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D J Monger

Publications and source records attributed to D J Monger.

9 recordsLinked to original sources
PubMed1989

Fatty acid metabolism in human keratinocytes cultivated at an air-medium interface.

Stratum corneum lipids, which provide the mammalian permeability barrier, display a distinctive fatty acid profile with a predominance of long chain, saturated fatty acids. In addition, linoleic acid (18:2) is present in substantial quantities, implying that it is an important structural component. To investigate selectivity of fatty acid incorporation into epidermal lipids, we examined the metabolism of exogenous fatty acids in cultured human keratinocytes, grown at the air-medium interface to enhance differentiation. Keratinocytes were pulsed with [3H] oleic, [14C] stearic, [14C] palmitic, or [14C] linoleic acids; lipids were extracted and fractionated by thin layer chromatography. All fatty acids were taken up and incorporated into complex lipids in a dose-dependent manner that was linear over the first 60 min. These fatty acids were incorporated predominantly into phospholipids and triacylglycerols; their incorporation could be rank ordered: linoleic greater than oleic greater than or equal to palmitic greater than stearic acid. Less than 2% of each fatty acid taken up by keratinocytes was oxidized to CO2; therefore, these differences in utilization cannot be ascribed to differences in rates of beta-oxidation. In pulse-chase studies fatty acids incorporated initially into triacylglycerols, subsequently chased into phospholipids. [14C]Palmitic acid and [14C] acetate were incorporated into sphingolipids more efficiently than the other fatty acids studied. These studies demonstrate that 1) keratinocytes have the ability to incorporate exogenous fatty acids preferentially into complex lipids; 2) triacylglycerols provide a pool of fatty acids for phospholipid synthesis; and 3) palmitate and de novo synthesized fatty acid are preferably utilized for sphingolipid synthesis.

Air↗
PubMed1988

Lipid content and metabolism of human keratinocyte cultures grown at the air-medium interface.

The differentiation of human keratinocytes in most culture systems is incomplete; e.g., lamellar bodies, the characteristic lipid-delivery organelles of epidermis, are not present. Moreover, their lipid profile does not reflect the distinctive composition found in cornifying epidermis. In contrast, keratinocytes that grow at an air-medium interface exhibit more complete differentiation. In this study, we compared the elaboration of lamellar bodies, the lipid content, and the lipid metabolism of human keratinocytes, cultured both under standard immersed conditions and after lifting to an air-medium interface. Whereas submerged cultures neither elaborated lamellar bodies nor displayed a lipid distribution characteristic of cornifying epidermis, lifted cultures displayed advanced cornification, elaborated lamellar bodies which were deposited in intercellular domains, and a lipid profile more typical of cornifying epidermis. Moreover, lipid biosynthesis was 5-10-fold more active in lifted than in immersed cultures, and was not inhibited by exogenous lipoproteins. These findings are consistent with recent studies that demonstrate both high rates of lipogenesis in differentiating layers of the epidermis as well as autonomy of lipogenesis from the influence of circulating lipoproteins. Thus, the lipid content and metabolism of human keratinocyte cultures, grown at an air-medium interface, demonstrate features that simulate the epidermis.

Cell Differentiation↗
PubMed1988

Neutral lipid storage disease with ichthyosis: lipid content and metabolism of fibroblasts.

Neutral lipid storage disease with ichthyosis is a newly recognized heritable disorder characterized by widespread cellular triglyceride storage. Lipid metabolism in fibroblasts cultured from three affected family members was studied. The stored lipid is triglyceride composed of an unremarkable fatty acid profile and derived from both exogenously-supplied and endogenously-synthesized fatty acids. Lipid storage could not be corrected by prolonged culture in lipid-depleted media. Acetyl CoA carboxylase activity and beta-oxidation of palmitate were both normal. Taken together, these studies exclude a primary defect of fatty acid uptake, over-synthesis or impaired beta-oxidation. Moreover, triacylglycerol lipase activity of homogenates of fibroblasts from patients with NLSDI examined over the range of pH 3.5-8.5 was normal.

Fatty Acids↗
PubMed1988

Inhibitors of cholesterol biosynthesis. Further studies of the metabolism of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one in rat liver preparations.

5 alpha-Cholest-8(14)-en-3 beta-ol-15-one is a potent inhibitor of sterol biosynthesis in mammalian cells in culture and has significant hypocholesterolemic activity upon oral administration to rodents and non-human primates. The conversion of the 15-ketosterol to cholesterol upon incubation with the 10,000 x g supernatant fraction of rat liver homogenate preparations under aerobic conditions has been reported (D.J. Monger, E.J. Parish and G.J. Schroepfer, Jr. (1980) J. Biol. Chem. 255, 11122-11129). Presented herein are results of studies of the metabolism of [2,4-3H]5 alpha-cholest-8(14)-en-3 beta-ol-15-one obtained upon incubation with the microsomal, cytosolic and the 10,000 x g supernatant fractions of liver homogenates of female rats under a variety of conditions. The results of these studies indicated metabolism of the 15-ketosterol to materials with the chromatographic properties of fatty acid esters of the 15-ketosterol, fatty acid esters of C27-monohydroxysterols, a component similar to the 15-ketosterol (possibly an isomer of the delta 8(14)-15-ketosterol), and a polar component. Detailed studies of the C27-monohydroxysterols obtained from incubation of the 15-ketosterol under anaerobic conditions indicated the formation of labeled 5 alpha-cholesta-8,14-dien-3 beta-ol and 5 alpha-cholest-7-en-3 beta-ol which were characterized by their behavior on silicic acid column chromatography, by the behavior of their acetate derivatives on medium pressure liquid chromatography on alumina-AgNO3 columns, and by co-crystallization of the labeled sterols with authentic unlabeled standards. The identification of 5 alpha-cholesta-8,14-dien-3 beta-ol and 5 alpha-cholest-7-en-3 beta-ol as metabolites of the 15-ketesterol, coupled with previous studies of the metabolism of 5 alpha-cholesta-8,14-dien-3 beta-ol and of 5 alpha-cholest-8(14)-ene-3 beta, 15 alpha-diol and 5 alpha-cholest-8(14)-ene-3 beta, 15 beta-diol has permitted the formulation of a scheme for the overall metabolism of the 15-ketosterol to cholesterol.

Animals↗
PubMed1988

Localization of sites of lipid biosynthesis in mammalian epidermis.

The end-product of epidermal differentiation is a stratified layer of corneocytes whose extracellular lipid bilayers provide a permeability barrier. It is generally accepted that the epidermis synthesizes most if not all of the lipids found in this tissue and that extra-epidermal tissues contribute very little to this lipid content. Moreover, the individual epidermal strata in which epidermal lipid biosynthesis occurs are not known. To address this question, we examined [3H]H2O incorporation into nonsaponifiable and saponifiable lipids in individual epidermal cell layers 3 hr after intraperitoneal injection into neonatal mice, and compared this to protein and DNA synthesis using intraperitoneal [3H]leucine and [3H]thymidine incorporation, respectively. Lipid biosynthesis was also assessed by [14C]acetate incorporation into lipid fractions in organ cultured skin and in epidermal subpopulations. The in vivo studies demonstrated that the biosynthetic activity of both saponifiable and nonsaponifiable lipids was comparable to, if not greater, in the stratum granulosum (SG) than in basal/spinous (SB + SS) layer, despite significantly lower levels of both protein and DNA synthesis in the SG. On a mass basis, the SG accounts for about four times the biosynthetic activity of the combined SB + SS layers. The lipid biosynthetic activity in vitro also was two- to fivefold higher in the SG, regardless of whether the epidermis was separated into individual cell layers before or after incubations with radiolabel. Moreover, this difference could not be ascribed to increased acetate pools or to elevated metabolism in the SG versus the SB + SS since the rates of CO2 production were much lower in the SG fraction. The increase in lipid biosynthesis in SG over SB + SS was greatest for phospholipids, followed by glycosphingolipids, and free sterols but was observed in almost all lipid classes. These studies show not only that mammalian epidermis is an active site of de novo lipid biosynthesis, but also that this activity remains high in the stratum granulosum, while other forms of metabolic activity are diminishing. These observations are consistent with the knowledge that lipids extruded from the stratum granulosum layer provide the hydrophobic permeability barrier, and further suggest that elevated synthetic activity in the stratum granulosum would allow rapid replenishment in the event that the barrier is damaged.

Animals↗
PubMed1982

Control of juvenile hormone biosynthesis. Evidence for phosphorylation of the 3-hydroxy-3-methylglutaryl coenzyme A reductase of insect corpus allatum.

The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a key enzyme of juvenile hormone biosynthesis, have been measured in the supernatants of homogenates (10,000 x g) prepared from the corpora allata of the adult tobacco hornworm moth, Manduca sexta. Enzyme activity was inhibited 80% by 50 mM NaF, a known phosphoprotein phosphatase inhibitor, if present during extirpation of the glands and all subsequent workup of the tissue. Reductase activity was also significantly decreased (20-30%) in homogenates preincubated with 4 mM MgCl2 and 2 mM ATP. These results provide evidence that reductase in the insect undergoes phosphorylation and dephosphorylation similar to that occurring with reductase of mammalian liver. If so, this would provide a rapid method for modulating juvenile hormone synthesis.

Adenosine Triphosphate↗
PubMed1980

15-Oxygenated sterols. Enzymatic conversion of [2,4]3H]5 alpha-cholest-8(14)-en-3 beta-ol-15-one to cholesterol in rat liver homogenate preparations.

5 alpha-Cholest-8(14)-en-3 beta-ol-15-one is a very potent inhibitor of sterol biosynthesis in animal cells in culture (G. J. Schroepfer, Jr., E. J. Parish, H. W. Chen, and A. A. Kandutsch (1977) J. Biol. Chem. 252, 8975-8980) and has significant hypocholesterolemic activity upon dietary administration to normal animals (G. J. Schroepfer, Jr., D. Monger, A. S. Taylor, J. S. Chamberlain, E. J. Parish, A. Kisic, and A. A. Kandutsch (1977) Biochem. Biophys. Res. Commun. 78, 1227-1233). In the present study we have prepared [2,4-3H]5 alpha-cholest-8(14)-en-3 beta-ol-15-one by chemical synthesis. This compound has been shown to be convertible to cholesterol upon incubation with the 10,000 X g supernatant fraction of rat liver homogenates under aerobic conditions. The efficiency of this conversion was higher in preparations derived from male rats than in those obtained from female rats. Labeled 5 alpha-cholest-7-en-3 beta-ol was also isolated and characterized as a product of the metabolism of the labeled delta 8(14)-15-ketosterol. The demonstration of the enzymatic conversion of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one to cholesterol in liver preparations suggests that previous estimates of the biological potency of this compound may be much lower than the true values and/or that metabolites of this compound may be the active species responsible for its biological actions.

Aerobiosis↗