Neutral Lipid Production Research Articles

Corticotropin-Releasing Hormone Skin Signaling is Receptor-Mediated and is Predominant in the Sebaceous Glands

There is increasing evidence that the sebaceous gland expresses receptors for several neuropeptides and is involved in responses to stress. Among them, corticotropin-releasing hormone (CRH) was currently found to be produced also in the skin. In this study, the distribution of CRH, CRH receptors 1 and 2 (CRH-R1 and CRH-R2), and CRH binding protein (CRH-BP) in cultured human (SZ95) sebocytes was further characterized. Moreover, the effects of CRH and CRH-like peptides on proliferation and inflammatory signaling of CRH receptor-expressing SZ95 sebocytes IN VITRO were investigated. Urocortin (Uct), urotensin and sauvagine are recently described members of the family of structurally related CRH-like peptides, whereas Uct shares a 45% homology with CRH. CRH and Uct inhibited SZ95 sebocyte proliferation with CRH also stimulating interleukin-6 (IL-6) and interleukin-8 (IL-8) release from SZ95 sebocytes. However, CRH had no effect on interleukin-1alpha and interleukin-1beta production in these cells. alpha-Helical-CRF, a CRH antagonistic peptide, annulled the CRH effect on SZ95 sebocyte proliferation and interleukin secretion, while the non-peptidic CRH-R1 selective antagonist antalarmin inhibited the increased production of neutral lipids caused by CRH. In conclusion, CRH, and to a lesser extent Uct, may be involved in signaling of stress pathophysiology in the skin. However, further investigations into the downstream effects of CRH and Uct are required to elucidate the mechanism by which these neuropeptides could establish a stress-related pathophysiological condition in the skin.

Regulation of Triglyceride Metabolism. I. Eukaryotic neutral lipid synthesis: “Many ways to skin ACAT or a DGAT”

Esterification of sterols, fatty acids and other alcohols into biologically inert forms conserves lipid resources for many cellular functions. Paradoxically, the accumulation of neutral lipids such as cholesteryl ester or triglyceride, is linked to several major disease pathologies. In a remarkable example of genetic expansion, there are at least eleven acyltransferase reactions that lead to neutral lipid production. In this review, we speculate that the complexity and apparent redundancy of neutral lipid synthesis may actually hasten rather than impede the development of novel, isoform-specific, therapeutic interventions for acne, type 2 diabetes, obesity, hyperlipidemia, fatty liver disease, and atherosclerosis.

Decline of hormones with increasing age regulates the biological activity of human sebocytes

Hormone deficiency occurring with age leads among other systemic disorders to deterioration of the skin quality. Systemic substitution with estradiol in females and GH in males reversed the observed alterations and retarded the skin aging process in single controlled studies. In order to evaluate the importance of sex steroids and growth factors on skin aging in vitro, human sebocytes were treated with GH, IGF‐I, 17β‐estradiol, DHEA, testosterone and progesterone in combination and as single agents in concentrations corresponding to those circulating in young (f20) and postmenopausal women (f60). Cell proliferation and lipid synthesis were measured by means of the 4‐methylumbelliferyl heptanoate fluorescence assay and nile‐red microassay/fluorescence microscopy, respectively. Cells incubated with all hormones mentioned above at f60 showed significantly lower content of sebaceous lipids (P < 0.001) vs. cells at f20 relating to in vivo observations, which have documented a decline of epidermal lipids in aged skin. While progesterone and DHEA, as single agents, showed no effect on lipid synthesis, after treatment with IGF‐I for 48 h at f20 and f60 concentrations, a significant dose‐dependent increase of neutral (P < 0.001) and polar lipids (P < 0.001) was observed at both concentrations tested. Cell size was also increased. On the other hand, GH at f20 and f60 enhanced significantly the production of neutral (P < 0.01 and P < 0.05, accordingly) and polar lipids (P < 0.05 and P < 0.01, accordingly) in sebocytes but not in such an extent as IGF‐I. 17β‐estradiol induced only polar lipid synthesis (P < 0.01) and cell size. No treatment affected the cell proliferation. In conclusion, sex steroids and growth factors and their decline with age play a key role in the regulation of the lipid synthesis in human sebocytes. Furthermore, among all hormones tested, the GH/IGF‐I axis seems to be the most potent regulator of skin lipids.

Comparative toxicity of oleic and linoleic acid on human lymphocytes

Commercially available lipid emulsions for parenteral nutrition are mainly composed by long chain triacylglycerol containing a high proportion of linoleic acid (LA) or oleic acid (OA). The immunological impact of such therapy is particularly important because parenteral diets are often administered to critically ill patients as a mechanism to supply adequate nutrition during catabolic stress conditions. The comparative toxicity of OA and LA on human lymphocytes and the type of cell death induced by these fatty acids were determined in vitro. Parameters of cell death were investigated by flow cytometry–cell viability, DNA fragmentation, phosphatidylserine externalization, mitochondrial depolarization, neutral lipid accumulation and production of reactive oxygen species–and by fluorescence microscopy—chromatin condensation. Additionally a spectrofluorometric assay was employed to determine the activities of caspase—3, 6 and 8. Evidence is presented herein that OA is less toxic to human lymphocytes than LA. However, both fatty acids promoted apoptosis and necrosis of these cells. The mechanism of cell death induced by OA involved activation of caspase 3 while the mechanism of death induced by LA involved mitochondrial depolarization and ROS production. Importantly, neutral lipid accumulation may be a mechanism to protect lymphocytes against the toxicity induced by OA. OA may offer an immunological less problematic alternative to LA with respect to fatty acid composition of parenteral nutritional emulsions.

On the biogenesis of lipid bodies in ancient eukaryotes: synthesis of triacylglycerols by a Toxoplasma DGAT1-related enzyme

In mammalian cells, the main stored neutral lipids are triacylglycerol and cholesteryl esters, which are produced by two related enzymes, acyl-CoA:diacylglycerol acyltransferase (DGAT) and acyl-CoA:cholesterol acyltransferase (ACAT), respectively. Very little is known about the metabolism, intracellular storage and function of neutral lipids in many pathogenic lower eukaryotes. In this paper, we have characterized the activity of an important triacylglycerol synthetic enzyme in the protozoan Toxoplasma gondii. A full-length cDNA and gene encoding a T. gondii DGAT1-related enzyme were identified and designated TgDGAT1. The gene is composed of 15 exons and 14 introns, and encodes a protein with a predicted M(r) 63.5kDa, containing signature motifs characteristic of the DGAT1 family. The native protein migrates at 44kDa under reducing conditions. TgDGAT1 is an integral membrane protein localized to the parasite cortical and perinuclear endoplasmic reticulum, with the C-terminus oriented to the lumen of the organelle. When a Saccharomyces cerevisiae mutant strain lacking neutral lipid production is transformed with TgDGAT1 cDNA, a significant DGAT activity is reconstituted, resulting in triacylglycerol synthesis and biogenesis of cytosolic lipid inclusions, resembling lipid bodies in T. gondii. No production of steryl esters is observed upon TgDGAT1 expression in yeast. In contrast to human DGAT1 lacking fatty acid specificity, TgDGAT1 preferentially incorporates palmitate. Our results indicate that parasitic protozoa are also neutral lipid accumulators and illustrate the first example of the existence of a functional DGAT gene in an ancient eukaryote, demonstrating that diacylglycerol esterification is evolutionarily conserved.

Effects of milk diets containing beef tallow or coconut oil on the fatty acid metabolism of liver slices from preruminant calves

Coconut oil (CO) induces a triacylglycerol infiltration in the hepatocytes of preruminant calves when given as the sole source of fat in the milk diet over a long-term period. Metabolic pathways potentially involved in this hepatic triacylglycerol accumulation were studied by in vitro methods on liver slices from preruminant Holstein x Friesian male calves fed a conventional milk diet containing CO (n 5) or beef tallow (BT, n 5) for 19 d. Liver slices were incubated for 12 h in the presence of 0.8 mm-[14C]oleate or -[14C]laurate added to the medium. Fatty acid oxidation was determined by measuring the production of CO2 (total oxidation) and acid-soluble products (partial oxidation). Production of CO2 was 1. 7-3.6-fold lower (P 0.0490) and production of acid-soluble products tended to be lower (P = 0.0625) in liver slices of CO- than BT-fed calves. Fatty acid esterification as neutral lipids was 2.6- to 3. 1-fold higher (P = 0.0088) in liver slices prepared from calves fed the CO diet compared with calves fed the BT diet. By contrast with what occurs in the liver of rats fed CO, the increase in neutral lipid production did not stimulate VLDL secretion by the hepatocytes of calves fed with CO, leading to a triacylglycerol accumulation in the cytosol. It could be explained by the reduction of fatty acid oxidation favouring esterification in the form of triacylglycerols, in association with a limited availability of triacylglycerols and/or apolipoprotein B for VLDL packaging and subsequent secretion.

Seasonal variation in lipid and fatty acid composition of ice algae from the Barents Sea

The fatty acid compositions of neutral lipid, glycolipid and phospholipid fractions from ice algae sampled from the Barents Sea in spring and autumn were examined for seasonal differences. The ice-algal assemblages were dominated by diatoms. In spring, Nitzschia frigida was the most common species whereas resting stages of Thalassiosira bioculata and Actinocyclus cf curvatulus predominated in autumn. With the exception of one spring sample, neutral lipids predominated over glycolipids and phospholipids in all algal samples. The lipid fractions displayed characteristic fatty acid compositions. In the spring samples the major fatty acids of the neutral lipid fraction were 16:0, 16:1(n-7) and 20:5(n-3) whilst the glycolipid fraction was characterised by higher levels of 20:5(n-3) and C16 polyunsaturated fatty acids, particularly 16:4(n-1). Phospholipids contained higher levels of 22:6(n-3) than the other two lipid fractions although 20:5(n-3) was still the major polyunsaturated fatty acid. In the autumn samples, the neutral lipid fraction contained higher proportions of saturated fatty acids and 16:1(n-7) than the two polar lipid fractions and 22:6(n-3) was most abundant in phospholipids. As with the spring samples, 20:5(n-3) was the major polyunsaturated fatty acid in all lipid fractions of the autumn algae. Overall, the fatty acid compositions of the lipid fractions from spring and autumn algal samples were similar and are consistent with diatoms being the predominant group in the ice algae studied. The high level of neutral lipids observed in both spring and autumn samples suggests that the production of neutral lipids is characteristic of ice algae regardless of season. Nevertheless, some species-specific differences in lipid production may exist since the neutral lipid content of autumn samples containing mainly A. curvatulus was substantially higher than those in which T. bioculata predominated.

Androgens markedly stimulate the accumulation of neutral lipids in the human prostatic adenocarcinoma cell line LNCaP.

Microscopic evaluation of LNCaP cells stained with the lipophilic dye Oil red O revealed that androgens induce a marked stimulation of lipid droplet accumulation. As determined by quantitative analysis of the Oil red O extracted from the stained cells, stimulatory effects of the synthetic androgen R1881 became apparent at concentrations as low as 10(-11) M. Maximal induction (15-fold) was reached at 10(-8) M. Increases were observed 2 days after hormone addition and were maximal 1 day later. Accumulation of lipid droplets was also induced by mibolerone (another synthetic androgen) and by the natural androgens testosterone and dihydrotestosterone. In agreement with the aberrant ligand specificity of the mutated androgen receptor in LNCaP cells, stimulation of lipid accumulation was also apparent after treatment with progesterone and estradiol. Cortisol and the synthetic glucocorticoid dexamethasone were ineffective. The androgen antagonist Casodex (bicalutamide) abolished the stimulatory effect of R1881, further supporting the involvement of the androgen receptor. In agreement with this conclusion, no changes in lipid accumulation were observed after androgen treatment of the androgen receptor-negative prostate tumor lines PC-3 and DU-145. To investigate the nature of the lipids affected by androgens, lipid extracts were analyzed by TLC, complemented with enzymatic lipid analyses. Androgens were shown to have major effects on the content of triglycerides and cholesterol esters (33- and 7-fold stimulation, respectively), the two main classes of lipids stained by Oil red O. Phospholipid and cholesterol contents were increased by a factor of 2. Incorporation studies with [2-14C]acetate revealed that androgens caused a major stimulation of 2-14C incorporation into triglycerides and cholesterol esters (11- and 13-fold, respectively), suggesting that androgens act at least in part at the level of lipid synthesis. Taken together, these findings indicate that androgens, besides affecting proliferation and protein secretion, also markedly stimulate the production and accumulation of neutral lipids, revealing a novel interesting aspect of androgen regulation of LNCaP cells.

The Influence of Dissolved Oxygen Tension on the Production of Cocoa Butter Equivalents and Gamma-linolenic Acid by Mucor circinelloides

Summary The influence of different dissolved oxygen tension (DOT) values ranging from 5 to 35% of saturation on the production of cocoa butter equivalents and gamma-linolenic acid by Mucor circinelloides f. circinelloides CBS 108.16 was investigated in nitrogen limited, fed-batch, pH-stat cultures with acetic acid as carbon source. DOT values of 8% and 10% were optimal for crude oil and neutral lipid production. The lipid composition at low and high DOT values differed extensively. The content of gamma-linolenic acid gradually decreased from 12.4% to approximately 4.6% of the neutral lipid fraction when the DOT was decreased from 35% to 5%. The stearic acid (18: 0) content increased from 15.6% of the neutral lipid fatty acids at a DOT of 35% to approximately 27% at a DOT of 5%. Similarly, a dramatic increase in symmetrical triacylglycerols occurred when the DOT was decreased from 35% to 5%. The DOT, therefore, effected major quantitative changes in lipid desaturation and cocoa butter equivalent production, modifying the microbial oil composition also in the DOT range where the respiration rate, and therefore probably growth, was not limited by the level of dissolved oxygen.

The effect of low density lipoproteins, cholesterol, and 25-hydroxycholesterol on apolipoprotein B gene expression in HepG2 cells.

The purpose of the present study was to examine the effects of exogenous cholesterol on the apolipoprotein (Apo) B gene expression in HepG2 cells. Pure cholesterol had no significant effect on either the cellular content of cholesteryl esters or the net accumulation of neutral lipids and ApoB in the culture medium. By contrast, addition of 25-hydroxycholesterol increased the net accumulation of cholesteryl esters in cells and medium by 2-3-fold and decreased that of unesterified cholesterol by 50% in both compartments. A 33% reduction in the cellular content of triglycerides was commensurate with a 40% increase in their accumulation in the medium. A significant 3-fold increase in the net accumulation of ApoB in the medium was predominantly due to enhanced secretion of newly synthesized ApoB as established by pulse-chase studies. The stimulation in ApoB secretion was accompanied by a 55% increase in cellular ApoB mRNA. Under these experimental conditions, the low density lipoprotein receptor activity was decreased by only 12-20%. Addition of progesterone prevented the effects of 25-hydroxycholesterol. The changes in the concentration of neutral lipids and ApoB were reflected in the composition of secreted "low-density" lipoproteins. These particles had increased percentage contents of cholesteryl esters and ApoB and a decreased percentage content of unesterified cholesterol in comparison with lipoproteins produced by control cells. The rate of ApoB production was not correlated with the triglyceride mass in the cells but was positively correlated with the cellular and secreted cholesteryl esters and secreted triglycerides. With the exception of unchanged cellular unesterified cholesterol and ApoB mRNA levels, plasma low density lipoprotein had similar, although less pronounced, effects on the production of neutral lipids and ApoB. These results demonstrate that in HepG2 cells the synthesis and secretion of ApoB and cholesteryl esters are tightly coupled and that 25-hydroxycholesterol increased the concentration of ApoB-containing lipoproteins primarily by stimulating their production rather than reducing their catabolism.

Effects of a first exposure to ethanol on the compositions of neutral and polar lipids in Euglena gracilis Z, taken as a hepatic cell model: Equilibration by citrulline-malate

In comparison to the lipid composition of Euglena cells fed with lactate, a first exposure of the cells to ethanol favors the production of neutral lipids containing mainly unsaturated fatty acids. The ethanol diminishes drastically the proportion of PC and weakly that of PE. In contrast, it increases slightly the proportion of DPG. The ethanol induces important changes in the fatty acid distributions of each lipid class, suggesting modifications of the elongation-desaturation system. On the one hand the proportion of unsaturated fatty acids is increased and, on the other hand, the last double bond is predominantly situated in the delta 6 position in place of delta 3. The addition of the complex citrulline-malate corrects most of these changes.

Growth and production of cell constituents in batch cultures of botryococcus sudeticus

The growth of, and the production of neutral lipids, carbohydrates and proteins by, the alga Botryococcus sudeticus in batch culture is described. The algal mass contains, during the stationary phase of growth, about 4.5% protein, 7.5% carbohydrate and 22.0% neutral lipid on a dry weight basis. Some physiological characteristics of this species are discussed.

The acute effects of streptozotocin-induced diabetes on rat liver glycerolipid biosynthesis

Streptozotocin-induced diabetes produced a significant rise in rat serum and liver triacylglycerol content and hepatic triacylglycerol biosynthesis measured in vivo. Microsomes, isolated from the livers of streptozotocin-exposed animals (2–72 h), exhibited an increased capacity to incorporate sn-[1,3- 14C]glycerol 3-phosphate into neutral lipid (diacylglycerol and triacylglycerol) in the presence of ATP, CoA and palmitate. The streptozotocin-induced elevation of microsomal neutral lipid production was accompanied by a corresponding rise in the activity of microsomal phosphatidate phosphohydrolase (4-fold after 72 h of streptozotocin exposure). Diabetic-dependent increases in acylglycerol formation, phosphatidate phosphohydrolase activity and serum triacylglycerol and fatty acid levels were reversed by administering insulin (10 units protamine zinc/kg) at 16-h intervals (three separate doses) beginning 24 h after streptozotocin exposure. However, the diabetic-related rise in hepatic triacylglycerol content was only partially corrected by insulin administration. Streptozotocin-related increases in liver triacylglycerol biosynthesis and phosphatidate phosphohydrolase activity were associated with alterations in plasma factors, since homogenates of hepatocyte monolayers exposed (18 h) to plasma isolated from diabetic (72 h exposure to streptozotocin) animals exhibit an increased capacity to incorporate sn-[1,3- 14C]glycerol 3-phosphate into triacylglycerol compared to homogenates of cells exposed to plasma from control (non-fasted) animals. The importance of these plasma factors in altering hepatic acylglycerol formation was also supported by the observation that hepatocyte monolayers exposed to a mixture of plasma isolated from normal (non-fasted) animals and plasma components elevated in diabetes (glucagon, glucose, oleate and ketones) showed increases in triacylglycerol formation which were similar to those produced by exposure to diabetic plasma. Additional studies demonstrated that fatty acids (oleate) appeared to be the agent primarily responsible for the diabetic plasma-induced rise in monolayer triacylglycerol biosynthesis and phosphatidate phosphohydrolase activity.

Neutral lipid biosynthesis in Mycobacterium smegmatis

The biosynthesis of neutral lipids in Mycobacterium smegmatis was studied using cell-free extracts. Maximum neutral lipid production was obtained when the reaction mixture (400 μl) consisted of 0.25 M potassium phosphate buffer (pH 7.5), 0.125 mM oleoyl-CoA, 3.75 mM sn-glycerol-3-P, 10 mM MgCl 2 and 1.85 mg bovine serum albumin. No magnesium dependency for the acylation of sn-glycerol-3-P was observed. A slight stabilizing effect seemed to occur due to this ion. The enzyme phosphatidate phosphohydrolase, on the other hand, was shown to be magnesium dependent. The activity of this enzyme also appeared to be stimulated by high concentration (0.75 to 1.25 mM) of ATP which enhanced lipid formation at all concentrations tested (0.25 to 3.75 mM). A heat-stable protective factor having a molecular weight less than 16 000 which caused a stimulatory effect on sn-glycerol 3-phosphate acyltransferase activity was found in the cell-free extracts. Preliminary experiments suggest that the factor might be polysaccharide in nature.

INVESTIGATIONS INTO THE PRODUCTION OF LIPIDS BY SUBMERGED CULTURES OF THE MUSHROOM TRICHOLOMA NUDUM: I. FATTY ACID COMPOSITION OF NEUTRAL LIPIDS AND PHOSPHOLIPIDS AS A FUNCTION OF TIME

The production of neutral lipids and phospholipids by submerged cultures of the mushroom Tricholoma nudum, as well as the fatty acid composition of these two fractions, was studied as a function of time. The bulk of the neutral lipids was produced after 2 days when the organism appeared to be in a non-proliferative phase. The major fatty acids of the neutral lipids were palmitic, oleic, and linoleic acid (23–35% each); stearic acid was a minor component (8–13%); myristic, palmitoleic, and linolenic acid were present in small amounts (0.5–4.8%). The major fatty acid of the phospholipids was linoleic acid (55–70%); palmitic (15–19%), stearic (1.8–4.6%), and oleic (7–19%) acid were minor components; myristic, palmitoleic, and linolenic (0–2.3%) were present in small amounts. Linolenic acid was a major fatty acid (26–30%) only in the early stages of growth.A preliminary investigation was carried out with a 4-day-old culture to establish the identity of the various components of the neutral lipids and phospholipids. The neutral lipids were mainly triglycerides (92%). Small amounts of ergosterol esters (1%), free fatty acids (< 1%), ergosterol (1.7%), and unidentified non-saponifiable compounds were also present. The phospholipids contained phosphatidyl choline (59%) as the major component; phosphatidyl ethanolamine (26%), phosphatidyl serine and phosphatidic acid (7.8%), and an inositol containing phospholipid were minor components.Some of the techniques applied were specially developed for the present type of studies and are described in detail.

INVESTIGATIONS INTO THE PRODUCTION OF LIPIDS BY SUBMERGED CULTURES OF THE MUSHROOM TRICHOLOMA NUDUM: I. FATTY ACID COMPOSITION OF NEUTRAL LIPIDS AND PHOSPHOLIPIDS AS A FUNCTION OF TIME

The production of neutral lipids and phospholipids by submerged cultures of the mushroom Tricholoma nudum, as well as the fatty acid composition of these two fractions, was studied as a function of time. The bulk of the neutral lipids was produced after 2 days when the organism appeared to be in a non-proliferative phase. The major fatty acids of the neutral lipids were palmitic, oleic, and linoleic acid (23–35% each); stearic acid was a minor component (8–13%); myristic, palmitoleic, and linolenic acid were present in small amounts (0.5–4.8%). The major fatty acid of the phospholipids was linoleic acid (55–70%); palmitic (15–19%), stearic (1.8–4.6%), and oleic (7–19%) acid were minor components; myristic, palmitoleic, and linolenic (0–2.3%) were present in small amounts. Linolenic acid was a major fatty acid (26–30%) only in the early stages of growth.A preliminary investigation was carried out with a 4-day-old culture to establish the identity of the various components of the neutral lipids and phospholipids. The neutral lipids were mainly triglycerides (92%). Small amounts of ergosterol esters (1%), free fatty acids (< 1%), ergosterol (1.7%), and unidentified non-saponifiable compounds were also present. The phospholipids contained phosphatidyl choline (59%) as the major component; phosphatidyl ethanolamine (26%), phosphatidyl serine and phosphatidic acid (7.8%), and an inositol containing phospholipid were minor components.Some of the techniques applied were specially developed for the present type of studies and are described in detail.