to prepare a bioartificial liver to treat acute hepatic failure using allo- and xenogeneic hepatocytes, immunoprotected by macroencapsulation and transplanted into the peritoneal cavity. The goal of this study was to prepare a large amount of isolated porcine hepatocytes, to encapsulate them within biocompatible membranes for transplant in allo- and xenogeneic combinations and to examine the viability and functionality of the cells 6 weeks later. Hepatocyte isolation was performed in 12 kg pigs (n= 15) by dissociation of the liver with collagenase D (1 g) without oxygenation. Encapsulation of the hepatocyte suspension (107/mL) was performed in hydrogel membranes AN69; hollow fibers (2 m x 0,8 mm) and flaskes (1,8 cm), and transplanted to Yucatan pigs (n = 4) and Lewis rats (n = 12). Six weeks later, they were removed to study the cell viability by histological examination, and the production of albumin by immunonephelometry.

We examined the effect of insulin on nuclear factor kappa B (NF-kappa B) activity in Chinese ovary (CHO) cells overexpressing wild-type (CHO-R cells) or -defective insulin receptors mutated at Tyr1162 and Tyr1163 autophosphorylation sites (CHO-Y2 cells). In CHO-R cells, insulin caused a specific, time-, and concentration-dependent activation of NF-kappa B. The insulin-induced DNA-binding complex was identified as the p50/p65 heterodimer. Insulin activation of NF-kappa B: 1) was related to insulin receptor number and tyrosine kinase activity since it was markedly reduced in parental CHO cells which proved to respond to insulin growth factor-1 and phorbol 12-myristate 13-acetate (PMA) activation, and was dramatically decreased in CHO-Y2 cells; 2) persisted in the presence of cycloheximide and was blocked by pyrrolidine dithiocarbamate, aspirin and sodium salicylate, three compounds interfering with I kappa B degradation and/or NF-kappa B.I kappa B complex dissociation; 3) was independent of both PMA-sensitive and atypical (zeta) protein kinases C; and 4) was dependent on Raf-1 kinase activity since insulin-stimulated NF-kappa B DNA binding activity was inhibited by 8-bromo-cAMP, a Raf-1 kinase inhibitor. Moreover, insulin activation of NF-kappa B-driven luciferase reporter gene expression was blocked in CHO-R cells expressing a Raf-1 dominant negative mutant. This is the first evidence that insulin activates NF-kappa B in mammalian cells through a post-translational mechanism requiring both insulin receptor tyrosine kinase and Raf-1 kinase activities.

We previously reported that insulin activates nuclear factor kappaB (NF-kappaB) in Chinese hamster ovary (CHO)-R cells overexpressing wild-type insulin receptors (IRs) through a pathway requiring IR tyrosine kinase and Raf-1 kinase activities. We now investigated whether the activation of NF-kappaB by insulin could serve an antiapoptotic function. Insulin (10(-9)-10(-7) M) inhibited apoptosis induced by serum withdrawal in CHO-R cells in a concentration-dependent manner. Insulin antiapoptotic signaling: (i) was dependent on IR number and IR tyrosine kinase activity since it was reduced in parental CHO cells and was abolished in CHO-Y2 cells overexpressing IRs mutated at Tyr1162/1163; (ii) was, like insulin activation of NF-kappaB, dependent on Raf-1 but not on mitogen-activated protein kinase activity since both processes were decreased by the dominant-negative Raf-1 mutant Raf-C4 whereas they persisted in mitogen-activated protein kinase-depleted cells; and (iii) required NF-kappaB activation since it was decreased by proteasome inhibitors and the dominant-negative IkappaB-alpha (A32/36) mutant and was mimicked by overexpression of the NF-kappaB c-Rel subunit. We also show that insulin antiapoptotic signaling but not insulin activation of NF-kappaB involved phosphatidylinositol 3-kinase (PI 3-kinase), as supported by the inhibition of the former but not of the latter process by the PI 3-kinase inhibitor LY294002. Inhibition of both NF-kappaB and PI 3-kinase totally abolished insulin antiapoptotic signaling. Thus insulin exerts a specific antiapoptotic function which is dependent on IR tyrosine kinase activity and is mediated by both a Raf-1-dependent pathway that leads to NF-kappaB activation and a PI 3-kinase-dependent pathway.

The hypothesis that insulin action involves a membrane proteolytic step was further explored, by using isolated rat adipocytes and liver plasma membranes. (1) The maximal insulin stimulation of 2-deoxyglucose transport and lipogenesis in fat-cells was selectively inhibited (73-88%) by N alpha-p-tosyl-L-lysine chloromethyl ketone (Tos-Lys-CH2Cl; active-site inhibitor of trypsin; 30-125 microM), p-nitrophenyl p'-guanidinobenzoate (active-site inhibitor of serine proteinases; 30-125 microM) and p-tosyl-L-arginine methyl ester (arginine ester substrate analogue of proteinases; 1-2 mM), under conditions where neither the basal rate of each metabolic process nor insulin binding nor cellular ATP content were affected. In contrast, N-acetyl-L-alanyl-L-alanyl-L-alanine methyl ester (alanine ester substrate analogue of proteinases; 1-2 mM) was ineffective. (2) Endoproteinase Arg-C (0.25-40 micrograms/ml) exerted dose-dependent insulin-like effects on both 2-deoxyglucose transport and lipogenesis in fat-cells, whereas endoproteinase Lys-C (5-100 micrograms/ml) was ineffective. The maximal activation by endoproteinase Arg-C of both processes (200 and 177% of control values respectively) was shown to occur under conditions where membrane integrity (assessed by measurement of lactate dehydrogenase leakage and passive glucose diffusion) was preserved. This effect was inhibited by Tos-Lys-CH2Cl (125 microM) and was not additive with the maximal insulin effect. (3) Insulin (1-100 ng/ml) produced a dose-dependent increase in the trichloroacetic acid-soluble 125I radioactivity released after a 30 min incubation at 37 degrees C of 125I-labelled liver plasma membranes, but was ineffective on 125I-labelled bovine serum albumin. Insulin effects on both radio-labelled proteins were reproduced by wheat-germ agglutinin (20 micrograms/ml), an insulin mimicker shown to act through the insulin receptor. These data provide further evidence for the hypothesis that insulin bioeffects involve the activation of a membrane serine proteinase with arginine specificity.

Background.: Hepatocyte transplantation could be an alternative to whole liver transplanlation. Allogeneic hepatocytes are rejected if transplanted without immunosuppression. The aim of this study was to transplant allogeneic hepatocyles in the peritoneum and to protect them from rejection by encapsulation in a new semipermeable membrane. Methods.: Rat hepatocytes were encapsulated in hydrogel-based hollow fibers, obtained from AN69 copolymer, before being transplated into the peritoneum of rats. Outcome of allogeneic hepatocytes encapsulated in hollow fibers was compared with that of syngeneic hepatocytes encapsulated in hollow fibers, with that of free allogeneic hepatocytes, and with allogeneic hepatocytes encapsulated in hollow fibers left open. Cell viability was assessed by erythrosin exclusion, structure by electron microscopy, and function by albumin release. Results.: Up to 90 days, viability of allogeneic hepatocytes in hollow fibers was greater than 80%. The structure remained normal at electron microscopy. Albumin release was 16.5+/-0.3 @mg/24 hr/10^6 hepatocytes (day 15), 14.2+/-2.0 @mg/24 hr/10^6 hepatocytes (day 30), 8.8+/-0.1 @mg/24 hepatocyles and hepatocytes in hollow fibers left open did not survive at day 15. Conclusions.: Viability and function of encapsulated allogeneic hepatocytes were maintained up to 90 days after transplantation, without immunosuppresion.