Examination of 'lipotoxicity' in skeletal muscle of high-fat fed and ob/ob mice.

Excess lipid accumulation resulting from an elevated supply of plasma fatty acids is linked to the pathogenesis of the metabolic syndrome and heart disease. The term 'lipotoxicity' was coined to describe how lipid accumulation leads to cellular dysfunction and death in non-adipose tissues including the heart, pancreas and liver. While lipotoxicity has been shown in cultured skeletal muscle cells, the degree of lipotoxicity in vivo and the functional consequences are unresolved. We studied three models of fatty acid overload in male mice: 5 h Intralipid((R)) and heparin infusion, prolonged high fat feeding (HFF) and genetic obesity induced by leptin deficiency (ob/ob mice). Markers of apoptosis, proteolysis and autophagy were assessed as readouts of lipotoxicity. The Intralipid((R)) infusion increased caspase 3 activity in skeletal muscle, demonstrating that enhancing fatty acid flux activates pro-apoptotic pathways. HFF and genetic obesity increased tissue lipid content but did not influence apoptosis. Gene array analysis revealed that HFF reduced the expression of 31 pro-apoptotic genes. Markers of autophagy (LC3beta and beclin-1 expression) were unaffected by HFF and were associated with enhanced Bcl(2) protein expression. Proteolytic activity was similarly unaffected by HFF or in ob/ob mice. Thus, contrary to our previous findings in muscle culture in vitro and in other non-adipose tissues in vivo, lipid overload did not induce apoptosis, autophagy or proteolysis in skeletal muscle. A broad transcriptional suppression of pro-apoptotic proteins may explain this resistance to lipid-induced cell death in skeletal muscle.

Psychiatric illness in female physicians. Are high rates of depression an occupational hazard?

More than half of female physicians may experience a psychiatric illness during their lifetime. Depression is by far the most common such disorder, and the suicide rate is alarmingly high. However, female physicians appear to be at lower risk for substance abuse than male physicians. The medical profession could benefit from increased awareness of depression among female physicians and removal of barriers to treatment, such as stigma and discrimination against those with psychiatric illness.

Reversal of chloroquine resistance in falciparum malaria.

Control of falciparum malaria infections has been increasingly hampered by the emergence of parasites resistant to chloroquine, pyrimethomine and other standard anti-malarials. Chloroquine-resistant strains of Plasmodium falciparum, for example, which originally appeared in South-East Asia and South America are now found in East Asia and sub-Saharan Africa(1). Attempts to combat this alarming development have to date taken two main forms: (1) the judicious use of existing ontimalarials, preferably in combinations, in an attempt to delay the emergence of resistance; and (2) on aggressive research effort aimed at identifying a new generation of antimalarial drugs. But what i f it became possible to administer an antimalarial drug together with a second drug capable of overcoming resistance to the first? A recent report from Samuel Martin and co-workers at The Walter Reed Army Institute of Research in Washington DC raises just such an intriguing possibility.

Rat liver and intestinal mucosa differ in the developmental pattern and hormonal regulation of carbamoyl-phosphate synthetase I and ornithine carbamoyl transferase gene expression.

cDNA probes were employed to measure levels of carbamoyl-phosphate synthetase I (CPS) and ornithine carbamoyltransferase (OCT) mRNAs in fetal and neonatal livers and intestines. In the fetal liver, significant levels of OCT mRNA were present at 15-days gestation while CPS mRNA could not be detected until day 17 of fetal development. Apart from a small decline just after birth, amounts of both mRNAs increased steadily to reach adult levels in postnatal life. In contrast to the situation in liver, CPS and OCT mRNA levels in the fetal intestine rose rapidly to peak at day 21 of gestation and then declined steadily in the first seven days after birth. Using the methyl-sensitive restriction isoschizomeric pair, MspI/HpaII, the 5' ends of both the CPS and OCT genes were shown to undergo demethylation during development. In the case of the OCT gene, however, the hypomethylation characteristic of the adult liver and intestinal mucosa was not observed in the 15-day-old fetal liver, where significant levels of gene expression had already been established. Levels of CPS and OCT mRNA in livers of adults responded to glucagon in normal animals (1.5-fold and 2.2-fold increases, respectively) and to dexamethasone in experimentally induced diabetic animals (3-fold increase in CPS mRNA with no change in OCT mRNA). These treatments were all without effect on the levels of CPS and OCT mRNA in intestinal mucosa.

Expression of nuclear genes encoding the urea cycle enzymes, carbamoyl-phosphate synthetase I and ornithine carbamoyl transferase, in rat liver and intestinal mucosa.

RNA dot-blot, quantitative electron microscope immunocytochemistry, and electrophoretic immunoblotting techniques were employed to investigate the expression of carbamoyl-phosphate synthetase I (CPS) and ornithine carbamoyl transferase (OCT) genes in rat liver and intestinal mucosa. Comparing only those cell types in the two tissues which express these enzymes, we show that the concentration of CPS and OCT in hepatocyte mitochondria is 2.3-times and 1.2-times greater, respectively, than in intestinal epithelial cell mitochondria. As a percentage of total tissue protein, however, liver homogenates contain 10-20 times more CPS and 5-10 times more OCT than is found in intestinal mucosa. These relatively large differences in enzyme protein levels between the two tissues are not reflected by differences in their mRNA levels. As a percentage of total translational activity in vitro (based on incorporation of [35S]methionine), total liver mRNA directed synthesis of about twice as much precursor CPS (pCPS) and precursor OCT (pOCT) than did equivalent amounts of mRNA from intestinal mucosa. The ratio of pCPS and pOCT mRNA levels between the two tissues (2:1, liver:intestinal mucosa) was confirmed by dot-blot and Northern hybridizations employing specific cDNA probes. The sizes of the respective mRNAs were the same for the two tissues: about 6000 residues for pCPS mRNA and about 1700 residues for pOCT mRNA.

Expression of carbamoyl-phosphate synthetase I mRNA in Reuber hepatoma H-35 cells. Regulation by glucocorticoid and insulin.

Reuber hepatoma H-35 cells actively synthesize the urea cycle enzyme, carbamoyl-phosphate synthetase I. Treatment of H-35 cells with dexamethasone (0.14 microM), however, enhanced synthesis of the enzyme (as measured by incorporation of [35S]methionine) by 4-5-fold. Insulin (0.18 microM) completely inhibited dexamethasone-dependent stimulation of enzyme synthesis. In vitro translation and cDNA hybridization assays were employed to measure effects of dexamethasone plus or minus insulin on levels of mRNA encoding the biosynthetic precursor of carbamoyl-phosphate synthetase I (pCPS) in Reuber H-35 cells. Both measurements yielded similar results: dexamethasone increased pCPS mRNA levels by 4-5-fold and insulin suppressed this response, but only by 50%. Specific cDNA hybridization assays also demonstrated that Reuber H-35 cells, even after hormone treatments, contain only very low levels of albumin mRNA, and no detectable ornithine carbamoyl-transferase mRNA.

Regulation and expression of carbamyl phosphate synthetase I mRNA in developing rat liver and Morris hepatoma 5123D.

A cDNA clone complementary to mRNA encoding the precursor (Mr = 165,000) to the rat liver mitochondrial matrix enzyme carbamyl phosphate synthetase I (Mr = 160,000) was employed to compare relative amounts of the messenger in adult and fetal liver and in Morris hepatoma 5123D and 3924A cells. Northern blot analysis gave a size estimate for the messenger of 6,500-6,700 nucleotides. Carbamyl phosphate synthetase mRNA levels in 15-day-old fetal liver were less than 10% of adult levels; 5123D cells expressed the messenger at levels about 2-fold higher than normal adult liver, but the messenger was undetectable in 3924A cells. Albumin mRNA was also expressed in the former but not in the latter. Maintaining rats for 5 days on a diet containing 60% casein augmented the relative amount of carbamyl phosphate synthetase mRNA by about 2-fold, while a protein-free diet resulted in reduced levels of the mRNA (about 50% compared to animals on a normal diet). Finally, the pattern of hybridization of carbamyl phosphate synthetase cDNA to HindIII-digested genomic DNA showed no differences between normal liver and its corresponding hepatoma; however, a HindIII site polymorphism was observed between Buffalo and ACI rats.

Production and preliminary characterization of monoclonal antibodies to human liver-specific lipoprotein (LSP).

Monoclonal antibodies (mc/anti-LSP) have been prepared by polyethylene glycol fusion of P3/NS1/Ag4-1 myeloma cells with spleen cells from Balb/c mice hyperimmunized with human liver-specific membrane lipoprotein (LSP). Ten hybridomas, cloned by limiting dilution, produced mc/anti-LSP reacting (by ELISA) with human LSP but not with normal human plasma proteins nor with a variety of other proteins likely to co-purify with LSP. Three of these ( A15 /7, A9/63 and B20 ), producing high-titre IgG1 mc/anti-LSP, were biosynthetically radiolabelled and used as index antibodies. By competitive inhibition of binding of the index antibodies to LSP in an immunoradiometric assay, the ten hybridoma products were classified into four distinct groups according to their specificities for different epitopes in LSP. None of the index antibodies reacted, on ELISA, with glutaraldehyde-fixed PLC/PRF/5, Chang, Daudi or HSB-2 cell lines nor with human peripheral blood leucocytes. However, A15 /27 (but not A9/63 or B20 ) reacted with saponin-permeabilized PLC/PRF/5 and Chang cells and also with rabbit LSP. The results emphasize the polyantigenic nature of LSP and indicate that at least one of the mc/anti-LSP ( A15 /27) recognises a species cross-reactive antigen that is present in liver-derived cell lines.