The bovine vitreous-derived lipid factor (bVLF) is a powerful inhibitor of retinal pigmented epithelial (hRPE) cell proliferation

Human retinal pigmented epithelial cell (hRPE) proliferation plays a significant role in various proliferative diseases associated to the retina that leads to loss of vision, such as proliferative vitreoretinopathy. In the current study, the role of the bovine vitreous lipid factor (bVLF) in hRPE cell proliferation has been investigated. bVLF is a bioactive lipid isolated from the bovine vitreous body with strong Ca(2+)-mobilizing activity in fibroblast. In the first approach, the effects of bVLF on Ca(2+)-mobilizing activity were investigated in hRPE. The results showed that bVLF induced, in a dose-dependent manner, a Ca(2+) mobilization from PA-sensitive intracellular stores [non-Ins(1,4,5)P(3)-sensitive stores], in which extracellular Ca(2+) participated. The increase in intracellular Ca(2+) was associated with a dose-dependent inhibiting effect on cell proliferation. At a dose of 10 microg/mL, bVLF caused a 26% or a 44% inhibition in hRPE cell proliferation during the 3- or the 6-day culture periods, respectively. These effects appear to be specific in hRPE cells, since EFGR-T17 fibroblast cells treated with equivalent amounts of bVLF did not show any inhibiting effects. This inhibitory action was not associated to apoptotic/necrotic processes. Furthermore, bVLF inhibited EGF-, bFGF-, IGF-I-, PDGF-, HGF- and VEGF-induced proliferation of the hRPE cells. Moreover, this inhibitory response was also observed in FBS-induced hRPE cell proliferation. bVLF, at a concentration of 10 microg/mL, induced 16% inhibition of proliferation during a culture period of 3 days. This inhibitory action was greater during the 6-day culture period, exceeding 40%. With regard to this action, the results showed that bVLF has a potent inhibitory effect on ERK1/2 activation, and plays a key role in the control of hRPE cell proliferation. These observations contribute to the knowledge of inhibitory factors responsible for keeping antiproliferative environment that preserve the RPE-associated activities in normal states. It advances the interesting possibility that this factor or a factor with characteristics common to bVLF might be involved in the pathogenesis of abnormal proliferative eye processes.     

Signaling Cascade of Diacylglycerol Kinase �� in the Pituitary Intermediate Lobe: Dopamine D2 Receptor/Phospholipase C��4/Diacylglycerol Kinase ��/Protein Kinase C��

The pituitary gland dynamically changes its hormone output under various pathophysiological conditions. One of the pathways implicated in the regulatory mechanism of this gland is a dopaminergic system that operates the phosphoinositide (PI) cycle to transmit downstream signal through second messengers. We have previously shown that diacylglycerol kinase β (DGKβ) is coexpressed with dopamine D1 and D2 receptors in medium spiny neurons of the striatum, suggesting a plausible implication of DGKβ in dopaminergic transmission. However, it remains elusive whether DGKβ is involved in the dopaminergic system in the pituitary gland. The aim of this study is to investigate the expression and localization of DGK in the pituitary gland, together with the molecular components involved in the PI signaling cascade, including dopamine receptors, phospholipase C (PLC), and a major downstream molecule, protein kinase C (PKC). Here we show that DGKβ and the dopamine D2 receptor are coexpressed in the intermediate lobe and localize to the plasma membrane side by side. In addition, we reveal that PLCβ4 and PKCα are the subtypes expressed in the intermediate lobe among those families. These findings will substantiate and further extend our understanding of the molecular-anatomical pathway of PI signaling and the functional roles of DGK in the pituitary intermediate lobe. (J Histochem Cytochem 58:119–129, 2010)     

Nuclear phosphoinositide kinases and inositol phospholipids

The presence of inositol phospholipids in the nuclei of mammalian cells has by now been well established, as has the presence of the enzymes responsible for their metabolism. However, our understanding of the role of these nuclear phosphoinositides in regulating cellular events has lagged far behind that for its cytosolic counterpart. It is clear, though, that the nuclear phosphoinositide pool is independent of the cytosolic pool and is, therefore, likely to be regulating a unique set of cellular events. As with its cytosolic phosphoinositides, many nuclear phosphoinositides and their metabolic enzymes are located at distinct sub-cellular structures. This arrangement spatially limits the production and activity of inositol phospholipids and is believed to be a major mechanism for regulating their function. Here, we will introduce the components of nuclear inositol phospholipid signal transduction and discuss how their spatial arrangement may dictate which nuclear functions they are modulating.     

The lipogenic enzymes DGAT1, FAS, and LPL in adipose tissue: effects of obesity, insulin resistance, and TZD treatment

Acyl-coenzyme A:diacylglycerol transferase (DGAT), fatty acid synthetase (FAS), and LPL are three enzymes important in adipose tissue triglyceride accumulation. To study the relationship of DGAT1, FAS, and LPL with insulin, we examined adipose mRNA expression of these genes in subjects with a wide range of insulin sensitivity (SI). DGAT1 and FAS (but not LPL) expression were strongly correlated with SI. In addition, the expression of DGAT1 and FAS (but not LPL) were higher in normal glucose-tolerant subjects compared with subjects with impaired glucose tolerance (IGT) (P < 0.005). To study the effects of insulin sensitizers, subjects with IGT were treated with pioglitazone or metformin for 10 weeks, and lipogenic enzymes were measured in adipose tissue. After pioglitazone treatment, DGAT1 expression was increased by 33 +/- 10% (P < 0.05) and FAS expression increased by 63 +/- 8% (P < 0.05); however, LPL expression was not altered. DGAT1, FAS, and LPL mRNA expression were not significantly changed after metformin treatment. The treatment of mice with rosiglitazone also resulted in an increase in adipose expression of DGAT1 by 2- to 3-fold, as did the treatment of 3T3 F442A adipocytes in vitro with thiazolidinediones. These data support a more global concept suggesting that adipose lipid storage functions to prevent peripheral lipotoxicity.     

Identification of novel phospholipid binding proteins in Saccharomyces cerevisiae

A proteomics approach was used to search for novel phospholipid binding proteins in Saccharomyces cerevisiae. Phospholipids were immobilized on a solid support and the lipids were probed with soluble yeast protein extracts. From this, the phosphatidic acid binding proteins were eluted and identified by mass spectrometry. Thirteen proteins were identified and 11 of these were previously unknown lipid binding proteins. The protein-lipid interactions identified would not have been predicted using bioinformatics approaches as none possessed a known lipid binding motif. A subset of the identified proteins was purified to homogeneity and determined to directly bind phospholipids immobilized on a solid support or organized into liposomes. This simple approach could be systematically applied to perform an exhaustive screen for soluble lipid binding proteins in S. cerevisiae or other organisms.     

Stimulation of arachidonic acid release by vasopressin in A7r5 vascular smooth muscle cells mediated by Ca2+-stimulated phospholipase A2

Arachidonic acid (AA) regulates many aspects of vascular smooth muscle behaviour, but the mechanisms linking receptors to AA release are unclear. In A7r5 vascular smooth muscle cells pre-labelled with (3)H-AA, vasopressin caused a concentration-dependent stimulation of 3H-AA release that required phospholipase C and an increase in cytosolic [Ca2+]. Ca2+ release from intracellular stores and Ca2+ entry via L-type channels or the capacitative Ca2+ entry pathway were each effective to varying degrees. Selective inhibitors of PLA2 inhibited the 3H-AA release evoked by vasopressin, though not the underlying Ca2+ signals, and established that cPLA2 mediates the release of AA. We conclude that in A7r5 cells vasopressin stimulates AA release via a Ca2+-dependent activation of cPLA2.     

Liver fat reduction with niacin is influenced by DGAT-2 polymorphisms in hypertriglyceridemic patients

Niacin reduces plasma triglycerides, but it may increase free fatty acids and insulin resistance during long-term treatment. We examined the effect of extended-release niacin on liver fat content in Chinese patients with dyslipidemia and whether the common diacylglycerol acyltransferase-2 (DGAT2) polymorphisms influenced this effect. The 39 patients (baseline liver fat content: 12.8 ± 7.6%, triglycerides: 3.30 ± 1.67 mmol/l) were treated with niacin, gradually increasing the dose to 2 g/day for a total of 23 weeks. The liver fat content and visceral/subcutaneous fat was measured before and after treatment. Subjects were genotyped for the DGAT2 rs3060 and rs101899116 polymorphisms. There were significant (P < 0.001) reductions in plasma triglycerides (-34.9 ± 37.6%), liver fat content (-47.2 ± 32.8%), and visceral fat (-6.3 ± 15.8%, P < 0.05) after niacin treatment. Mean body weight decreased by 1.46 ± 2.7% (1.17 ± 2.44 kg, P < 0.001) during the study, but liver fat changes remained significant after adjustment for age, gender, and body weight changes [mean absolute change (95% CI): -6.1% (-8.0, -4.3), P < 0.001]. The DGAT2 variant alleles were associated with a smaller reduction in liver fat content in response to niacin after adjustment for other covariates (P < 0.01). These findings suggest that niacin treatment may reduce liver fat content in Chinese patients with dyslipidemia and that the mechanism may involve inhibition of DGAT2. However, the findings might have been confounded by the small but significant reductions in body weight during the study. Future large randomized controlled trials are needed to verify these findings.     

Intestinal DGAT1 deficiency reduces postprandial triglyceride and retinyl ester excursions by inhibiting chylomicron secretion and delaying gastric emptying

Acyl CoA:diacylglycerol acyltransferase (DGAT) 1 catalyzes the final step of triglyceride (TG) synthesis. We show that acute administration of a DGAT1 inhibitor (DGAT1i) by oral gavage or genetic deletion of intestinal Dgat1 (intestine-Dgat1^−/−) markedly reduced postprandial plasma TG and retinyl ester excursions by inhibiting chylomicron secretion in mice. Loss of DGAT1 activity did not affect the efficiency of retinol esterification, but it did reduce TG and retinoid accumulation in the small intestine. In contrast, inhibition of microsomal triglyceride transfer protein (MTP) reduced chylomicron secretion after oral fat/retinol loads, but with accumulation of dietary TG and retinoids in the small intestine. Lack of intestinal accumulation of TG and retinoids in DGAT1i-treated or intestine-Dgat1^−/− mice resulted, in part, from delayed gastric emptying associated with increased plasma levels of glucagon-like peptide (GLP)-1. However, neither bypassing the stomach through duodenal oil injection nor inhibiting the receptor for GLP-1 normalized postprandial TG or retinyl esters excursions in the absence of DGAT1 activity. In summary, intestinal DGAT1 inhibition or deficiency acutely delayed gastric emptying and inhibited chylomicron secretion; however, the latter occurred when gastric emptying was normal or when lipid was administered directly into the small intestine. Long-term hepatic retinoid metabolism was not impacted by DGAT1 inhibition.     

The BUME method: a novel automated chloroform-free 96-well total lipid extraction method for blood plasma

Lipid extraction from biological samples is a critical and often tedious preanalytical step in lipid research. Primarily on the basis of automation criteria, we have developed the BUME method, a novel chloroform-free total lipid extraction method for blood plasma compatible with standard 96-well robots. In only 60 min, 96 samples can be automatically extracted with lipid profiles of commonly analyzed lipid classes almost identically and with absolute recoveries similar or better to what is obtained using the chloroform-based reference method. Lipid recoveries were linear from 10-100 μl plasma for all investigated lipids using the developed extraction protocol. The BUME protocol includes an initial one-phase extraction of plasma into 300 μl butanol:methanol (BUME) mixture (3:1) followed by two-phase extraction into 300 μl heptane:ethyl acetate (3:1) using 300 μl 1% acetic acid as buffer. The lipids investigated included the most abundant plasma lipid classes (e.g., cholesterol ester, free cholesterol, triacylglycerol, phosphatidylcholine, and sphingomyelin) as well as less abundant but biologically important lipid classes, including ceramide, diacylglycerol, and lyso-phospholipids. This novel method has been successfully implemented in our laboratory and is now used daily. We conclude that the fully automated, high-throughput BUME method can replace chloroform-based methods, saving both human and environmental resources.     

Re-evaluating lipotoxic triggers in skeletal muscle: relating intramyocellular lipid metabolism to insulin sensitivity

Ectopic fat accumulation has been linked to lipotoxic events, including the development of insulin resistance in skeletal muscle. Indeed, intramyocellular lipid storage is strongly associated with the development of type 2 diabetes. Research during the last two decades has provided evidence for a role of lipid intermediates like diacylglycerol and ceramide in the induction of lipid-induced insulin resistance. However, recently novel data has been gathered that suggest that the relation between lipid intermediates and insulin resistance is less straightforward than has been previously suggested, and that there are several routes towards lipid-induced insulin resistance. For example, research in this field has shifted towards imbalances in lipid metabolism and lipid droplet dynamics. Next to imbalances in key lipogenic and lipolytic proteins, lipid droplet coat proteins appear to be essential for proper intramyocellular lipid storage, turnover and protection against lipid-induced insulin resistance.Here, we discuss the current knowledge on lipid-induced insulin resistance in skeletal muscle with a focus on the evidence from human studies. Furthermore, we discuss the available data that provides supporting mechanistic information.