Stimulation Of Lipid Synthesis Research Articles

Protein Kinase C Isoforms Have Differential Roles in the Regulation of Human Sebocyte Biology

Protein kinase C (PKC) isoforms have crucial roles in cutaneous signaling. Interestingly, we lack information about their involvement in human sebaceous gland biology. Therefore, in this current study, we investigated the functions of the PKC system in human immortalized SZ95 sebocytes. Using molecular biological approaches, imaging, and functional assays, we report that SZ95 sebocytes express the conventional cPKCα; the novel nPKCδ, ɛ, and η; and the atypical aPKCζ. Activation of the PKC system by phorbol 12-myristate 13-acetate (PMA) stimulated lipid synthesis (a hallmark of differentiation) and resulted in translocation and then downregulation of cPKCα and nPKCδ. In good accord with these findings, the effect of PMA was effectively abrogated by inhibitors and short interfering RNA-mediated "silencing" of cPKCα and nPKCδ. Of further importance, molecular or pharmacological inhibition of nPKCδ also prevented the lipogenic and apoptosis-promoting action of arachidonic acid. Finally, we also found that "knockdown" of the endogenous aPKCζ activity markedly increased basal lipid synthesis and apoptosis, suggesting its constitutive activity in suppressing these processes. Collectively, our findings strongly argue for the fact that certain PKCs have pivotal, isoform-specific, differential, and antagonistic roles in the regulation of human sebaceous gland-derived sebocyte biology.

Hepatic endoplasmic reticulum stress in obesity: Deeper insights into processes, but are they relevant to nonalcoholic steatohepatitis?

Abstract The endoplasmic reticulum (ER) is the main site of protein and lipid synthesis, membrane biogenesis, xenobiotic detoxification and cellular calcium storage, and perturbation of ER homeostasis leads to stress and the activation of the unfolded protein response. Chronic activation of ER stress has been shown to have an important role in the development of insulin resistance and diabetes in obesity. However, the mechanisms that lead to chronic ER stress in a metabolic context in general, and in obesity in particular, are not understood. Here we comparatively examined the proteomic and lipidomic landscape of hepatic ER purified from lean and obese mice to explore the mechanisms of chronic ER stress in obesity. We found suppression of protein but stimulation of lipid synthesis in the obese ER without significant alterations in chaperone content. Alterations in ER fatty acid and lipid composition result in the inhibition of sarco/endoplasmic reticulum calcium ATPase (SERCA) activity and ER stress. Correcting the obesity-induced alteration of ER phospholipid composition or hepatic Serca over-expression in vivo both reduced chronic ER stress and improved glucose homeostasis. Hence, we established that abnormal lipid and calcium metabolism are important contributors to hepatic ER stress in obesity. (Hepatology 2011

Aberrant lipid metabolism disrupts calcium homeostasis causing liver endoplasmic reticulum stress in obesity

The endoplasmic reticulum (ER) is the main site of protein and lipid synthesis, membrane biogenesis, xenobiotic detoxification and cellular calcium storage, and perturbation of ER homeostasis leads to stress and the activation of the unfolded protein response. Chronic activation of ER stress has been shown to have an important role in the development of insulin resistance and diabetes in obesity. However, the mechanisms that lead to chronic ER stress in a metabolic context in general, and in obesity in particular, are not understood. Here we comparatively examined the proteomic and lipidomic landscape of hepatic ER purified from lean and obese mice to explore the mechanisms of chronic ER stress in obesity. We found suppression of protein but stimulation of lipid synthesis in the obese ER without significant alterations in chaperone content. Alterations in ER fatty acid and lipid composition result in the inhibition of sarco/endoplasmic reticulum calcium ATPase (SERCA) activity and ER stress. Correcting the obesity-induced alteration of ER phospholipid composition or hepatic Serca overexpression in vivo both reduced chronic ER stress and improved glucose homeostasis. Hence, we established that abnormal lipid and calcium metabolism are important contributors to hepatic ER stress in obesity.

Transfer of the glycosylphosphatidylinositol-anchored 5'-nucleotidase CD73 from adiposomes into rat adipocytes stimulates lipid synthesis.

In addition to predominant localization at detergent-insoluble, glycolipid-enriched plasma membrane microdomains (DIGs), glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-proteins) have been found associated with lipid droplets (LDs) and adiposomes. Adiposomes are vesicles that are released from adipocytes in response to anti-lipolytic and lipogenic signals, such as H(2)O(2), palmitate and the antidiabetic sulfonylurea drug, glimepiride, and harbour (c)AMP-degrading GPI-proteins, among them the 5-nucleotidase CD73. Here the role of adiposomes in GPI-protein-mediated information transfer was studied. Adiposomes were incubated with isolated rat adipocytes under various conditions. Trafficking of CD73 and lipid synthesis were analysed. Upon blockade of GPI-protein trafficking, CD73 specifically associated with DIGs of small, and to a lower degree, large, adipocytes. On reversal of the blockade, CD73 appeared at cytosolic LD in time- adiposome concentration- and signal (H(2)O(2) > glimepiride > palmitate)-dependent fashion. The salt- and carbonate-resistant association of CD73 with structurally intact DIGs and LD was dependent on its intact GPI anchor. Upon incubation with small and to a lower degree, large adipocytes, adiposomes increased lipid synthesis in the absence or presence of H(2)O(2), glimepiride and palmitate and improved the sensitivity toward these signals. Upregulation of lipid synthesis by adiposomes was dependent on the translocation of CD73 with intact GPI anchors from DIGs to LD. The signal-induced transfer of GPI-anchored CD73 from adiposomes via DIGs to LD of adipocytes mediates paracrine upregulation of lipid synthesis within the adipose tissue.

In Vitro Metabolic and Mitogenic Signaling of Insulin Glargine and Its Metabolites

BackgroundInsulin glargine (Lantus®) is a long-acting basal insulin analog that demonstrates effective day-long glycemic control and a lower incidence of hypoglycemia than NPH insulin. After subcutaneous injection insulin glargine is partly converted into the two main metabolites M1 ([GlyA21]insulin) and M2 ([GlyA21,des-ThrB30]insulin). The aim of this study was to characterize the glargine metabolites in vitro with regard to their insulin receptor (IR) and IGF-1 receptor (IGF1R) binding and signaling properties as well as their metabolic and mitogenic activities.MethodsThe affinity of human insulin, insulin glargine and its metabolites to the IR isoforms A and B or IGF1R was analyzed in a competitive binding assay using SPA technology. Receptor autophosphorylation activities were studied via In-Cell Western in CHO and MEF cells overexpressing human IR-A and IR-B or IGF1R, respectively. The metabolic response of the insulins was studied as stimulation of lipid synthesis using primary rat adipocytes. Thymidine incorporation in Saos-2 cells was used to characterize the mitogenic activity.ConclusionsThe binding of insulin glargine and its metabolites M1 and M2 to the IR were similar and correlated well with their corresponding autophosphorylation and metabolic activities in vitro. No differences were found towards the two IR isoforms A or B. Insulin glargine showed a higher affinity for IGF1R than insulin, resulting in a lower EC50 value for autophosphorylation of the receptor and a more potent stimulation of thymidine incorporation in Saos-2 cells. In contrast, the metabolites M1 and M2 were significantly less active in binding to and activation of the IGF1R and their mitogenicity in Saos-2 cells was equal to human insulin. These findings strongly support the idea that insulin glargine metabolites contribute with the same potency as insulin glargine to blood glucose control but lead to significantly reduced growth-promoting activity.

Insulin therapy stimulates lipid synthesis and improves endocrine functions of adipocytes in dietary obese C57BL/6 mice

To evaluate whether insulin intervention could affect the metabolic and endocrine functions of adipose tissue. C57BL/6 mice were fed on a high-fat-diet for 12-16 weeks to induce insulin resistance. Insulin intervention was administered in the high-fat-diet mice for 4 weeks at 12 weeks (early insulin treatment) or 16 weeks (late insulin treatment). Intraperitoneal glucose tolerance tests were performed before and after insulin treatment. Expression levels of factors involved in the triglyceride synthesis and endocrine functions of adipose tissue including phosphoenolpyruvate carboxykinase (PEPCK-C), fatty acid synthase (FAS), aquaporin 7 (AQP7), adiponectin, visfatin, and interleukin-6 (IL-6) were determined by Western blot. In the obese mice, glucose tolerance was impaired; triglyceride content was increased in the liver tissue; protein expression of FAS and adiponectin was decreased; expression of visfatin was increased in adipose tissue. After 4-week insulin treatment, glucose tolerance was improved; triglyceride content was decreased in the liver and skeletal muscle; expression of PEPCK-C, FAS, and adiponectin was increased in the adipose tissue; IL-6 and AQP7 expression was reduced in the fat. Early insulin treatment had better effect in increasing the expression of FAS and PEPCK-C and decreasing the expression of IL-6. These results indicate that insulin can target adipocytes for improvement of insulin sensitivity through stimulating triglyceride synthesis and partly improving endocrine functions.

CFTR knockdown stimulates lipid synthesis and transport in intestinal Caco-2/15 cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel highly expressed in epithelial cells of the gastrointestinal tract. Mutations in the CFTR gene cause cystic fibrosis (CF), a disease characterized by pancreatic insufficiency, fat malabsorption, and steatorrhea. Despite the administration of pancreatic enzymes to normalize malabsorption, CF patients still experienced lipid fecal loss, nutritional deficiencies, and abnormalities in serum lipid profile, suggesting the presence of intrinsic defects in the intestinal handling of nutrients. The objective of the present study was to assess the impact of CFTR gene knockdown on intracellular lipid metabolism of the intestinal Caco-2/15 cell line. Partial CFTR gene inactivation led to cellular lipid accretion of phospholipids, triglycerides, and cholesteryl esters. Likewise, secretion of these lipid fractions was significantly increased following CFTR gene manipulation. As expected from these findings, the output of triglyceride-rich lipoproteins showed the same increasing pattern. Investigation of the mechanisms underlying these changes revealed that CFTR knockdown resulted in raised levels of apolipoproteins in cells and media and microsomal transfer protein activity, two important factors for the efficient assembly and secretion of lipoproteins. Similarly, scrutiny of the enzymatic monoacylglycerol acyltransferase and diacylglycerol acyltransferase, which exhibit dynamic function in triacylglycerol resynthesis and chylomicron formation in enterocytes, revealed a significant augmentation in their activity. Conversely, cholesterol uptake mediated by Niemann-Pick C1 like 1, Scavenger Receptor Class B Type I, and ATP-binding cassette G8 remains unaffected by genetic modification of CFTR. Collectively, these results highlight the role played by CFTR in intestinal handling of lipids and may suggest that factors other than defective CFTR are responsible for the abnormal intracellular events leading to fat malabsorption in CF patients.

Propionibacterium acnes and Sebaceous Lipogenesis: A Love–Hate Relationship?

In this issue, Iinuma et al. show that Propionibacterium acnes (P. acnes)-conditioned medium and formalin-killed P. acnes augment intracellular lipid formation in hamster sebocytes by increasing the de novo synthesis of triacylglycerols. This commentary summarizes the current knowledge of the association of P. acnes with sebaceous lipogenesis, inflammation, and innate immunity, and points out the concurrent evidence that P. acnes-induced lipids may represent a recruitment of allies and/or enemies of the human skin.

Mechanisms for the Hypotriglyceridemic Effect of Marine Omega-3 Fatty Acids

A mechanism to explain the hypotriglyceridemic effects of marine omega-3 fatty acids in humans has not been clarified. A working model can be developed at the gene transcriptional level, which involves >or=4 metabolic nuclear receptors. These include liver X receptor, hepatocyte nuclear factor-4alpha (HNF-4alpha), farnesol X receptor, and peroxisome proliferator-activated receptors (PPARs). Each of these receptors is regulated by sterol receptor element binding protein-1c (SREBP-1c), the main genetic switch controlling lipogenesis. Omega-3 fatty acids elicit hypotriglyceridemic effects by coordinately suppressing hepatic lipogenesis through reducing levels of SREBP-1c, upregulating fatty oxidation in the liver and skeletal muscle through PPAR activation, and enhancing flux of glucose to glycogen through downregulation of HNF-4alpha. The net result is the repartitioning of metabolic fuel from triglyceride storage toward oxidation, thereby reducing the substrate available for very-low-density lipoprotein (VLDL) synthesis. By simultaneously downregulating genes encoding proteins that stimulate lipid synthesis and upregulating genes encoding proteins that stimulate fatty acid oxidation, omega-3 fatty acids are more potent hypotriglyceridemic agents than are omega-6 fatty acids, on a carbon-for-carbon basis. Additionally, peroxidation of omega-3 fatty acids may reduce VLDL secretion through stimulating apolipoprotein B degradation. Omega-3 fatty acids may act by enhancing postprandial chylomicron clearance through reduced VLDL secretion and by directly stimulating lipoprotein lipase activity. These combined effects support the use of omega-3 fatty acids as a valuable clinical tool for the treatment of hypertriglyceridemia.

Basis for Improved Permeability Barrier Homeostasis Induced by PPAR and LXR Activators: Liposensors Stimulate Lipid Synthesis, Lamellar Body Secretion, and Post-Secretory Lipid Processing

Previously, we demonstrated that topical applications of peroxisome proliferator-activated receptors (PPARs) and liver X receptor (LXR) activators improve permeability barrier homeostasis. We showed further that stimulation of epidermal differentiation provides one mechanism that could account for such improvement. Here, we studied the effects of these agents on the lipid matrix of the stratum corneum. Hairless mice were treated topically with activators of PPARalpha (WY14643), PPARdelta (GW1514), PPARgamma (ciglitazone), and LXR (22(R)-cholesterol or TO901317) or vehicle twice daily for 3 days. All activators significantly increased epidermal cholesterol, fatty acid, and sphingolipid synthesis, including the production of barrier-specific ceramide species. In addition, lamellar body (LB) formation, secretion, and post-secretory processing accelerated significantly following acute barrier disruption in PPAR/LXR-activator-treated animals. Finally, the activity of epidermal beta-glucocerebrosidase, a key lipid-processing enzyme, increased in PPAR/LXR-activator-treated animals. Thus, topical PPAR and LXR activators stimulate epidermal lipid synthesis, increase LB secretion, and accelerate extracellular lipid processing, providing additional mechanisms that further account for their ability to improve epidermal permeability barrier homeostasis. Since the liposensors are activated by endogenous lipid metabolites, they may serve as unique regulators of barrier homeostasis.

Neuroendocrine regulation of the sebaceous gland

The sebaceous gland is the organ conferring upon the skin an endocrine function. It also seems to be involved in responses to stress expressing receptors for neuropeptides such as corticotropin‐releasing hormone (CRH), α‐melanocyte‐stimulating hormone (α‐MSH), β‐endorphin, vasoactive intestinal polypeptide, neuropeptide Y, and calcitonin gene‐related peptide. CRH is the most proximal element of the hypothalamic‐pituitary‐adrenal (HPA) axis, and it acts as central coordinator for neuroendocrine and behavioral responses to stress. The CRH/CRH‐binding protein/CRH receptor loop is expressed in human sebocytes in vivo and in vitro and is regulated and biologically active in cultured sebocytes. CRH may be an autocrine sebocyte factor that exerts homeostatic lipogenic activity by controlling the expression of Δ5‐3β‐hydroxysteroid dehydrogenase, whereas testosterone and growth hormone induce CRH negative feedback. α‐MSH and calcitonin gene‐related peptide are not produced by human sebocytes and downregulate the enhanced IL‐8 synthesis in IL‐1β‐challenged cells, therefore exhibiting an anti‐inflammatory effect. β‐endorphin is also a paracrine neuropeptide for human sebocytes which suppresses their proliferation induced by epidermal growth factor in Ca2+‐rich medium. Moreover, substance P‐immune reactive nerves are localized near the sebaceous glands and can stimulate undifferentiated sebocytes of the outmost sebaceous gland layer to express neutral endopeptidase, a substance P‐inactivatimg molecule. In addition, substance P stimulates lipid synthesis in human sebocytes. In conclusion, sebaceous glands may be involved in a hypothalamus‐pituitary‐skin pathway conceptually similar to that of the HPA axis. The current data implicate neuropeptides to possibly play a strategic role in the regulation of sebaceous gland activity in close coordination with other hormones making likely that they are involved in the development of various skin diseases.

Lipid storage metabolism is limited by the prevailing low oxygen concentrations within developing seeds of oilseed rape.

The aim of this study was to investigate whether endogenous restrictions in oxygen supply are limiting for storage metabolism in developing oilseed rape (Brassica napus) seeds. Siliques were studied 30 d after flowering, when rapid lipid accumulation is occurring in the seeds. (a). By using microsensors, oxygen concentrations were measured within seeds and in the silique space between seeds. At ambient external oxygen (21% [v/v]) in the light, oxygen fell to 17% (v/v) between and 0.8% (v/v) within seeds. A step-wise reduction of the external oxygen concentration led within 2 h to a further decrease of internal oxygen concentrations, and a step-wise increase of the external oxygen concentration up to 60% (v/v) resulted in an increase in internal oxygen that rose to 30% (v/v) between and 8% (v/v) within seeds. (b). The increase in oxygen levels in the seeds was accompanied by a progressive increase in the levels of ATP, UTP, and the ATP to ADP and UTP to UDP ratios over the entire range from 0% to 60% (v/v) external oxygen. (c). To investigate metabolic fluxes in planta, 14C-sucrose was injected into seeds, which remained otherwise intact within their siliques. The increase in oxygen in the seeds was accompanied by a progressive increase in the rate of lipid (including triacylglycerol), protein and cell wall synthesis, and an increase in glycolytic flux over a range from sub- to superambient oxygen concentrations. In contrast to lipid synthesis, starch synthesis was not significantly increased at superambient oxygen levels. The levels of fermentation products such as lactate and glycerol-3P increased only at very low (0%-4% [v/v]) external oxygen concentrations. (d). When 14C-acetate or 14C-acetyl-coenzyme A (CoA) was injected into seeds, label incorporation into triacylglycerol progressively increased over the whole range of external oxygen concentrations from 0% to 60% (v/v). (e). Stimulation of lipid synthesis was accompanied by an increase in sugar levels and a decrease in the levels of hexose-phosphates and acetyl-CoA, indicating sucrose unloading and the use of acetyl-CoA as possible regulatory sites. (f). Increased lipid synthesis was also accompanied by an increase in the maximal activities of invertase and diacylglycerol acyltransferase. (g). The developmental shift from starch to lipid storage between 15 and 45 d after flowering was accompanied by an increase in the seed energy state. (h). The results show that at ambient oxygen levels, the oxygen supply is strongly limiting for energy metabolism and biosynthetic fluxes in growing rape seeds, affecting lipid synthesis more strongly than starch synthesis. The underlying mechanisms and implications for strategies to increase yield and storage product composition in oilseed crops are discussed.

Ontogenetic study of the effects of energetic nutrients on amino acid metabolism of rat cerebral cortex.

We studied the effect of various energetic nutrients on metabolism of L-[U-14C]leucine and [1-14C]glycine in cerebral cortex of rats at different ages. At gestational age, glucose and lactate stimulated protein synthesis from L-[U-14C]leucine and [1-14C]glycine and from L-[U-14C]leucine, respectively; glucose, beta-OH-butyrate and lactate stimulated lipid synthesis from L-[U-14C]leucine. At 10 days of age, glucose, mannose, and fructose stimulated protein synthesis, and glucose and mannose stimulated oxidation to CO2 as well as lipid synthesis from L-[U-14C]leucine. In adult rats, glucose, mannose, and fructose stimulated protein synthesis from L-[U-14C]leucine and [1-14C]glycine; glutamine also markedly decreased the oxidation of L-[U-14C]leucine and [1-14C]glycine in 10-day-old and adult rats.

Insulin selectively increases SREBP-1c mRNA in the livers of rats with streptozotocin-induced diabetes.

Sterol regulatory element binding proteins (SREBPs) enhance transcription of genes encoding enzymes of cholesterol and fatty acid biosynthesis and uptake. In the current experiments, we observed a decline in the mRNA encoding one SREBP isoform, SREBP-1c, in the livers of rats that were rendered diabetic by treatment with streptozotocin. There was no change in the mRNA encoding SREBP-1a, which is derived from the same gene as SREBP-1c but uses a different promoter. The ratio of SREBP-1c:1a transcripts fell 25-fold from 5:1 in control rats to 0.2:1 in the diabetic animals. The SREBP-1c mRNA rose nearly to normal, and the 1c:1a ratio increased 17-fold when the diabetic rats were treated for 6 h with insulin. These treatments produced no change in the mRNA for SREBP-2, which is encoded by a separate gene. The SREBP-1c mRNA also fell selectively in freshly isolated rat hepatocytes and rose when the cells were treated with insulin. Considered together with recent data on hepatocytes [Foretz, M., Pacot, C., Dugal, I., et al. (1999) Mol. Cell. Biol. 19, 3760-3768], the current in vivo studies suggest that insulin may stimulate lipid synthesis in the liver by selectively inducing transcription of the SREBP-1c gene.

Increased Levels of Nuclear SREBP-1c Associated with Fatty Livers in Two Mouse Models of Diabetes Mellitus

Hepatic steatosis is common in non-insulin-dependent diabetes and can be associated with fibrosis and cirrhosis in a subset of individuals. Increased rates of fatty acid synthesis have been reported in livers from rodent models of diabetes and may contribute to the development of steatosis. Sterol regulatory element-binding proteins (SREBPs) are a family of regulated transcription factors that stimulate lipid synthesis in liver. In the current studies, we measured the content of SREBPs in livers from two mouse models of diabetes, obese ob/ob mice and transgenic aP2-SREBP-1c436 (aP2-SREBP-1c) mice that overexpress nuclear SREBP-1c only in adipose tissue. The aP2-SREBP-1c mice exhibit a syndrome that resembles congenital generalized lipodystrophy in humans. Both lines of mice develop hyperinsulinemia, hyperglycemia, and hepatic steatosis. Nuclear SREBP-1c protein levels were significantly elevated in livers from ob/ob and aP2-SREBP-1c mice compared with wild-type mice. Increased nuclear SREBP-1c protein was associated with elevated mRNA levels for known SREBP target genes involved in fatty acid biosynthesis, which led to significantly higher rates of hepatic fatty acid synthesis in vivo. These studies suggest that increased levels of nuclear SREBP-1c contribute to the elevated rates of hepatic fatty acid synthesis that leads to steatosis in diabetic mice.

Chloromethyl ketones are insulin-like stimulators of lipid synthesis in locust fat body.

N alpha-tosyl-L-lysine chloromethyl ketone (TLCK) stimulates lipid synthesis in locust fat body in vitro, and is able to reverse the inhibitory effects of AKH-I on lipid synthesis. Effective stimulatory concentrations of TLCK were in the range of 0.2-1.0 mM. Similar stimulatory effects were also achieved with phenylalanine chloromethyl ketone (PheCK) and leucine chloromethyl ketone (LeuCK), but not with tosyl-phenylalanine chloromethyl ketone (TPCK), dansyl-glu-gly-arg-CK, chloroacetone, chloroacetic acid, chloroacetamide, chloroacetaldehyde, chloroacetyl-L-leucine or acetylated or fluorescamine-labelled TLCK, PheCK, and LeuCK. The level of stimulation caused by TLCK was dependent on incubation time, so that after a 5-h preincubation of fat body tissue with TLCK the stimulated rate was severalfold higher than the control. TLCK also increased the rate of uptake of trehalose and uridine, but not glucose, deoxyglucose or glycine. Increasing concentrations of bovine serum albumin (BSA) in the incubation medium caused a reduction in the rate of TLCK-stimulated acetate uptake, such that levels of uptake were no higher with 1% BSA than in the controls. A range of more specific protease and kinase inhibitors was tested, but none caused stimulation; thus the mode of action of TLCK on the stimulation of acetate uptake has yet to be identified. Elucidation of the mode of action of TLCK may facilitate the development of novel compounds for insect pest control.

Retinoids stimulate lipid synthesis and accumulation in LNCaP prostatic adenocarcinoma cells

In a previous report we demonstrated that androgens markedly stimulate accumulation of lipid droplets in LNCaP cells. The effects were already evident at low concentrations of androgens optimal for proliferation but became much more pronounced at high concentrations optimal for differentiation. In the present report we explored whether other agonists acting by nuclear receptors and modulating LNCaP growth and differentiation also affect lipid accumulation. The agonists investigated were 1 α,25-dihydroxycholecalciferol (VD 3), all- trans-retinoic acid (atRA), and triiodothyronine (T 3). Lipid accumulation was evaluated by Oil Red O staining followed by image analysis of Oil Red O-stained cells or by extraction and measurement of absorbency. Only marginal effects were noted for VD 3 and T 3. The atRA, on the contrary, increased lipid staining 5–12-fold. This effect required high concentrations of retinoids (10 −6 M) and was accompanied by growth stimulation. Lipid accumulation was less pronounced than that observed with maximally effective concentrations of androgens (10 −3 M R1881). Thin layer chromatography (TLC) and enzymatic determination of the various lipid fractions demonstrated that retinoids increase triacylglycerides and an unidentified lipid fraction with a slightly higher mobility. In contrast with androgens, however, they did not stimulate the accumulation of cholesterol esters. Incorporation studies with [2- 14C]acetate revealed that the increased accumulation of the mentioned lipids is related both to increased synthesis and to decreased secretion. Retinoid-induced lipid accumulation is accompanied by increased steady-state levels of the mRNA encoding fatty acid synthase (FAS), a key enzyme involved in lipid synthesis, while the expression of HMG-CoA-reductase, an enzyme controlling cholesterol synthesis is only marginally affected. It is concluded that retinoids share the ability of androgens to increase lipid accumulation in LNCaP cells. The nature of the lipids affected by both agonists, however, differs at least in part suggesting that the underlying mechanisms may also be different. For the studied compounds (androgens, VD 3, atRA, and T 3) no simple and consistent relationship could be observed between their ability to decrease proliferation and increase differentiation on the one hand and their ability to promote lipid accumulation on the other hand.

Analysis of lipid metabolism in adipocytes using a fluorescent fatty acid derivative. I. Insulin stimulation of lipogenesis

Stimulation of lipid synthesis (lipogenesis) is one of the most pronounced metabolic actions of insulin. Here we demonstrate insulin-stimulated lipogenesis in isolated rat adipocytes using a fatty acid derivative which carries a fluorophore. Three major fluorescent lipid products (lipids 1, 2, 3) are generated as revealed by TLC analysis and subsequent fluorescent scanning or imaging. Lipolytic digestion and labeling studies suggest monoacylglycerol-3-phosphate and diacylglycerol (-3-phosphate) structures harboring a single fluorescent fatty acyl residue each for lipids 1 and 3 (2), respectively. Fluorescent triglycerides are not generated. Assaying acylation with isolated microsomes using the purified lipids 1 and 3 indicates that incorporation of one fluorescent fatty acyl residue into glycerol(-3-phosphate) interferes with subsequent esterification. Pretreatment of the adipocytes with insulin significantly stimulates synthesis of lipids 1 and 2, only. The insulin concentration–response relationship (EC 50=0.5 nM) and the maximal insulin response for synthesis of lipid 1 (stimulation factor=14- to 20-fold at low glucose and 3- to 7-fold at high glucose) are comparable with those for incorporation of [3- 3H]glucose into total adipocyte lipids. Thus this fluorescence-based assay may be useful for studying insulin action and lipogenesis.

Global Brain Ischemia and Reperfusion

Brain damage accompanying cardiac arrest and resuscitation is frequent and devastating. Neurons in the hippocampus CA 1 and CA 4 zones and cortical layers III and V are selectively vulnerable to death after injury by ischemia and reperfusion. Ultrastructural evidence indicates that most of the structural damage is associated with reperfusion, during which the vulnerable neurons develop disaggregation of polyribosomes, peroxidative damage to unsaturated fatty acids in the plasma membrane, and prominent alterations in the structure of the Golgi apparatus that is responsible for membrane assembly. Reperfusion is also associated in vulnerable neurons with prominent production of messenger RNAs for stress proteins and for the proteins of the activator protein-1 complex, but these vulnerable neurons fail to efficiently translate these messages into the proteins. The inhibition of protein synthesis during reperfusion involves alteration of translation initiation factors, specifically serine phosphorylation of the α-subunit of eukaryotic initiation factor-2 (eIF-2α). Growth factors—in particular, insulin—have the potential to reverse phosphorylation of eIF-2α , promote effective translation of the mRNA transcripts generated in response to ischemia and reperfusion, enhance neuronal defenses against radicals, and stimulate lipid synthesis and membrane repair. There is now substantial evidence that the insulin-class growth factors have neuron-sparing effects against damage by radicals and ischemia and reperfusion. This new knowledge may provide a fundamental basis for a rational approach to "cerebral resuscitation" that will allow substantial amelioration of the often dismal neurologic outcome now associated with resuscitation from cardiac arrest. [White BC, Grossman LI, O'Neil BJ, DeGracia DJ, Neumar RW, Rafols JA, Krause GS: Global brain ischemia and reperfusion. Ann Emerg Med 1996;27:588-594.]

Evidence for an altered lipid metabolic state in circulating blood monocytes under conditions of hyperlipemia in swine and its implications in arterial lipid metabolism.

Circulating blood monocytes were isolated from normal and hypercholesterolemic swine, and the monocyte lipid compositions and lipid biosynthesis profiles were assessed. The data indicate that monocytes freshly isolated from hyperlipemic swine have increased phospholipid and cholesterol contents and have increased biosynthetic capability for synthesizing phospholipids, triglycerides, and cholesteryl esters, but not cholesterol. The profile of the stimulated lipid synthesis capability is similar to that of the swine aortic intima undergoing atherogenic change. These studies indicate that circulating blood monocytes in hyperlipemic swine, which are known to give rise to intimal foam cells in the early fatty streak lesion, can contribute to altered vessel lipid metabolism without a requirement for in situ modification by wall factors.