Laparoscopic Liver Resection: A South American Experience with 2887 Cases.

BACKGROUND: Laparoscopic liver resections (LLR) have been increasingly performed in recent years. Most of the available evidence, however, comes from specialized centers in Asia, Europe and USA. Data from South America are limited and based on single-center experiences. To date, no multicenter studies evaluated the results of LLR in South America. The aim of this study was to evaluate the experience and results with LLR in South American centers. METHODS: From February to November 2019, a survey about LLR was conducted in 61 hepatobiliary centers in South America, composed by 20 questions concerning demographic characteristics, surgical data, and perioperative results. RESULTS: Fifty-one (83.6%) centers from seven different countries answered the survey. A total of 2887 LLR were performed, as follows: Argentina (928), Brazil (1326), Chile (322), Colombia (210), Paraguay (9), Peru (75), and Uruguay (8). The first program began in 1997; however, the majority (60.7%) started after 2010. The percentage of LLR over open resections was 28.4% (4.4-84%). Of the total, 76.5% were minor hepatectomies and 23.5% major, including 266 right hepatectomies and 343 left hepatectomies. The conversion rate was 9.7%, overall morbidity 13%, and mortality 0.7%. CONCLUSIONS: This is the largest study assessing the dissemination and results of LLR in South America. It showed an increasing number of centers performing LLR with the promising perioperative results, aligned with other worldwide excellence centers.

Altered gene expression in hippocampus and depressive-like behavior in young adult female mice by early protein malnutrition.

Perinatal development represents a critical period in the life of an individual. A common cause of poor development is that which comes from undernutrition or malnutrition. In particular, protein deprivation during development has been shown to have deep deleterious effects on brain's growth and plasticity. Early-life stress has also been linked with an increased risk to develop different psychopathologies later in life. We have previously shown that perinatal protein malnutrition in mice leads to the appearance of anxiety-related behaviors in the adulthood. We also found evidence that the female offspring was more susceptible to the development of depression-related behaviors. In the present work, we further investigated this behavior together with its molecular bases. We focused our study on the hippocampus, as it is a structure involved in coping with stressful situations. We found an increase in immobility time in the forced swimming test in perinatally malnourished females, and an alteration in the expression of genes related with neuroplasticity, early growth response 1, calcineurin and c-fos. We also found that perinatal malnutrition causes a reduction in the number of neurons in the hippocampus. This reduction, together with altered gene expression, could be related to the increment in immobility time observed in the forced swimming test.

p38MAPK inhibition: a new combined approach to reduce neuroblastoma resistance under etoposide treatment.

Neuroblastoma (NB) is the second most common solid pediatric tumor and is characterized by clinical and biological heterogeneity, and stage-IV of the disease represents 50% of all cases. Considering the limited success of present chemotherapy treatment, it has become necessary to find new and effective therapies. In this context, our approach consists of identifying and targeting key molecular pathways associated with NB chemoresistance. This study has been carried out on three stage-IV NB cell lines with different status of MYCN amplification. Cells were exposed to a standard chemotherapy agent, namely etoposide, either alone or in combination with particular drugs, which target intracellular signaling pathways. Etoposide alone induced a concentration-dependent reduction of cell viability and, at very high doses, totally counteracted cell tumorigenicity and neurosphere formation. In addition, etoposide activated p38 mitogen-activated protein kinase (MAPK), AKT and c-Jun N-terminal kinase. Pre-treatment with SB203580, a p38MAPK inhibitor, dramatically sensibilized NB cells to etoposide, strongly reducing the dosage needed to inhibit tumorigenicity and neurosphere formation. Importantly, SB203580-etoposide cotreatment also reduced cell migration and invasion by affecting cyclooxygenase-2, intercellular adhesion molecule-1, C-X-C chemokine receptor-4 and matrix metalloprotease-9. Collectively, our results suggest that p38MAPK inhibition, in combination with standard chemotherapy, could represent an effective strategy to counteract NB resistance in stage-IV patients.

GEBR-7b, a novel PDE4D selective inhibitor that improves memory in rodents at non-emetic doses.

BACKGROUND AND PURPOSE: Strategies designed to enhance cerebral cAMP have been proposed as symptomatic treatments to counteract cognitive deficits. However, pharmacological therapies aimed at reducing PDE4, the main class of cAMP catabolizing enzymes in the brain, produce severe emetic side effects. We have recently synthesized a 3-cyclopentyloxy-4-methoxybenzaldehyde derivative, structurally related to rolipram, and endowed with selective PDE4D inhibitory activity. The aim of the present study was to investigate the effect of the new drug, namely GEBR-7b, on memory performance, nausea, hippocampal cAMP and amyloid-ß (Aß) levels. EXPERIMENTAL APPROACH: To measure memory performance, we performed object recognition tests on rats and mice treated with GEBR-7b or rolipram. The emetic potential of the drug, again compared with rolipram, was evaluated in rats using the taste reactivity test and in mice using the xylazine/ketamine anaesthesia test. Extracellular hippocampal cAMP was evaluated by intracerebral microdialysis in freely moving rats. Levels of soluble Aß peptides were measured in hippocampal tissues and cultured N2a cells by elisa. KEY RESULTS: GEBR-7b increased hippocampal cAMP, did not influence Aß levels and improved spatial, as well as object memory performance in the object recognition tests. The effect of GEBR-7b on memory was 3 to 10 times more potent than that of rolipram, and its effective doses had no effect on surrogate measures of emesis in rodents. CONCLUSION AND IMPLICATIONS: Our results demonstrate that GEBR-7b enhances memory functions at doses that do not cause emesis-like behaviour in rodents, thus offering a promising pharmacological perspective for the treatment of memory impairment.

Phosphatidylinositol 3-kinase and Ras/mitogen-activated protein kinase signaling pathways are required for the regulation of 5-aminolevulinate synthase gene expression by insulin.

Insulin regulates the expression of several hepatic genes. Although the general definition of insulin signaling has progressed dramatically, the elucidation of the complete signaling pathway from insulin receptor to transcription factors involved in the regulation of a specific gene remains to be established. In fact, recent works suggest that multiple divergent insulin signaling pathways regulate the expression of distinct genes. 5-Aminolevulinate synthase (ALAS) is a mitochondrial matrix enzyme that catalyzes the first and rate-limiting step of heme biosynthesis. It has been reported that insulin caused the rapid inhibition of housekeeping ALAS transcription, but the mechanism involved in this repression has not been explored. The present study investigates the role of phosphatidylinositol 3-kinase (PI3-kinase) and mitogen-activated protein kinase pathways in insulin signaling relevant to ALAS inhibition. To explore this, we combined the transient overexpression of regulatory proteins involved in these pathways and the use of small cell permeant inhibitors in rat hepatocytes and HepG2 cells. Wortmannin and LY294002, PI3-kinase inhibitors, as well as lovastatin and PD152440, Ras farnesylation inhibitors, and MEK inhibitor PD98059 abolished the insulin repression of ALAS transcription. The inhibitor of mTOR/p70(S6K) rapamycin had no effect whatsoever upon hormone action. The overexpression of vectors encoding constitutively active Ras, MEK, or p90(RSK) mimicked the inhibitory action of insulin. Conversely, negative mutants of PKB, Ras, or MEK impaired insulin inhibition of ALAS promoter activity. Furthermore, inhibition of one of the pathways blocks the inhibitory effect produced by the activation of the other. Our findings suggest that factors involved in two signaling pathways that are often considered to be functionally separate during insulin action, the Ras/ERK/p90(RSK) pathway and the PI3K/PKB pathway, are jointly required for insulin-mediated inhibition of ALAS gene expression in rat hepatocytes and human hepatoma cells.

Transforming growth factor beta-1 enhances Smad transcriptional activity through activation of p8 gene expression.

We report that exposure of mouse embryonic fibroblasts to transforming growth factor beta-1 (TGFbeta-1) (5 ng/ml) results in a strong activation of p8 mRNA expression that precedes the induction of cell growth. Involvement of the p8 promoter in the regulation was demonstrated by using a p8-chloramphenicol acetyltransferase construct. We therefore speculated that p8 might be a mediator of TGFbeta-1 in these cells. The incorporation of [(3)H]thymidine on treatment with TGFbeta-1 was indeed significantly higher in p8(+/+) fibroblasts than in p8(-/-) fibroblasts. Smad transcriptional activity was used as marker of the TGFbeta-1 signalling pathway, to probe the lower p8(-/-) response to TGFbeta-1. Two Smad-binding elements (SBEs)-luciferase constructs were transfected into p8(-/-) and p8(+/+) embryonic fibroblasts before treatment with TGFbeta-1. A lower level of Smad transactivation was observed in p8(-/-) embryonic fibroblasts, under basal conditions and after stimulation with TGFbeta-1. To test whether Smad underexpression in p8(-/-) cells was actually due to p8 depletion, p8(-/-) embryonic fibroblasts were transfected with a human p8 expression plasmid together with an SBE-luciferase construct. The expression of p8 restored Smad transactivation in unstimulated and TGFbeta-1-treated cells to the level found in p8(+/+) cells. We concluded that TGFbeta-1 activates p8 expression, which in turn enhances the Smad-transactivating function responsible for TGFbeta-1 activity.

5-Aminolaevulinate synthase gene promoter contains two cAMP-response element (CRE)-like sites that confer positive and negative responsiveness to CRE-binding protein (CREB).

The first and rate-controlling step of the haem biosynthetic pathway in mammals and fungi is catalysed by the mitochondrial-matrix enzyme 5-aminolaevulinate synthase (ALAS). The purpose of this work was to explore the molecular mechanisms involved in the cAMP regulation of rat housekeeping ALAS gene expression. Thus we have examined the ALAS promoter for putative transcription-factor-binding sites that may regulate transcription in a cAMP-dependent protein kinase (PKA)-induced context. Applying both transient transfection assays with a chloramphenicol acetyltransferase reporter gene driven by progressive ALAS promoter deletions in HepG2, and electrophoresis mobility-shift assays we have identified two putative cAMP-response elements (CREs) at positions -38 and -142. Functional analysis showed that both CRE-like sites were necessary for complete PKA induction, but only one for basal expression. Co-transfection with a CRE-binding protein (CREB) expression vector increased PKA-mediated induction of ALAS promoter transcriptional activity. However, in the absence of co-transfected PKA, CREB worked as a specific repressor for ALAS promoter activity. A CREB mutant deficient in a PKA phosphorylation site was unable to induce expression of the ALAS gene but could inhibit non-stimulated promoter activity. Furthermore, a DNA-binding mutant of CREB did not interfere with ALAS promoter basal activity. Site-directed-mutagenesis studies showed that only the nearest element to the transcription start site was able to inhibit the activity of the promoter. Therefore, we conclude that CREB, through its binding to CRE-like sites, mediates the effect of cAMP on ALAS gene expression. Moreover, we propose that CREB could also act as a repressor of ALAS transcription, but is able to reverse its role after PKA activation. Dephosphorylated CREB would interfere in a spatial-disposition-dependent manner with the transcriptional machinery driving inhibition of gene expression.

Human p8 is a HMG-I/Y-like protein with DNA binding activity enhanced by phosphorylation.

We have studied the biochemical features, the conformational preferences in solution, and the DNA binding properties of human p8 (hp8), a nucleoprotein whose expression is affected during acute pancreatitis. Biochemical studies show that hp8 has properties of the high mobility group proteins, HMG-I/Y. Structural studies have been carried out by using circular dichroism (near- and far-ultraviolet), Fourier transform infrared, and NMR spectroscopies. All the biophysical probes indicate that hp8 is monomeric (up to 1 mm concentration) and partially unfolded in solution. The protein seems to bind DNA weakly, as shown by electrophoretic gel shift studies. On the other hand, hp8 is a substrate for protein kinase A (PKA). The phosphorylated hp8 (PKAhp8) has a higher content of secondary structure than the nonphosphorylated protein, as concluded by Fourier transform infrared studies. PKAhp8 binds DNA strongly, as shown by the changes in circular dichroism spectra, and gel shift analysis. Thus, although there is not a high sequence homology with HMG-I/Y proteins, hp8 can be considered as a HMG-I/Y-like protein.

Recent measles viral activity in Uruguai: serological and genetic approaches.

In the summer 1999, a measles outbreak occurred in Uruguai. During this outbreak 58 cases were recorded, 36 of which were laboratory confirmed as positive for measles virus (MV) IgM. The cases occurred in touristic places (Montevideo and Maldonado) predominantly among health facilities and tourist service personnel. Urine specimens collected between days 1 and 4 after the onset of the rash from seven cases were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and nested PCR with primers specific for the carboxyl-terminal region of the nucleoprotein (N) gene. Three of these specimens/cases were positive for MV. Sequencing of 300 nucleotides (nt) of PCR products corresponding to a part of the carboxyl-terminal region of the MV N gene detected in these specimens MV of D6 genotype. The same nucleotide sequences and the same genotype were also previously observed for MV isolates from the 1997 epidemic in Brazil and the 1998 epidemic in Argentina, demonstrating that the D6 genotype was, and may be still circulating in South America.

Transcriptional regulation of 5-aminolevulinate synthase by phenobarbital and cAMP-dependent protein kinase.

5-Aminolevulinate synthase (ALA-S) is a mitochondrial matrix enzyme that catalyzes the first and rate-limiting step of the heme biosynthesis. There are two ALA-S isozymes encoded by distinct genes. One gene encodes an isozyme that is expressed exclusively in erythroid cells, and the other gene encodes a housekeeping isozyme that is apparently expressed in all tissues. In this report we examine the mechanisms by which phenobarbital and cAMP regulate housekeeping ALA-S expression. We have determined that cAMP and phenobarbital effects are additive and the combined action is necessary to observe the cAMP effect on ALA-S mRNA in rat hepatocytes. The role of the cAMP-dependent protein kinase (PKA) has been examined. A synergism effect on ALA-S mRNA induction is observed in rat hepatocytes treated with pairs of selective analogs by each PKA cAMP binding sites. A 870-bp fragment of ALA-S 5'-flanking region is able to provide cAMP and phenobarbital stimulation to chloramphenicol O-acetyltranferase fusion vectors in transiently transfected HepG2 cells. ALA-S promoter activity is induced by cotransfection with an expression vector containing the catalytic subunit of PKA. Furthermore, cotransfection with a dominant negative mutant of the PKA regulatory subunit impairs the cAMP analog-mediated increase, but the phenobarbital-mediated induction is not modified. Our data suggest that the transcription factor cAMP-response element binding protein (CREB) is probably involved in PKA induction of ALA-S gene expression. Finally, heme addition greatly decreases the basal and phenobarbital or cAMP analog-mediated induction of ALA-S promoter activity. The present work provides evidence that cAMP, through PKA-mediated CREB phosphorylation, and phenobarbital induce ALA-S expression at the transcriptional level, while heme represses it.

Structural and functional characterization of the mouse p8 gene: promotion of transcription by the CAAT-enhancer binding protein alpha (C/EBPalpha) and C/EBPbeta trans-acting factors involves a C/EBP cis-acting element and other regions of the promoter.

Rat p8 mRNA was discovered because of its strong activation in pancreas during the acute phase of pancreatitis. We report here structural and functional data on the mouse p8 gene. The mouse p8 polypeptide is 80 amino acids long and shows 91% and 75% identity with its rat and human counterparts respectively. The p8 gene is organized into three exons interrupted by two introns. Promoter regions involved in the regulation of p8 gene expression in NIH 3T3 cells were analysed. Chloramphenicol acetyltransferase (CAT) reporter assays with progressive deletions of the 5' flanking region of the mouse p8 gene revealed four silencer elements located from nucleotides -5000 to -1472, -1471 to -671, -670 to -473, and -239 to -117 respectively. One positive element was identified between nucleotides -117 and -72. We identified a CAAT-enhancer binding protein (C/EBP) cis-acting element at position -111. Site-directed mutagenesis of this consensus site decreased promoter activity to 5% of that of the wild-type. An electrophoretic mobility-shift assay, using an oligonucleotide probe corresponding to the C/EBP consensus and nuclear extracts of NIH 3T3 cells transfected with C/EBPalpha or C/EBPbeta expression vectors, generated specific DNA-protein complexes that were supershifted with specific antibodies against C/EBPalpha and C/EBPbeta. Co-transfection with C/EBPalpha or C/EBPbeta expression vectors and the p-116/+36p8-CAT construct increased the reporter gene activity in a dose-dependent fashion. Surprisingly, overexpression of C/EBPalpha or C/EBPbeta still increased the promoter activity of both pC/EBPmut-116/+36p8-CAT (which contains the C/EBP mutated site) and the p-71/+36-CAT construct (which does not contain the C/EBP site). Collectively, these results show that C/EBPalpha and C/EBPbeta trans-acting factors can promote transcription of the mouse p8 gene (i) by direct binding to the C/EBP consensus site, and (ii) by enhancing the activity of other trans-acting factors interacting with the p8 promoter.

Cloning and expression of the human p8, a nuclear protein with mitogenic activity.

We have previously identified a new rat mRNA, provisionally named p8, which showed a strong, but transient, induction in the pancreas in response to acute pancreatitis. We report here the cloning and sequencing of the human p8 cDNA. The human p8 polypeptide is 82 amino acids long and shows an overall identity of 74% with the rat counterpart. The complete structure of the p8 gene was also established. The p8 gene comprises three exons separated by two introns and the gene was mapped to chromosome 16. Analysis of the p8 primary structure suggested the presence of a bipartite motif of nuclear targeting, indicating that p8 may function within the nucleus. This presumption has been confirmed by transfection of COS-7 cells with the p8 cDNA followed by immunostaining with specific antibodies. p8 mRNA expression is induced in some, but not all, cells by serum starvation and ceramide. To analyze the p8 function, we stably transfected HeLa cells with a p8 expression plasmid, and measured their growth and their sensitivity to apoptosis. Response to TNF alpha and staurosporine-induced apoptosis was not modified by p8 expression. However, cells expressing p8 grew significantly more rapidly.

Insulin inhibits delta-aminolevulinate synthase gene expression in rat hepatocytes and human hepatoma cells.

Insulin has been known to regulate intracellular metabolism by modifying the activity or location of many enzymes but it is only in the past few years that the regulation of gene expression is recognized to be a major action of this hormone. The present work provides evidences that insulin inhibits delta-aminolevulinate synthase (ALA-S) gene expression, the enzyme which governs the rate-limiting step in heme biosynthesis. The addition of 5 nM insulin to hepatocytes culture led to a significant decrease of both basal and phenobarbital-induced ALA-S mRNA in a dose-dependent manner, as measured by Northern and slot-blot analysis. Several clues as to how insulin regulates ALA-S transcription were determined. The inhibitory effect is achieved at physiological concentrations but much higher proinsulin doses are needed. Insulin's effect is rapid, quite specific, and protein synthesis is not required. Moreover, ALA-S mRNA half-life is not modified by the presence of the peptidic hormone. Our results demonstrate that the insulin effect is dominant; it overrides 8-CPT-cAMP plus phenobarbital-mediated induction. Also, insulin requires the activation of protein kinase C to exert its full effect. On the other hand, a 870-bp fragment of the ALA-S promoter region is able to sustain the inhibition of CAT expression in plasmid-transfected HepG2 cells. Thus, these results indicate that insulin plays an important role in regulating ALA-S expression by inhibiting its transcription.

Evidence that protein kinase C is involved in delta-aminolevulinate synthase expression in rat hepatocytes.

There are many factors that regulate the rate of synthesis of delta-aminolevulinate synthase (ALA-S), the enzyme which governs the rate-limiting step in heme biosynthesis. In rat hepatocytes, phenobarbital increases ALA-S gene transcription and dibutyryl cAMP potentiates this induction, whereas insulin and glucose have the opposite effect. The present report provides evidence that protein kinase C (PKC) activation negatively influences ALA-S mRNA levels, as measured by Northern and slot-blot analysis. The addition of 1,2-dioctanoyl-sn-glycerol (DOG) or 12-O-tetradecanoylphorbol 13-acetate (TPA), a PKC activator that mimics diacylglycerol function, to cultures led to a significant decrease of both basal and phenobarbital-induced ALA-S mRNA levels in a dose-dependent manner. This TPA effect depends on the specific activation of PKC because the analog 4 alpha-phorbol 12,13-diacetate, a nonstimulatory PKC phorbol ester, is unable to inhibit ALA-S mRNA. Furthermore, the effect of TPA is blocked by the PKC inhibitors staurosporine and calphostin C. Desensitization of the PKC pathway by prolonged exposure to TPA abolished the subsequent action of the phorbol ester. On the other hand, neither TPA nor DOG modified the half-life of ALA-S mRNA. The study of the combinatorial action of TPA and cAMP revealed that the inhibitory effect of TPA overcomes dibutyryl cAMP induction. Thus, these results indicate that PKC plays an essential role in regulating ALA-S expression, probably at a transcriptional level.

Glucose inhibits phenobarbital-induced delta-aminolevulinate synthase expression in normal but not in diabetic rat hepatocytes.

In the present work, we demonstrate the presence of a glucose inhibitory effect on the phenobarbital-mediated induction of the delta-aminolevulinate synthase mRNA in normal rat hepatocytes, consistent with the results obtained with the delta-aminolevulinate synthase activity previously reported. This "glucose effect" can be prevented by adding cAMP, adenylate cyclase activators, or a phosphodiesterase inhibitor. Delta-Aminolevulinate synthase mRNA half-life is not modified in the presence of phenobarbital or glucose. When the same experiments are performed using diabetic cells, no glucose effect is observed, even when the endogenous cAMP content is lowered to normal levels. The results obtained in this study suggest that glucose decreases delta-aminolevulinate synthase biosynthesis by acting at a pretranslational step. Assuming that the glucose effect operates by a repression mechanism exerted by metabolites derived from or related to glucose, the present results may reflect a derangement in the formation of these metabolites as a result of the abnormal metabolism operating in the diabetic state.

cAMP regulation of phenobarbital-mediated induction of delta-aminolevulinate synthase mRNA in hepatocytes from normal and experimental diabetic rats.

We examined the mechanism underlying the effect of cAMP on delta-aminolevulinate synthase mRNA biosynthesis in isolated hepatocytes from normal and experimental diabetic rats. We have demonstrated that the potentiation by dibutyryl cAMP of the phenobarbital-mediated induction of delta-aminolevulinate synthase enzyme activity, observed in our previously reported studies, reflects an increased amount of its mRNA. The inducing effect exerted by phenobarbital on the biosynthesis of delta-aminolevulinate synthase mRNA in diabetic hepatocytes is greater than that observed in normal cells. This enhanced response to the increased level of endogenous cAMP in diabetic hepatocytes is apparently sufficient for a maximum activation of the cAMP-dependent protein kinase. The present results suggest that in rat liver dibutyryl cAMP modulates delta-aminolevulinate synthase mRNA biosynthesis by acting predominantly, if not exclusively, at the level of gene transcription.

Regulation of phenobarbital-induced ferrochelatase mRNA activity by dibutyryl cAMP and glucose in normal and diabetic rat hepatocytes.

The induction of ferrochelatase activity by phenobarbital and its potentiation by dibutyryl cAMP assayed in normal rat hepatocytes are associated with increased activity of ferrochelatase mRNA. Glucose inhibits this stimulatory effect. This inhibition can be reversed with increasing concentrations of dibutyryl cAMP. The inducing effect exerted by phenobarbital on the activity of ferrochelatase mRNA in diabetic hepatocytes is greater than that observed in normal cells. This enhanced response in diabetic rat hepatocytes is neither potentiated by adding dibutyryl cAMP nor repressed by glucose. The absence of a glucose effect persists even when the endogenous cAMP content is lowered to normal levels. The results obtained in this study are consistent with those reported in other published studies of ferrochelatase activity. This adds more experimental evidence to support the concept that ferrochelatase is inducible. The results obtained suggest that ferrochelatase is more susceptible to induction with phenobarbital in diabetic rat hepatocytes than in normal rat hepatocytes.

Identification of viruses in a study of acute respiratory tract infection in children from Uruguay.

For the purpose of identifying viral agents associated with acute respiratory tract infections (ARI) in children less than 5 years old, a longitudinal community study was undertaken in Montevideo, Uruguay, from May 1985 to December 1987. This report includes results obtained by cell culture and immunofluorescence techniques for detection of respiratory syncytial virus (RSV), influenza A and B viruses, parainfluenza 1 and 3 viruses, and adenovirus. Two populations were studied: children visited at home by pediatricians (group 1) and children with an ARI episode who attended an outpatient clinic (group 2). Nasopharyngeal aspirates were obtained at the time of an ARI episode: 858 from group 1 and 488 from group 2. Viruses were identified in 15.3% of group 1 specimens and in 17.6% of group 2 specimens. RSV was the most frequently recovered agent, accounting for 67.9% and 58.1%, respectively, of all viruses detected. The sensitivity and specificity of RSV isolation by cell culture are compared with detection by indirect immunofluorescence.

Studies on induction of delta-aminolevulinic acid synthase, ferrochelatase, cytochrome P-450 and cyclic AMP by phenformin. Chlorpropamide, allylisopropylacetamide and lead in hepatocytes from normal and experimental diabetic rats.

The present work demonstrates that phenformin exerted an inducing effect on delta-aminolevulinic acid synthase (ALA-S) and ferrochelatase activities and on cytochrome P-450 content in isolated hepatocytes from rats with experimental diabetes. Similar results were obtained with respect to ALA-S activity and cytochrome P-450 content when chlorpropamide was used. The inducing effect exerted by allylisopropylacetamide (AIA) on ALA-S and ferrochelatase activities in diabetic hepatic cells was markedly greater than that observed in normal hepatocytes. This stimulatory response was not enhanced by adding dibutyryl cyclic AMP (cAMP). When phenformin was added to isolated rat hepatocytes of normal rats, induction of ALA-S and ferrochelatase activities and cytochrome P-450 content was observed only in the presence of added dibutyryl cAMP. Addition of chlorpropamide to this in vitro system did not exert an inducing effect on the same enzymes even in the presence of dibutyryl cAMP. The present results add more experimental evidence about the lability of the heme pathway of diabetic hepatocytes.

Studies on regulatory mechanisms of heme biosynthesis in hepatocytes from normal and experimental-diabetic rats. Role of insulin.

In the present work we demonstrate that insulin decreases the phenobarbital-induced activities of delta-aminolevulinic acid synthase and ferrochelatase in isolated hepatocytes from normal and experimental-diabetic rats. Insulin concentrations required to produce significant inhibition in diabetic hepatocytes were higher than in normal cells. Under similar experimental conditions, insulin decreased the basal activities of delta-aminolevulinic acid synthase and ferrochelatase in hepatocytes from normal rats; no inhibitory effect was observed on the basal activity of delta-aminolevulinic acid synthase in hepatocytes from diabetic rats. Cytochrome P-450 content of both normal and diabetic cells was not affected by insulin in absence or presence of phenobarbital. The inhibitory action of insulin was exerted even when effective concentrations of glucagon, dexamethasone, or 8-(p-chlorophenylthio)-cAMP were present.