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.

Apo AIV and Citrulline Plasma Concentrations in Short Bowel Syndrome Patients: The Influence of Short Bowel Anatomy.

INTRODUCTION: Parenteral nutrition (PN) dependence in short bowel syndrome (SBS) patients is linked to the functionality of the remnant small bowel (RSB). Patients may wean off PN following a period of intestinal adaptation that restores this functionality. Currently, plasma citrulline is the standard biomarker for monitoring intestinal functionality and adaptation. However, available studies reveal that the relationship the biomarker with the length and function of the RSB is arguable. Thus, having additional biomarkers would improve pointing out PN weaning. AIM: By measuring concomitant changes in citrulline and the novel biomarker apolipoprotein AIV (Apo AIV), as well as taking into account the anatomy of the RSB, this exploratory study aims to a better understanding of the intestinal adaptation process and characterization of the SBS patients under PN. METHODS: Thirty four adult SBS patients were selected and assigned to adapted (aSBS) and non-adapted (nSBS) groups after reconstructive surgeries. Remaining jejunum and ileum lengths were recorded. The aSBS patients were either on an oral diet (ORAL group), those with intestinal insufficiency, or on oral and home parenteral nutrition (HPN group), those with chronic intestinal failure. Apo AIV and citrulline were analyzed in plasma samples after overnight fasting. An exploratory ROC analysis using citrulline as gold standard was performed. RESULTS: Biomarkers, Apo AIV and citrulline showed a significant correlation with RSBL in aSBS patients. In jejuno-ileocolic patients, only Apo AIV correlated with RSBL (rb = 0.54) and with ileum length (rb = 0.84). In patients without ileum neither biomarker showed any correlation with RSBL. ROC analysis indicated the Apo AIV cut-off value to be 4.6 mg /100 mL for differentiating between the aSBS HPN and ORAL groups. CONCLUSIONS: Therefore, in addition to citrulline, Apo AIV can be set as a biomarker to monitor intestinal adaptation in SBS patients. As short bowel anatomy is shown to influence citrulline and Apo AIV plasma values, both biomarkers complement each other furnishing a new insight to manage PN dependence.

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.

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.

Clinical approach to intestinal maturation in neonates prenatally exposed to alcohol.

AIM: The need for a non-invasive diagnosis of the effects of ethanol in utero on the development of the intestine in humans led us to look for a serum marker of the structural integrity of the intestine. We propose apolipoprotein A-IV (apoA-IV) as a possible candidate. In humans this protein is synthesized only by intestinal mucosa, it is expressed in the enterocyte of the foetus from 20 weeks of gestation, and it is released to the blood stream after synthesis. METHODS: We measured the levels of apoA-IV in the umbilical cord serum of neonates whose mothers had consumed alcohol during pregnancy and neonates born to women who had not (controls). The gestational age at delivery of the cases studied ranged from 36 to 42 weeks. ELISA and Western blot analysis were used. RESULTS: There was no difference in the mean body weight of neonates from either group. Nevertheless, exposure to ethanol in utero significantly reduced (by about 30%) the apoA-IV levels in serum at birth, regardless of body weight. CONCLUSION: Our findings suggest that circulating apoA-IV levels could be used as a clinical marker of the prenatal effects of ethanol on the structural integrity of the intestine. Neonatal diagnosis of these intestinal effects could improve post-natal outcome.

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.

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.