Lipolytic Products Research Articles

169 - Oxidized neutral lipid lipolysis regulates insulin signaling during acute stress

Acute hyperglycemia and systemic insulin resistance (IR) often develop after injury or surgery. This stress response, appropriately known as critical illness diabetes, leads to increased post-operative complications and mortality. Insulin administration reduces this hyperglycemia in some patients but has a risk of dangerous hypoglycemia. The underlying mechanisms driving IR are unknown, which limits the development of alternative therapies and emphasizes the need to elucidate the pathophysiology. It is, however, known that surgical animal models rapidly develop adipose IR, and impaired insulin action in adipose alone can result in whole-body IR. Therefore, we focused on how adipose contributes to stress-induced IR. We have previously demonstrated using a mouse model of surgery that the development of IR and hyperglycemia are dependent on adipose lipolysis. Furthermore, β-adrenergic stimulation of adipocytes causes inhibition and dissociation of mTORC1 and mTORC2, key steps in the insulin cascade, in a lipolysis-dependent manner. Therefore, we hypothesized that a product of adipose lipolysis was responsible for the development of IR. We found that lipid extracts isolated from forskolin-stimulated 3T3-L1 adipocytes inhibited the activity of mTORC in vitro, however “traditional” lipolytic products (DAG, MAG, fatty acids) had no effect. Interestingly, stimulating peroxidation of fatty acids further exacerbated mTORC inhibition, while antioxidants reversed this effect. This suggests that the active species is an oxidized fatty acid. In fact, incubation of mTORC with in vitro auto-oxidized fatty acids is sufficient to inhibit kinase activity. Therefore, we propose that lipolysis breaks down oxidized TAGs into oxidized fatty acids, which cause mTORC dissociation and attenuation of insulin signaling during the stress response. Our work will elucidate a novel role for oxidized TAGs and their lipolytic products in the regulation of insulin signaling. Defining the mechanism by which catecholamine-stimulated lipolysis attenuates insulin signaling will provide novel therapeutic targets for improved post-operative glucose homeostasis.

Application of in vitro lipolysis for the development of oral self-emulsified delivery system of nimodipine

The objective of the current study was to optimize for the first time the formulation variables of self-emulsified drug delivery system (SEDDS) based on drug solubilization during lipolysis under a biorelevant condition of digestion such as lipase activity, temperature, pH, fed-fasting state, etc. Nimodipine (ND), a BCS class II, was used as a model drug to prepare the SEDDS. Various oils, surfactants, and cosurfactants were screened for their solubilization potential of ND. Area of self-emulsification was identified using various ternary phase diagrams. Box-Behnken design was employed to investigate effects of formulation variables on various dispersion, emulsification, and lipolysis characteristics of SEDDS. Among 26 candidate formulations, highest ND solubility of 12.72%, 11.09% and 11.2% w/w were obtained in peppermint oil as the oily phase, Cremphor EL as the surfactant and PEG400 as the cosurfactant, respectively. Cremphor EL was the most significant factor to decrease SEDDS droplet size to 30.16 nm. On the other hand, increasing the oil concentration was found to significantly increase the polydispersity index up to 0.31. A faster emulsification rate of 3.37%/min was obtained at higher Cremphor El/PEG 400 ratio. Increasing the percentage of lipid components of SEDDS resulted in lower rate of lipolysis with less recovery of ND in aqueous phase. Under fed state, percentage of lipolysis of optimized formulation was less than that observed under fasted state. However, lowest rate and percentage of lipolysis were observed in lipolysis media without phospholipids and bile salts. Hence, this study demonstrated that in vitro lipolysis could be used as a surrogate approach to distinguish effects of formulation variables on fate of SEDDS upon digestion. Further studies are in progress to identify the lipolytic products of the employed excipients by LC-MS/MS.

Studying the real-time interplay between triglyceride digestion and lipophilic micronutrient bioaccessibility using droplet microfluidics. 2 application to various oils and (pro)vitamins

The kinetics of micellar solubilization of lipophilic micronutrients (bioaccessibility) in relation with triglyceride digestion remains poorly known. To study this interplay in real-time, a droplet microfluidic method was designed and used as reported in the first part of this article series. In this second part, the interplay between the micellar solubilization of (pro)vitamins (beta-carotene or retinyl palmitate) and the digestion of triglyceride oils (tricaprylin TC, or high-oleic sunflower seed oil HOSO, or fish oil FO) during simulated gastrointestinal digestion was investigated. The relation between the release of both micronutrients and of triglyceride lipolytic products was found to be non-linear. The kinetics of beta-carotene was found to follow the kinetics of lipolytic products, depending on the oil type (TC > HOSO > FO). The effect of the gastric phase on the intestinal phase was also found to follow this order, mostly due to partial lipolysis during the gastric phase.

Improving Correlations Between Drug Solubilization and In Vitro Lipolysis by Monitoring the Phase Partitioning of Lipolytic Species for Lipid-Based Formulations

Solution proton nuclear magnetic resonance analysis was used in conjunction with in vitro lipolysis to elucidate the time-dependent speciation and release of lipolytic products during the digestion of lipid-loaded inorganic particles, allowing correlations to be made between the phase partitioning of lipolytic products and an encapsulated poorly soluble drug. Silicon dioxide, montmorillonite, and laponite were used to encapsulate medium chain triglycerides into solid-state lipid-based formulations (LBFs), and coumarin 102 was selected as a model poorly soluble compound. The specific inorganic carrier material used to encapsulate medium chain triglycerides significantly impacted the release and partitioning of the solubilizing lipolytic products, that is, diglycerides, monoglycerides, and fatty acids. A strong linear correlation was obtained between drug solubilization and fatty acid release to the aqueous phase (R2 = 0.996), indicating fatty acids to be the most important lipid species for enabling solubilization and potential drug absorption in vivo. This method was developed to improve upon the use of pH-stat titration for characterizing LBF digestion during in vitro lipolysis studies and is demonstrated herein to provide useful insights into how the selected inorganic carrier material impacts LBF performance when solid-state LBF powders are fabricated via adsorption.

Effect of sex on glucose handling by adipocytes isolated from rat subcutaneous, mesenteric and perigonadal adipose tissue.

BackgroundAdult rat epididymal adipocytes are able to convert large amounts of glucose to lactate and glycerol. However, fatty acid efflux is much lower than that expected from glycerol levels if they were the product of lipolysis. Use of glucose for lipogenesis is limited, in contrast with the active glycolysis-derived lactate (and other 3-carbon substrates). In this study, we analyzed whether white adipose tissue (WAT) site and sex affect these processes.MethodsMature adipocytes from perigonadal, mesenteric and subcutaneous WAT of female and male rats were isolated, and incubated with 7 or 14 mM glucose during 1 or 2 days. Glucose consumption, metabolite efflux and gene expression of glycolytic and lipogenesis-related genes were measured.ResultsThe effects of medium initial glucose concentration were minimal on most parameters studied. Sex-induced differences that were more extensive; however, the most marked, distinct, effects between WAT sites, were dependent on the time of incubation. In general, the production of lactate was maintained during the incubation, but glycerol release rates increased with time, shifting from a largely glycolytic origin to its triacylglycerol (TAG) lipolytic release. Glycerol incorporation was concurrent with increased TAG turnover: lipolytic glycerol was selectively secreted, while most fatty acids were recycled again into TAG. Fatty acid efflux increased with incubation, but was, nevertheless, minimal compared with that of glycerol. Production of lactate and glycerol from glucose were maximal in mesenteric WAT.DiscussionFemale rats showed a higher adipocyte metabolic activity than males. In mesenteric WAT, gene expression (and substrate efflux) data suggested that adipocyte oxidation of pyruvate to acetyl-CoA was higher in females than in males, with enhanced return of oxaloacetate to the cytoplasm for its final conversion to lactate. WAT site differences showed marked tissue specialization-related differences. Use of glucose for lipogenesis was seriously hampered over time, when TAG turnover-related lipolysis was activated. We postulate that these mechanisms may help decrease glycaemia and fat storage, producing, instead, a higher availability of less-regulated 3-carbon substrates, used for energy elsewhere.

Inorganic surface chemistry and nanostructure controls lipolytic product speciation and partitioning during the digestion of inorganic-lipid hybrid particles

HypothesisSolid-state lipid formulations, whereby liquid lipids are encapsulated in inorganic particle matrices, have attracted significant interest for drug/nutrient delivery in recent years. We hypothesized that the surface chemistry of the inorganic material used to encapsulate lipids impacts the lipase-mediated digestion and partitioning of lipolytic species between the solubilized aqueous and insoluble pellet phases. ExperimentsMedium chain triglycerides were spray dried with silica nanoparticles, montmorillonite or laponite platelets to form inorganic-lipid hybrid particles. In vitro lipolysis studies were conducted under gastric (pH 1.6) and intestinal (pH 7.5) conditions, and the speciation and partitioning of lipolytic products between the aqueous and pellet phases was characterized using solution-state proton nuclear magnetic resonance and fourier transform infrared spectroscopy. FindingsUnder gastric conditions, greater than 80% of all lipid species remained adsorbed within each lipolysis pellet after 60 min. Approximately 40%, 50–60% and 80–90% of all lipid species were adsorbed from solution by silica-, montmorillonite- and laponite-based particle matrices during intestinal lipolysis. Monoglycerides were preferentially adsorbed by silica, whereas triglycerides and fatty acids were adsorbed by montmorillonite and laponite. Adsorption of lipolytic products from solution is expected to impact significantly on drug/nutrient solubilization and absorption in vivo. To the best of our knowledge, this is the first report characterizing the speciation and phase behavior of lipolytic products released from solid-state lipid formulations during in vitro lipolysis studies.

Solubilisation behaviour of poorly water-soluble drugs during digestion of solid SMEDDS

Lipid based-formulations can enhance the bioavailability of poorly water-soluble lipophilic drugs through enhanced solubilisation of drugs in the gastrointestinal (GI) tract during digestion. This study investigates the solubilisation behaviour of poorly water-soluble drugs upon digestion of solid self-microemulsifying drug delivery system (S-SMEDDS). The S-SMEDDS were prepared using two different core lipids, Gelucire® 44/14 (GEL) or glyceryl monooleate (GMO), and were loaded with two model drugs, fenofibrate (FEN) and cinnarizine (CINN). S-SMEDDS formulations were characterized using wide-angle X-ray scattering (WAXS) and Raman spectroscopy, and their structural behaviour and drug solubilisation behaviour were monitored using drug-related diffraction peaks during digestion under fasted and fed simulated intestinal conditions using time-resolved small and wide-angle X-ray scattering (SAXS/WAXS). The concentrations of FEN and CINN released into the aqueous phase (AP) during digestion were quantified using high-performance liquid chromatography (HPLC). Both model drugs, FEN and CINN, had greater solubility in the GMO-based S-SMEDDS formulations and were partially solubilised into lipid matrix and uniformly distributed in solid formulations. The extent of digestion was greater for the GEL-based systems (92–94%) than GMO-based systems (65–75%) as was the rate of digestion. GEL-based S-SMEDDS formulations formed a lamellar phase during digestion in the fasted state and formed mixed micelles in the fed state. In contrast, the GMO-based system formed the mixed micelles in both intestinal conditions. The time-resolved SAXS profiles revealed solubilisation of crystalline drugs into the lipolysis products. Synchrotron SAXS results were in correlation with the HPLC measurements, confirming the ability of the SAXS technique to monitor drug behaviour and showing that the digestion of S-SMEDDS can enhance drug solubilisation.

Abstract 5835: Adipocyte-released agents induce resistance of breast cancer cells to lapatinib

Abstract Adipose tissue is often reduced to its role in fat storage and metabolic support for tumor cells. However, several studies have shown an adipocyte-mediated resistance effect to various anticancer therapies. However, the responsible agent(s), the mechanism(s) and the signaling pathway(s) involved in this resistance remain unclear. In the present study, we assessed the cytotoxic effect of lapatinib on several breast cancer cell lines in the presence or absence of adipocyte-conditioned medium. We performed different physical and chemical treatments on these conditioned media in order to separate and identify the different molecules and agents involved. In parallel, we investigated the changes that occurs in breast tumor cells following the contact with adipocyte-conditioned medium and the exposure to lapatinib both at transcriptional and protein level. In order to validate our hypothesis in vivo, we created a mouse model reproducing the contact between adipose tissue and tumors. Our results showed that tumor cells exposed to adipocyte-conditioned medium were less sensitive to lapatinib-mediated cytotoxic effects and cell cycle blockade than cells grown in standard culture medium. For example, we found that the IC50 was approximately seven-fold increased with lapatinib for tumor cells exposed to adipocyte-conditioned medium compared to the cells kept in control medium. This resistance was observed with the conditioned medium obtained both from human and murine adipocytes and confirmed on different breast tumor cell lines overexpressing HER2. Using a xenograft of normal human adipose tissue with implantation of tumor cells in contact to this adipose tissue, we also confirmed the protective effect against the antitumoral activity of lapatinib in vivo. The nature of the responsible agents of the resistance has been partly elucidated as well as the mechanisms. Indeed, it seems that the presence of soluble factors released from adipocytes such as lipolysis and metabolism products are required to limit the effect of lapatinib on tumor cells. The exposure of tumor cells to adipocyte-conditioned medium triggered changes on the lapatinib-mediated cell cycle blockade and on the expression of cell cycle regulation dependent proteins such as AKT, P27 and cyclins. In conclusion, compounds released from adipocytes reduced the lapatinib induced cytotoxicity on breast cancer cells overexpressing HER2 by interfering with the AKT signaling pathway. These results suggest possible strategies to counteract tumor cell resistance to lapatinib, by targeting adipocyte-released mediators. Citation Format: Aline Geneste, Minh Ngoc Duong, Aurore Cleret, Sabine Beaumel, Kamel Chettab, Emmanuel Delay, Philippe Valet, Lars Petter Jordheim, Charles Dumontet. Adipocyte-released agents induce resistance of breast cancer cells to lapatinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5835.

Calcium Alters the Interfacial Organization of Hydrolyzed Lipids during Intestinal Digestion.

Calcium plays an important dual role in lipid digestion: promoting removal of long-chain fatty acids from the oil-water interface by forming insoluble calcium soaps while also limiting their bioaccessibility. This becomes more significant in food containing high calcium concentration, such as dairy products. Nevertheless, scarce attention has been paid to the effect of calcium on the interfacial properties during lipid digestion, despite this being largely an interfacial reaction. This study focused on the dynamics of the formation of calcium soaps at the oil-water interface during lipolysis by pancreatic lipase in the absence and presence of the two primary human bile salts (sodium glycocholate or sodium glycochenodeoxycholate). The competitive adsorption of lipase, bile salts, and lipolysis products, as well as the formation of calcium soaps in the presence of increasing concentrations of calcium were mainly characterized by recording the interfacial tension and dilatational modulus in situ. In the absence of bile salts, calcium complexes with fatty acids at the oil-water interface forming a relatively strong viscoelastic network of calcium soaps over time. The dilatational modulus of the calcium soap network is directly related to the interfacial concentration of lipolysis products and the calcium bulk concentration. Calcium soaps are also visualized forming a continuous rough layer on the surface of oil droplets immersed in simulated intestinal aqueous phase. Despite bile salts having different surface activity, they play a similar role on the interfacial competition with lipase and lipolysis products although altering their kinetics. The presence of bile salts disrupts the network of calcium soaps, as suggested by the decrease in the dilatational modulus and the formation of calcium soap islands on the surface of the oil droplets. The accelerant effect of calcium on lipolysis is probably because of fatty acid complexation and subsequent removal from the interface rather than reduced electrostatic repulsion between lipase and bile salt molecules and promoted lipase adsorption. The work shown here has implications for the delivery of oil-soluble bioactives in the presence of calcium.

Early life stress reduces in vivo lipolysis efficiency in female mice fed a high fat diet

Previously, we have shown that female mice exposed to maternal separation and early weaning (MSEW), a model of postnatal neglect, increases fat mass and worsens the cardiometabolic function in response to high fat diet (HF) feeding. We also found that β3 adrenergic receptor mRNA expression, involved in local lipolysis stimulation, was reduced in gonadal white adipose tissue (gWAT). Thus, the aim of this study was to test the hypothesis that female MSEW mice display reduced lipolysis in‐vivo and in‐vitro. MSEW was performed by separating the pups from the mother for periods of 4–8 hr during postnatal days 2–16. Mice were weaned at P17. Control mice remained undisturbed in the home cage all times and weaned at P21. At weaning, mice were placed on a low fat (10% kcal from fat) or high fat (60% kcal from fat) diet for 16 weeks. At week 14, an in‐vivo lipolysis test was performed by injecting with 1mg/kg BW of isoproterenol, IP. Free fatty acids (FFA) were measured in plasma obtained after 15 minutes of the injection. At week 16, an in‐vitro lipolysis test was performed in gWAT. As such, gWAT was removed from female control and MSEW mice, weighed, and washed with ice‐cold PBS. In‐vitro lipolysis was performed in the presence or absence of isoproterenol (10uM, Sigma Aldrich). Free glycerol kit (Sigma Aldrich) was used to measure lipolytic products and results were normalized to tissue weight. In‐vivo, FFAs were reduced in female MSEW mice fed a HF (102±13 uM/ml) when compared with controls (144±24 uM/ml, p=0.066). In‐vitro, we found similar free glycerol levels in control and MSEW mice fed a LF (9.2±0.8 vs. 10.5±1.4 mg/dl/g tissue, respectively). Although HF reduced isoproterenol‐induced free glycerol levels, control and MSEW mice showed not statistically significant differences (4.4±0.6 vs. 3.5±0.6 mg/dl/g tissue, respectively). Taken together, these data indicate that HF reduced lipolysis capacity either in control and MSEW mice. Also, MSEW reduced lipolysis when determined in‐vivo conditions, suggesting that neuroendocrine and autonomic factors may be implicated in the mechanism by which MSEW mice are prone to increase fat mass.Support or Funding InformationThis study was supported by funds from the NIH National Heart, Lung, and Blood Institute to A.S.L (R00 HL111354), start‐up funds from the University of Kentucky to A.S.L, and the pilot project from the University of Kentucky Center of Research in Obesity and Cardiovascular Disease COBRE P20 GM103527‐06 to A.S.L.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lipoprotein Lipase Deficiency Impairs Bone Marrow Myelopoiesis and Reduces Circulating Monocyte Levels.

Tissue macrophages induce and perpetuate proinflammatory responses, thereby promoting metabolic and cardiovascular disease. Lipoprotein lipase (LpL), the rate-limiting enzyme in blood triglyceride catabolism, is expressed by macrophages in atherosclerotic plaques. We questioned whether LpL, which is also expressed in the bone marrow (BM), affects circulating white blood cells and BM proliferation and modulates macrophage retention within the artery. We characterized blood and tissue leukocytes and inflammatory molecules in transgenic LpL knockout mice rescued from lethal hypertriglyceridemia within 18 hours of life by muscle-specific LpL expression (MCKL0 mice). LpL-deficient mice had ≈40% reduction in blood white blood cell, neutrophils, and total and inflammatory monocytes (Ly6C/Ghi). LpL deficiency also significantly decreased expression of BM macrophage-associated markers (F4/80 and TNF-α [tumor necrosis factor α]), master transcription factors (PU.1 and C/EBPα), and colony-stimulating factors (CSFs) and their receptors, which are required for monocyte and monocyte precursor proliferation and differentiation. As a result, differentiation of macrophages from BM-derived monocyte progenitors and monocytes was decreased in MCKL0 mice. Furthermore, although LpL deficiency was associated with reduced BM uptake and accumulation of triglyceride-rich particles and macrophage CSF-macrophage CSF receptor binding, triglyceride lipolysis products (eg, linoleic acid) stimulated expression of macrophage CSF and macrophage CSF receptor in BM-derived macrophage precursor cells. Arterial macrophage numbers decreased after heparin-mediated LpL cell dissociation and by genetic knockout of arterial LpL. Reconstitution of LpL-expressing BM replenished aortic macrophage density. LpL regulates peripheral leukocyte levels and affects BM monocyte progenitor differentiation and aortic macrophage accumulation.

Inhibition of perilipin 2 expression reduces pro-inflammatory gene expression and increases lipid droplet size.

Our lab previously demonstrated that triglyceride-rich lipoprotein (TGRL) lipolysis products induce lipid droplet formation and pro-inflammatory gene expression in monocytes. We hypothesized that the inhibition of perilipin 2 expression in THP-1 monocytes would reduce lipid droplet formation and suppress pro-inflammatory gene expression induced by TGRL lipolysis products. In the current study, we use microarray analysis to identify gene expression altered by TGRL lipolysis products in THP-1 monocytes. We confirmed the expression of selected genes by quantitative reverse transcription PCR and characterized lipid droplet formation in these cells after exposure to TGRL lipolysis products. Using siRNA inhibition of perilipin 2 expression, we examined the role of perilipin 2 in the response of THP-1 monocytes to TGRL lipolysis products. We found that perilipin 2 siRNA increased the intracellular triglyceride content, increased the size of lipid droplets, and reduced pro-atherogenic and pro-inflammatory gene expression. We saw a reduction of serum/glucocorticoid kinase 1, v-maf musculoaponeurotic fibrosarcoma oncogene homolog F (avian), chemokine (C-C motif) ligand 3, and interleukin 8 gene expression induced by TGRL lipolysis products. This study supports previous findings that reduction of perilipin 2 expression is protective against atherogenesis, while finding an unexpected increase in lipid droplet size with reduced perilipin 2 expression.

Periparturient lipolysis and oxylipid biosynthesis in bovine adipose tissues.

The periparturient period of dairy cows is characterized by intense lipolysis in adipose tissues (AT), which induces the release of free fatty acids (FFA) into circulation. Among FFA, polyunsaturated fatty acids are susceptible to oxidation and can modulate inflammatory responses during lipolysis within AT. Linoleic and arachidonic acid oxidized products (oxylipids) such as hydroxy-octadecadienoic acids (HODE) and hydroxy-eicosatetraenoic acids (HETE), were recently identified as products of lipolysis that could modulate AT inflammation during lipolysis. However, the effect of lipolysis intensity during the transition from gestation to lactation on fatty acid substrate availability and subsequent AT oxylipid biosynthesis is currently unknown. We hypothesized that in periparturient dairy cows, alterations in AT and plasma fatty acids and oxylipid profiles coincide with changes in lipolysis intensity and stage of lactation. Blood and subcutaneous AT samples were collected from periparturient cows at -27±7 (G1) and -10±5 (G2) d prepartum and at 8±3 d postpartum (PP). Targeted lipidomic analysis was performed on plasma and AT using HPLC-MS/MS. We report that FFA concentrations increased as parturition approached and were highest at PP. Cows exhibiting high lipolysis rate at PP (FFA>1.0 mEq/L) had higher body condition scores at G1 compared to cows with low lipolysis rate (FFA<1.0 mEq/L). Concentrations of plasma linoleic and arachidonic acids were increased at PP. In AT, 13-HODE, and 5-, 11- and 15-HETE were increased at PP compared to G1 and G2. Concentrations of beta hydroxybutyrate were positively correlated with those of 13-HODE and 15-HETE in AT. Plasma concentrations of 5- and 20-HETE were increased at PP. These data demonstrate that prepartum adiposity predisposes cows to intense lipolysis post-partum and may exacerbate AT inflammation because of increased production of pro-inflammatory oxylipids including 5- and 15-HETE and 13-HODE. These results support a role for certain linoleic and arachidonic acid-derived oxylipids as positive and negative modulators of AT inflammation during periparturient lipolysis.

IR spectroscopy analysis of pancreatic lipase-related protein 2 interaction with phospholipids: 3. Monitoring DPPC lipolysis in mixed micelles

Usual methods for the continuous assay of lipolytic enzyme activities are mainly based on the titration of free fatty acids, surface pressure monitoring or spectrophotometry using substrates labeled with specific probes. These approaches only give a partial information on the chemistry of the lipolysis reaction and additional end-point analyses are often required to quantify both residual substrate and lipolysis products. We used transmission infrared (IR) spectroscopy to monitor simultaneously the hydrolysis of phospholipids by guinea pig pancreatic lipase-related protein 2 (GPLRP2) and the release of lipolysis products. The substrate (DPPC, 1,2-Dipalmitoyl phosphatidylcholine) was mixed with sodium taurodeoxycholate (NaTDC) to form mixed micelles in D2O buffer at pD 6 and 8. After hydrogen/deuterium exchange, DPPC hydrolysis by GPLRP2 (100nM) was monitored at 35°C in a liquid cell by recording IR spectra and time-course variations in the CO stretching region. These changes were correlated to variations in the concentrations of DPPC, lysophospholipids (lysoPC) and palmitic acid (Pam) using calibration curves established with these compounds individually mixed with NaTDC. We were thus able to quantify each compound and its time-course variations during the phospholipolysis reaction and to estimate the enzyme activity. To validate the IR analysis, variations in residual DPPC, lysoPC and Pam were also quantified by thin-layer chromatography coupled to densitometry and similar hydrolysis profiles were obtained using both methods. IR spectroscopy can therefore be used to monitor the enzymatic hydrolysis of phospholipids and obtain simultaneously chemical and physicochemical information on substrate and all reaction products (H-bonding, hydration, acyl chain mobility).

Physical properties and biological fate of OSA-modified-starch-stabilized emulsions containing β-carotene: Effect of calcium and pH

Understanding the roles of the calcium ion in both the physical properties of emulsions and the biological fate of the emulsion during digestion is important for the rational design of emulsion-based delivery systems that can moderate lipid digestion and the bioaccessibility of bioactive compounds. The effect of CaCl2 on the zeta-potential, the particle size, and the microstructure of emulsions, the rate and extent of lipid digestion, and the bioaccessibility of β-carotene in emulsions stabilized by octenyl succinic anhydride (OSA)-modified starches was investigated in an in vitro mouth/stomach/intestine model system. The addition of CaCl2 to starch solutions at pH 7.0 before homogenization decreased the emulsifying ability of the OSA-modified starches. When the concentration of added CaCl2 increased, the particle size of the emulsions increased and the creaming stability decreased. At pH 3.0, CaCl2 had no influence on the physical properties of OSA-modified-starch-stabilized emulsions. However, the influence of calcium on the biological fate of emulsions containing β-carotene was independent of the emulsion pH. The addition of CaCl2 promoted the digestion of lipid but decreased the bioaccessibility of β-carotene in both emulsions made at pH 7.0 and emulsions made at pH 3.0. The effect of CaCl2 on the biological fate of the emulsions was attributed mainly to the interaction of the calcium ion with lipolytic products and bile salts, which seemed to offset the effect of structure and physical properties.

Bile salt stimulated lipase: Inhibition by phospholipids and relief by phospholipase A2

IntroductionBile salt stimulated lipase (BSSL; Enzyme Commission (EC) number 3.1.1.13) has been a candidate triglyceridase for improving enzyme therapy for pancreatic insufficiency; however, its efficacy is near absent. We hypothesise that similarly to pancreatic lipase, BSSL is inhibited by phospholipids and this inhibition is relieved by Phospholipase A2 (PLA2; EC 3.1.1.4), and the present study was undertaken to explore this possibility. Materials and methodsSynthetic emulsions of triglyceride and phosphatidylcholine (PC) or lysophosphatidylcholine (LPC)/bile salt mixed micelles were used as a model of intestinal digestion-media. The effect of PLA2 treatment of systems containing PC on BSSL activity was also explored. Automatic titration at constant pH (pH-stat) and nuclear magnetic resonance (NMR) spectroscopy were used to measure the rate and identify products of lipolysis. ResultsPC was inhibitory to BSSL activity, while LPC became inhibitory only above an LPC/bile salt concentration ratio of 0.3. PLA2 treatment relieved the inhibition only below this ratio, despite its complete phospholipid-hydrolysing action. Thus, LPC had an inhibitory effect at higher concentrations. ConclusionsThese results may implicate a change in the design of enzyme therapy in patients with pancreatic exocrine insufficiency. Supplementation of BSSL with PLA2 could improve patient health with adequate manipulation of phospholipid and lysophospholipid concentrations in the intestinal fluid.

P 198 - Role of ATF3 in mediating lipid-induced stress signaling in brain microvascular endothelial cells

Elevation of blood triglycerides, primarily triglyceride-rich lipoproteins (TGRL), is an independent risk factor for atherosclerotic cardiovascular disease and Vascular Dementia (VaD). The accumulating evidence indicates that the development of atherosclerosis and VaD are linked to vascular inflammation. Previous studies suggest a relationship of blood lipids and/or lipoproteins to vascular inflammation leading to dementia. The objective of this study is to characterize human brain microvascular endothelial cells (HBMVEC) responses to postprandial TGRL and determine the pathways that have potential to increase risk for vascular dementia. To address this question, we treated HBMVEC with TGRL lipolysis products and then we used NGS-based RNA-Seq methods for transcriptional profiling experiments. Our data demonstrated up-regulation of activating transcription factor 3 (ATF3)-dependent inflammatory pathways in HBMVEC. Additionally, system biology analysis exhibited putative lipid-related targets and corresponding isoforms of ATF3 that may be implicated in hypertriglyceridemia-induced vascular inflammation in brain. Furthermore, RNA-Seq analysis determined specific isoforms of key transcriptional regulator ATF3 and stress response signaling pathway in lipolysis products-mediated brain vascular inflammation which may lead to vascular dementia.

Effects of calcium on lipid digestion in nanoemulsions stabilized by modified starch: Implications for bioaccessibility of β-carotene

The aim of this study was to investigate the influence of calcium on and its mechanism in the digestion and bioaccessibility of β-carotene in nanoemulsions stabilized by modified starch, using an in vitro digestion model. Higher concentrations of calcium ions resulted in more irregular aggregates, a greater rate and extent of lipid digestion but a lower bioaccessibility of β-carotene. The major components of the mixed micelles were analyzed to explain the relationship between lipolysis and bioaccessibility. The lipolytic products and bile salts in the mixed micelles decreased with an increase in the calcium concentration because of the formation of calcium soaps and calcium–bile acid complexes. A correlation analysis showed that the bioaccessibility of β-carotene had a highly statistically significant positive linear correlation with both lipolytic products (r = 0.943) and bile salts (r = 0.896) in mixed micelles with calcium. These results illustrated that the lipolytic products and the bile salts in the micelles were the critical factors contributing to the bioaccessibility of β-carotene. This study could help in an understanding of the function of calcium in the digestion of delivery systems and the bioaccessibility of encapsulated lipophilic functional ingredients.

Energy regulation in newly diagnosed TB with chronic energy deficiency: free fatty acids and RBP4.

Energy metabolism may be dysfunctionally integral between host and infective agent in active tuberculosis, mediated by adipocytokines and free fatty acids (FFA) as the products of triglyceride lipolysis in fat, blood or other tissues. Retinol Binding Protein 4 (RBP4) and asymmetric dimethylarginine (ADMA) are candidate adipocytokines. The possibility of a deleterious metabolic nexus in chronic energy deficiency (CED) (BMI <18.5 kg/m2) is explored. Newly diagnosed patients with tuberculosis (n=63) were selected using consecutive random sampling at a Centre for the Care and Treatment of Lung Diseases in Makassar, Indonesia. Diagnosis of pulmonary TB required microscopy with Ziehl-Neelsen stain. Anthropometric measurements were taken. Venesection allowed glomerular filtration rate, FFA, serum glutamic oxaloacetic transaminase and glutamate-pyruvate transaminase to be assessed. CED was evident in 60.3%. For the well and lesser nourished, medians were, respectively, FFA 0.30 and 0.37 mmol/mL (p=0.960); RBP4 199730 ng/mL and 11721 ng/mL (p=0.009); GFR 106 ml/min and 113 ml/min (p=0.673); and ADMA 0.52 ng/mL and 0.51 ng/mL (p=0.172). BMI and serum RBP4 were correlated (ρ=0.52, p<0.001), with odds ratios (OR) 5.8 (CI 1.68-20.3). RBP4 in CED was lower than in better nourished patients. Serum FFA is not evidently associated with BMI in patients with active TB. RBP4 is some 6-fold lower when active TB patients have CED than when BMI >25 kg/m2. However, FFA was not associated with CED in these active TB patients which may be a type 2 error or represent an energy impasse where infection and the host's metabolic needs are in competition.

Incorporation of lipolysis in monolayer permeability studies of lipid-based oral drug delivery systems.

Lipid-based drug delivery systems, a well-tolerated class of formulations, have been evaluated extensively to enhance the bioavailability of poorly soluble drugs. However, it has been difficult to predict the in vivo performance of lipid dosage forms based on conventional in vitro techniques such as cell monolayer permeability studies because of the complexity of the gastrointestinal processing of lipid formulations. In the current study, we explored the feasibility of coupling Caco-2 and Madin-Darby canine kidney monolayer permeability studies with lipolysis, a promising in vitro technique to evaluate lipid systems. A self-emulsifying lipid delivery system was formulated using a blend of oil (castor oil), surfactant (Labrasol® or PL497), and co-surfactant (lecithin). Formulations demonstrating high drug solubility and rapid self-emulsification were selected to study the effect of lipolysis on in vitro cell permeability. Lipolysis of the formulations was carried out using pancreatin as the digestive enzyme. All the digested formulations compromised monolayer integrity as indicated by lowered trans-epithelial electrical resistance (TEER) and enhanced Lucifer yellow (LY) permeability. Further, the changes in TEER value and LY permeability were attributable to the digestion products of the formulation rather than the individual lipid excipients, drug, digestion enzyme, or the digestion buffer. The digested formulations were fractionated into pellet, oily phase, and aqueous phase, and the effect of each of these on cell viability was examined. Interestingly, the aqueous phase, which is considered important for in vivo drug absorption, was responsible for cytotoxicity. Because lipid digestion products lead to disruption of cell monolayer, it may not be appropriate to combine lipolysis with cell monolayer permeability studies. Additional in vivo studies are needed to determine any potential side effects of the lipolysis products on the intestinal permeability barrier, which could determine the suitability of lipid-based systems for oral drug delivery.