Alphonse Le Cam

Characterization of a natural inhibitor of the insulin receptor tyrosine kinase: cDNA cloning, purification, and anti-mitogenic activity

Amino acid sequence of the precursor of the phosphorylated N-glycoprotein (pp63) secreted by rat hepatocytes was deduced from the cDNA sequence. This polypeptide (Mr = 40,586) was rich in both cysteine and proline and contained three potential N-glycosylation sites. A single pp63 mRNA species (approximately 2000 bp), found in normal hepatocytes but not in FaO hepatoma cells, appeared to result from transcription of a single gene. pp63 purified by affinity chromatography inhibited insulin receptor tyrosine kinase and receptor autophosphorylation. Only the phosphorylated form of the protein was active. In additon, pp63 antagonized the growth-promoting action of insulin in FaO cells but did not affect hormone-mediated increase in amino acid transport capacity or tyrosine aminotransferase induction in these cells.

Glucagon stimulates the A system for neutral amino acid transport in isolated hepatocytes of adult rat

Summary The effect of glucagon on neutral amino acid transport systems was studied in freshly-prepared suspensions of isolated hepatocytes by measuring basal and hormone-stimulated α-aminoisobutyric acid (AIB) and cycloleucine uptake. Glucagon stimulated AIB uptake against a concentration gradient. Half-maximal stimulation occured with 0.4 nM glucagon. Glucagon stimulation was restricted to the A mediation of transport. The effect was rapid, occured without time lag, was abolished by cycloheximide and mimicked by dibutyryl cyclic AMP.

Effect of glucocorticoids on amino acid transport in isolated rat hepatocytes

The effects of glucocoiticoids on neutral amino acid transport were studied in freshly prepared suspensions of isolated hepatocytes from adult rat. Glucocorticoids stimulated the active transport of α-aminoisobutyric acid by increasing the influx. The onset of effect was preceded by a lag period of about 1 h. Cortisol (ED50 ~2 μM) was 10–20 times less potent than synthetic analogs. Glucocorticoids increased the Vmax of transport without affecting the Km. Actinomycin D and cycloheximide virtually abolished this effect. The glucocorticoid effect involved predominantly the A system of transport. The effect was additive with that of glucagon, insulin and catecholamines.

Primary structure of the rat gene encoding an inhibitor of the insulin receptor tyrosine kinase.

The gene (PP63) encoding the inhibitor (PP63) of the insulin receptor tyrosine kinase was isolated from a rat genomic library. The intron/exon organization was deduced from Southern-blot analysis and sequence data (i.e., the exons + the boundaries). The PP63 gene, which maps to chromosome 11, spans approx. 8 kb and contains seven exons separated by six introns of different sizes. All of the boundaries match the consensus GT/AG sequence for donor and acceptor splice sites. Primer extension and S1 mapping experiments were used to locate the transcription start point (tsp) 73 nt upstream from the translational initiator. Both in vitro transcription assays and transcription of a chimeric gene in intact hepatoma cells indicated that the sequence located immediately upstream from the tsp contained a promoter. Several putative cis-regulatory elements, including a TATA box and a C/EBP-binding site were found within the 250 bp preceding the tsp.

Insulin enhances protein phosphorylation in isolated hepatocytes by inhibiting an amiloride sensitive phosphatase

Abstract The effects of amiloride on basal and hormone-stimulated protein phosphorylation were studied in isolated rat hepatocytes labeled with 32P-phosphate. Two types of effect on basal phosphorylation were detected: 1. an increase in labeling of two proteins with molecular weights 93,000 and 18,000; 2. a decrease in labeling of proteins with molecular weights 46,000, 34,000, 22,000 and 13,000. All these effects were dose-dependent (maximum with 0,8-to 1 mM) and reached a maximum after 30 to 40 min treatment of the cells with the drug. Amiloride inhibited specifically all insulin effects whereas glucagon specific effects were largely unaffected. In pulse-chase experiments, amiloride increased and insulin decreased the rate of dephosphorylation of the same proteins (Mr 46,000, 34,000 and 22,000). The data support the conclusion that in hepatocytes insulin increases the degree of phosphorylation of proteins by inhibiting an amiloride-sensitive phosphatase.

Insulin and Insulin-like Growth Factor I: Effects on Protein Synthesis in Isolated Muscles from Lean and Goldthioglucose-Obese Mice

The effects of insulin and insulin-like growth factor I (IGF-I) on protein synthesis were compared in muscle isolated from lean and goldthioglucose (GTG)-obese mice. Two types of skeletal muscles, the red soleus and the white extensor digitorum longus (EDL) muscles, were studied. In muscles from lean mice, 6.7 nM insulin and 50 nM IGF-I caused a similar maximal stimulationof tyrosine incorporation in total proteins (40% increase). However, the potency of IGF-I was only 5–10% that of insulin both in soleus and in EDL muscles (EC50 ≅ 6 nM for IGF-Iand 0.5 nM for insulin). Basal rate of protein synthesis was identical in muscles from GTG-obese and lean mice. Similarly, a comparable increase in the rate of protein synthesis was obtained using maximally effective concentrations of insulin and IGF-I in both lean and GTG-obese animals. SDS-polyacrylamide gel electrophoresis analysis of proteins labeled with 35S-methionine confirmed that, in muscles from lean and GTG-obese animals, insulin and IGF-I increased overall protein synthesis in a similar manner. These results suggest that the protein synthesis machinery is not impaired in GTG-induced obesity, which is therefore not associated with resistance to insulin for its effect on protein metabolism.

Regulation of protein phosphorylation by polyamines in hepatocytes

The effects of natural aliphatic polyamines on basal and hormone-stimulated protein phosphorylations in hepatocytes were studied. Cells isolated from adult rats were incubated in suspension with [32P]orthophosphate, in the absence or presence of polyamines at varying concentrations and for different times; hepatocytes were then exposed to various hormones for 10 min. Phosphoproteins contained in total cell lysates were analyzed by one- and two-dimensional gel electrophoresis and autoradiography. Spermine, the most effective amine, decreased the basal level of phosphorylation of proteins with 46, 34 and 22 kDa, and increased that of a 18 kDa protein. These effects, maximal with an external concentration of 7.5-10 mM, were detectable after a lag period of about 10 min and reached a plateau after 45 min. Pretreatment of cells with the polyamine almost completely prevented stimulation of the phosphorylation of the 46 and 34 kDa proteins by insulin; in contrast, the effects of phenylephrine on the same proteins were only partly inhibited, whereas those of glucagon appeared largely unaffected. The major polyamine effect observed in intact cells (i.e., decreased phosphorylation) could be reproduced in a cell-free system where no kinase activity persisted. Indeed, spermine added directly to cell extracts strongly accelerated dephosphorylation of the 46 kDa protein and also of the 61 kDa protein identified as pyruvate kinase; furthermore, restoration of the activity of this enzyme occurred concomitantly with dephosphorylation of the 61 kDa protein in the presence of spermine.

Electroporation-Mediated Gene Transfer into Hepatocytes

The study of gene expression regulation relies on the introduction of foreign DNA into eukaryotic cells. A wide variety of DNA-transfer procedures have been developed that utilize retroviruses (1), polycations (2), liposomes (3), chromosomes (4), reconstituted viral envelopes (5) and other chemical reagents, such as calcium phosphate (6), DEAE-dextran (7), and lipopolyamines (8). DNA also can be transferred into cells by physical means, such as microinjection (9), laser beams (10), and electroporation (11). However, none of these methods work with high efficiency on every cell type, whether freshly isolated cells or established cell lines. Some of these techniques, such as microinjection and lasermediated transfection, require sophisticated apparatus and are technically difficult, whereas others, such as protoplast fusion, retroviral vectors, and liposome fusion, require time-consuming biochemical manipulations. Electroporation, which circumvents most of these problems, has emerged as an effective tool for the transfection of eukaryotic cells in suspensions (for a review, see ref. 12). We are interested in genes specifically expressed in normal parenchymal liver cells (i.e., hepatocytes) and controlled by growth hormone (GH), glucocorticoids (GC), and cytokines. Because none of the hepaticderived cell lines express their endogenous GH receptor, we chose the hepatocyte cell system to carry out gene-expression studies. Because

Comparative analysis of proteins labelled with [35S]methionine in the liver in vivo and in freshly isolated and short-term-cultured hepatocytes in vitro

[35S]Methionine-labelled liver proteins, analysed by one- or two-dimensional gel electrophoresis showed a strikingly similar pattern whether synthesized in vivo or by freshly isolated hepatocytes. In contrast, major qualitative and quantitative differences were observed with the patterns of labelled proteins found in cultured hepatocytes. The changes detectable very early (within 1 h) in culture affected preferentially the synthesis of cytoskeleton proteins (cytokeratins, actin, myosin), which was dramatically increased. Physical factors like cell attachment appear to be responsible for these changes which, however, occurred more rapidly in the presence of serum. Freshly isolated hepatocytes and short-term-cultured cells responded similarly to insulin and glucagon, which respectively increased and decreased the labelling of the whole set of cellular and exported proteins. Glucocorticoids caused either an increase or a decrease in the labelling of several proteins, but the effects were detectable only under chronic exposure of cultured hepatocytes. Based on these results, freshly isolated hepatocytes appear more representative of the liver in vivo than cultured hepatocytes, and therefore seem more suitable for short-term studies. However, cultured hepatocytes can be used for long-term studies since they maintain many specific liver functions and remain hormonally sensitive.

Synthesis of the growth hormone-regulated rat liver anti-protease GHR-P63 is inhibited by acute inflammation

Synthesis of the growth hormone‐regulated anti‐protease GHR‐P63 by rat hepatocytes was strongly reduced during acute inflammation. This decrease was detected 8 h after the onset of inflammation and reached a maximum after 24 h. A decrease in the GHR‐P63 mRNA level measured by in vitro translation and by hybridization mainly accounted for the alteration of GHR‐P63 synthesis. Besides this major pretranslational mechanism, inflammation also interfered with GHR‐P63 synthesis at a posttranslational level. This was indicated by the production of abnormal immunoprecipitable species at early stages of the acute‐phase response.Synthesis of the growth hormone-regulated anti-protease GHR-P63 by rat hepatocytes was strongly reduced during acute inflammation. This decrease was detected 8 h after the onset of inflammation and reached a maximum after 24 h. A decrease in the GHR-P63 mRNA level measured by in vitro translation and by hybridization mainly accounted for the alteration of GHR-P63 synthesis. Besides this major pretranslational mechanism, inflammation also interfered with GHR-P63 synthesis at a posttranslational level. This was indicated by the production of abnormal immunoprecipitable species at early stages of the acute-phase response.