The response to fasting and refeeding reveals functional regulation of lipoprotein lipase proteoforms.

Lipoprotein lipase (LPL) is responsible for the intravascular catabolism of triglyceride-rich lipoproteins and plays a central role in whole-body energy balance and lipid homeostasis. As such, LPL is subject to tissue-specific regulation in different physiological conditions, but the mechanisms of this regulation remain incompletely characterized. Previous work revealed that LPL comprises a set of proteoforms with different isoelectric points, but their regulation and functional significance have not been studied thus far. Here we studied the distribution of LPL proteoforms in different rat tissues and their regulation under physiological conditions. First, analysis by two-dimensional electrophoresis and Western blot showed different patterns of LPL proteoforms (i.e., different pI or relative abundance of LPL proteoforms) in different rat tissues under basal conditions, which could be related to the tissue-specific regulation of the enzyme. Next, the comparison of LPL proteoforms from heart and brown adipose tissue between adults and 15-day-old rat pups, two conditions with minimal regulation of LPL in these tissues, yielded virtually the same tissue-specific patterns of LPL proteoforms. In contrast, the pronounced downregulation of LPL activity observed in white adipose tissue during fasting is accompanied by a prominent reconfiguration of the LPL proteoform pattern. Furthermore, refeeding reverts this downregulation of LPL activity and restores the pattern of LPL proteoforms in this tissue. Importantly, this reversible proteoform-specific regulation during fasting and refeeding indicates that LPL proteoforms are functionally diverse. Further investigation of potential differences in the functional properties of LPL proteoforms showed that all proteoforms exhibit lipolytic activity and have similar heparin-binding affinity, although other functional aspects remain to be investigated. Overall, this study demonstrates the ubiquity, differential distribution and specific regulation of LPL proteoforms in rat tissues and underscores the need to consider the existence of LPL proteoforms for a complete understanding of LPL regulation under physiological conditions.

Lipoprotein lipase isoelectric point isoforms in humans.

Lipoprotein lipase (LPL) hydrolyzes circulating triacylglycerols (TAG) into free fatty acids and glycerol. It is present in almost all tissues and its tissue-specific regulation directs the flow of circulating TAG in the body. We demonstrated in a previous study that, in rat heart and post-heparin plasma (PHP), LPL consists of a pattern of more than 8 forms of the same apparent molecular weight, but different isoelectric point (pI). In the present study we describe, for the first time, the existence of at least nine LPL pI isoforms in human PHP, with apparent pI between 6.8 and 8.6. Separation and characterization of these forms was carried out by 2DE combined with Western blotting and mass spectrometry (MALDI-TOF/MS and LC-MS/MS). Further studies are needed to discover their molecular origin, the pattern of pI isoforms in human tissues, their possible physiological functions and possible modifications of their pattern in different pathologies.

Lipoprotein lipase is nitrated in vivo after lipopolysaccharide challenge.

Lipopolysaccharide (LPS) administration down-regulates lipoprotein lipase (LPL) activity at the posttranscriptional level. Hypertriglyceridemia is the main metabolic consequence of this fall in LPL activity and is presumably involved in the innate immune response to infection. Nitric oxide (NO) has been implicated in LPS-induced down-regulation of LPL activity, but whether its effects are direct or indirect remains unclear. Here we examined the potential nitration of LPL in vivo in response to LPS challenge in rats. We found hypertriglyceridemia, iNOS expression, NO overproduction, and a generalized decrease in LPL activity in tissues 6 h after LPS administration. LPL sensitivity to nitration was first explored by in vitro exposure of bovine LPL to peroxynitrite, a reactive nitrogen species (RNS). Nitration was confirmed by anti-nitrotyrosine Western blot and subsequent identification of specific nitrotyrosine-containing LPL sequences by tandem mass spectrometry. Further analysis by targeted mass spectrometry revealed three in vivo-nitrated tyrosine residues in heart LPL from LPS-challenged rats. This is the first study to identify nitrated tyrosine residues in LPL, both in vitro and in vivo, and it demonstrates that LPL is a target for RNS in endotoxemia. These results indicate that LPL nitration may be a new mechanism of LPL activity regulation in vivo.

Discovery of lipoprotein lipase pI isoforms and contributions to their characterization.

Lipoprotein lipase (LPL) plays a pivotal role in lipid metabolism and is implicated in several pathophysiological conditions. A large number of LPL studies have been performed in rat, although the amount of information derived from direct study of the protein in this species is limited. Here we attempted to examine possible modifications of LPL using proteomic tools. By combining high-resolution two-dimensional gel electrophoresis and Western blot with biological mass spectrometry we demonstrate the coexistence of multiple LPL pI isoforms in rat heart. We studied the origin of this pI heterogeneity by: (1) comparison with the 2D pattern of LPL from post-heparin rat plasma (as a source of mature LPL); (2) protein dephosphorylation; (3) protein deglycosylation; and (4) partial sequencing of LPL isoforms. The results reveal that LPL pI heterogeneity does not correspond to different stages of intracellular maturation or protein phosphorylation. It can be partially explained by glycosylation, although other post-translational modifications must also be involved. We also report the first partial sequence to be obtained from direct study of rat LPL protein. These findings should be the basis for further research aimed at identifying the molecular differences between LPL isoforms and exploring their potential functional implications.

Lipoprotein lipase expression in livers of morbidly obese patients could be responsible for liver steatosis.

BACKGROUND: Most patients with morbid obesity develop non-alcoholic fatty liver disease (NAFLD). The origins of lipid deposition in the liver and the effects of bariatric surgery in the obese with NAFLD are controversial. METHODS: We analyzed lipids and lipoprotein lipase (LPL) in both plasma and liver biopsies performed before and 12-18 months after Roux-en-Y gastric bypass surgery in 26 patients. RESULTS: In the livers of morbidly obese patients, the levels of LPL messenger RNA (mRNA) were higher (4.5-fold) before surgery than afterwards than control livers. In these patients, LPL activity was also significantly higher (91 +/- 7 mU/g) than in controls (51 +/- 3 mU/g, p = 0.0026) and correlated with the severity of the liver damage. All hepatic lipids were significantly increased in obese patients; however, after bariatric surgery, these lipids, with the exception of NEFA, tended to recover to normal levels. CONCLUSIONS: The liver of obese patients presented higher LPL activity than controls, and unlike the controls, this enzyme could be synthesized in the liver because it also present LPL mRNA. The presence of the LPL activity could enable the liver to capture circulating triacylglycerides, thus favoring the typical steatosis observed in these patients.

Nitric oxide and the release of lipoprotein lipase from white adipose tissue.

BACKGROUND/AIM: Lipoprotein lipase (LPL) is the main enzyme responsible for the distribution of circulating triacylglycerides in tissues. Its regulation via release from active sites in the vascular endothelium is poorly understood. In a previous study we reported that in response to acute immobilization (IMMO), LPL activity rapidly increases in plasma and decreases in white adipose tissue (WAT) in rats. In other stress situations IMMO triggers a generalized increase in nitric oxide (NO) production. METHODS/RESULTS: Here we demonstrate that in rats: 1) in vivo acute IMMO rapidly increases NO concentrations in plasma 2) during acute IMMO the WAT probably produces NO via the endothelial isoform of nitric oxide synthase (eNOS) from vessels, and 3) epididymal WAT perfused in situ with an NO donor rapidly releases LPL from the endothelium. CONCLUSION: We propose the following chain of events: stress stimulus / rapid increase of NO production in WAT (by eNOS) / release of LPL from the endothelium in WAT vessels. This chain of events could be a new mechanism that promotes the rapid decrease of WAT LPL activity in response to a physiological stimulus.

Application of proteomic tools to detect the nonspecificity of a polyclonal antibody against lipoprotein lipase.

Specific antibodies are essential tools for studying proteins. P66 is a chicken polyclonal antibody raised against bovine lipoprotein lipase (LPL) that has been used in earlier studies. Here, we developed a two-dimensional (2D) Western blot with reducing gels, using commercial bovine LPL (53 kDa) as a standard and P66 for detection. Our results revealed incomplete purification of commercial LPL and nonspecificity of P66, both undetectable in one-dimensional analysis. Antithrombin III (ATIII) was identified as both a major contaminant in commercial LPL and a cross-reacting protein with P66. Although LPL purification methods were presumably designed to eliminate ATIII, here we demonstrate that some procedures fell short of this objective and thus led to the production of a nonspecific antibody. Our results define 2D electrophoresis/Western blot and mass spectrometric protein identification as the most reliable procedure for validating LPL purity and the specificity of antibodies against this enzyme.

Social stress profoundly affects lipid metabolism: over-expression of SR-BI in liver and changes in lipids and lipases in plasma and tissues of stressed mice.

We examined the effect of chronic social stress, similar to that endured by humans, on lipid metabolism of mice. The activity of the lipoprotein lipase (LPL) enzyme increased in adrenals, while in plasma it diminished significantly. Hepatic lipase (HL) was strongly affected in liver and adrenal glands, increasing four-fold and three-fold, respectively. At the same time, scavenger receptor class-B type-I (SR-BI), which are considered the high-density lipoprotein (HDL) receptor in the liver, increased significantly. Although the adrenals do not synthesise HL, the increase in HL may facilitate the uptake of HDL cholesterol for the synthesis of corticoids, which increase significantly following chronic stress. The volume of adrenal glands in control animals was significantly higher than in stressed animals (1.23+/-0.12 mm3 versus 0.29+/-0.06 mm3, p<0.001), corresponding with the weight difference of these organs. Medulla volume was also different in the two groups (0.27+/-0.10 mm3 versus 0.04+/-0.02 mm3, p<0.05). Despite this, corticosterone in plasma was significantly higher in stressed animals. Our results shows, for the first time, the effect of chronic social stress on lipid metabolism in general, and in particular on the SR-BI receptor and HL, which is directly involved in cholesterol reverse transport.

Retroperitoneal white adipose tissue lipoprotein lipase activity is rapidly down-regulated in response to acute stress.

Tissue-specific regulation of LPL has been widely studied in rats. Previous studies reported that in vivo administration of adrenaline and acute stress cause an increase in plasma LPL activity coinciding with a decrease in white adipose tissue (WAT) LPL activity. We studied the speed of LPL activity changes during 30 min of stress by immobilization (IMMO) in rats. A first experimental approach in permanently cannulated rats permitted sequential blood sampling in the same animal during IMMO and the obtaining of hemodynamic parameters. In a second experimental approach, animals were euthanized at different times after the start of IMMO to determine LPL activity in tissues. Stress was characterized by rises in blood pressure, heart rate, plasma corticosterone, and available circulating energy substrates. Five min after the start of IMMO, LPL activity fell in retroperitoneal WAT and increased in plasma. These data show the quickest LPL activity change ever described in response to a physiological situation. The speed and simultaneity of these changes suggest that the release from endothelium to the bloodstream may constitute a fast nonexplored mechanism of tissue LPL activity regulation, involved in the lipid energy-substrate redistribution between tissues needed to prepare the "fight-or-flight" response.

Changes in lipoprotein lipase modulate tissue energy supply during stress.

We studied the variations caused by stress in lipoprotein lipase (LPL) activity, LPL-mRNA, and local blood flow in LPL-rich tissues in the rat. Stress was produced by body immobilization (Immo): the rat's limbs were taped to metal mounts, and its head was placed in a plastic tube. Chronic stress (2 h daily of Immo) decreased total LPL activity in mesenteric and epididymal white adipose tissue (WAT) and was accompanied by a weight reduction of these tissues. In limb muscle, heart, and adrenals, total LPL activity and mRNA levels increased, and, in plasma, LPL activity and mass also increased. Acute stress (30-min Immo) caused a decrease in total LPL activity only in retroperitoneal WAT and an increase in preheparin plasma active LPL, but the overall weight of this tissue did not vary significantly. We propose an early release of the enzyme from this tissue into the bloodstream by some unknown extracellular pathways or other local mechanisms. These changes in this key energy-regulating enzyme are probably induced by catecholamines. They modify the flow of energy substrates between tissues, switching the WAT from importer to exporter of free fatty acids and favoring the uptake by muscle of circulating triacylglycerides for energy supply. Moreover, we found that acute stress almost doubled blood flow in all WAT studied, favoring the export of free fatty acids.

An in situ perfusion protocol of rat epididymal adipose tissue useful in metabolic studies.

Experimental approaches involving the perfusion of tissues and organs offer the advantage of improved physiological relevance over the use of isolated tissues or cells while at the same time being much more controlled and tissue-specific than studies in vivo. Nevertheless, there have been few metabolic studies performed in perfused white adipose tissue, largely because of the difficulty of the surgical technique involved. Although some methods have been described, they are difficult to use as perfusion protocols and their reproducibility is poor. We have modified a rat perfusion method, based on a modification of the Ho and Meng technique, for use with epididymal white adipose tissue (eWAT), and we present it here as a protocol to be reproduced. We also offer surgical solutions for the most common variants of vessel distributions in rats. Using the protocol described here, the perfused adipose tissue is viable and metabolically active, as indicated by the maintenance of tissue ATP levels and adiponectin secretion and by endogenous lipolysis regulation. Moreover, there is a high level of lipoprotein lipase activity in the endothelium of the tissue, which is heparin-releasable. Thus, this method is a useful and reproducible tool that allows the perfusion of eWAT for use in metabolic studies.

Isoproterenol increases active lipoprotein lipase in adipocyte medium and in rat plasma.

White adipose tissue (WAT) lipoprotein lipase (LPL) activity channels diet fat towards storage in adipocytes. Adrenaline (ADR) is accepted to reduce WAT or adipocyte LPL activity (LPLa), but available data are not clear-cut regarding long exposure to ADR in vitro or in vivo. We studied the effects of long exposures to ADR or beta-adrenergic agonist on LPL: in isolated rat adipocytes (3 h) and in rats (>1 day). Isoproterenol (ISO) (1 microM) did not alter LPLmRNA nor LPLa in adipocytes, but increased LPLa in medium more than twofold (3.58 +/- 0.35 vs. 1.32 +/- 0.35 mU/10(6) adipocytes, P < 0.001). Effect was time (not present at 1 h, clear at 2 h) and concentration dependent (high sensitivity from 10 to 100 nM, max at 1 microM). Adenylate cyclase activator or cyclic AMP (cAMP) analogue produced a similar increase. Thus in adipocytes ISO produced an increase in LPLa release and/or a decrease in extracellular LPLa degradation. ADR or ISO treated rats had a two to fourfold decrease in WAT LPLa vs. unchanged LPLmRNA. This decrease was 10-fold in WAT heparin-releasable LPLa (5.7 +/- 0.6 vs. 57.3 +/- 10.2 mU/g, P < 0.001), which represents peri/extracellular LPLa. Plasma LPLa was increased 11-fold by ADR (3.30 +/- 0.58 vs. 0.32 +/- 0.08 mU/ml, P < 0.001) whereas only threefold by ISO (P > 0.01). We suggest that in vivo ADR increased release of active LPL to plasma from endothelial cells of LPL-rich tissue(s)-WAT was probably one of these tissues releasing LPL since it lost 90% of its peri/extracellular LPLa-and/or decreased degradation of plasma active LPL. Since liver LPLa was not increased, plasma active LPL might be kept away from hepatic degradation by binding to stabilising entities in plasma (fatty acids (FA), lipoproteins or soluble heparan sulphates (HS)). In conclusion, we believe this is the first report stating that: (a) ISO increases LPLa in isolated adipocyte medium, and (b) ADR administration to rats decreases WAT extracellular active LPL and increases preheparin plasma active LPL.

Inactive hepatic lipase in rat plasma.

Hepatic lipase activity is detectable in liver but also in adrenal glands, ovaries, and plasma. The subunit size of hepatic lipase in liver, adrenal glands, and nonheparin plasma was compared. Hepatic lipase in liver and adrenal glands appeared as a 55 kDa band. In liver, a faint band of lower size was also detected. In nonheparin plasma, hepatic lipase appeared as a doublet of 57 kDa and 59 kDa. When activity/mass ratio was calculated, similar values were obtained for liver and adrenal glands. In plasma this value was much lower. After heparin administration in vivo, hepatic lipase activity in plasma increased nearly 100-fold with appearance of an additional 55 kDa band in postheparin plasma. This band coeluted with activity after preparative polyacrylamide gel electrophoresis. Differences in size persisted after digestion with peptide-N-glycosidase F. A progressive increase in 57 kDa and 59 kDa in postheparin plasma followed disappearance of the 55 kDa band, suggesting that these larger bands originate from the smaller form. In plasma, both smaller and larger forms were associated with HDL, but not with LDL or VLDL. We conclude that rat plasma contains a larger form of hepatic lipase that is inactive in in vitro assay.

Acute regulation of hepatic lipase secretion by rat hepatocytes.

Hepatic lipase is involved in cholesterol uptake by the liver. Although it is known that catecholamines are responsible for the daily variation of enzyme activity, the mechanisms involved are poorly understood. Rat hepatocytes incubated with adrenaline or other Ca(2+)-mobilizing hormones were used as an experimental model. Adrenaline reduced in a similar proportion the secretion of both hepatic lipase and albumin. The effect of adrenaline disappeared completely in cells exposed to cycloheximide. Adrenaline decreased incorporation of [35S]Met into cellular and secreted proteins, but it affected neither degradation of [35S]Met-prelabeled proteins nor the abundance of total and specific (albumin, hepatic lipase, beta-actin) mRNA. Other Ca(2+)-mobilizing agents had the opposite effect on hepatic lipase secretion: it was decreased by vasopressin but was increased by epidermal growth factor. Vasopressin and epidermal growth factor had the opposite effect on [35S]Met incorporation into cellular and secreted proteins, but neither affected hepatic lipase mRNA. The acute effect of adrenaline, vasopressin, and epidermal growth factor on hepatic lipase secretion is the consequence of the effect of these hormones on protein synthesis and is therefore nonspecific.

Immobilization stress alters intermediate metabolism and circulating lipoproteins in the rat.

In humans, stress can increase the risk of cardiovascular disease by altering lipoprotein metabolism. Scarce experimental and clinical data are available on this effect. Therefore, we studied the metabolic response to acute and chronic stress following a model of immobilization (IMO) in rats and we evaluated the resulting circulating lipoprotein levels. Repeated IMO treatment (2 hours daily, always between 9:00 AM and 11:00 AM, for 2 periods of 5 and 4 consecutive days, separated by 2 days of rest) daily decreased body weight gain and food intake, increased adrenal weight, and slightly reduced liver glycogen and plasma insulin (without considerable variations of blood glucose), which is characteristic of chronic stress. A single IMO application (30 minutes of an unexpected IMO starting at 2:00 PM immediately before the animals were killed) significantly increased the circulating levels of corticosterone, glucose, insulin, glycerol, and ketone bodies, which is the typical response to acute stress. Both acute and chronic stress decreased the plasmatic triacylglycerol (TAG) concentration, as reflected by the reduction in the number of very-low-density lipoprotein (VLDL) particles. This may be due to an increase in the metabolization of TAG, as suggested by the slightly higher amounts of circulating LDLs. Chronic stress, but not acute stress, significantly increased both the number and the estimated size of circulating high-density lipoprotein (HDLs), as shown by the plasma cholesterol concentration. Acute stress did not have an additive effect over chronic stress on the lipoprotein parameters studied. The metabolic effects of these IMO-induced alterations on lipoprotein profiles are discussed, and future studies in lipidic metabolism are suggested.

Ultracentrifugation micromethod for preparation of small experimental animal lipoproteins.

Sequential flotation ultracentrifugation is commonly used in the preparation of plasma lipoproteins. However, protocols often require prolonged centrifugation time (48-72 h) and large plasma volumes (2-20 ml), which makes them unsuitable for studies on small laboratory animals. Although analytical techniques such as FPLC have often small sample requirements, further fraction analysis is often limited to the small fraction volume obtained. A sequential ultracentrifugation micromethod is described to obtain rat lipoprotein fractions from 400 microl of plasma in a cumulative centrifugation time of 7.5 h. Fraction volumes were determined and densities were adjusted to those of rat plasma lipoproteins. Polyacrylamide gel electrophoresis and enzymatic measurements of triglycerides, total cholesterol, and phospholipids were used to assess the purity of the lipoprotein fractions. The results were compared with those obtained from a classical sequential ultracentrifugation protocol. The micromethod presented here can be further adapted to other experimental animal species with little modifications.