Dietary Triacylglycerol Research Articles

Effect of feed supplementation with docosahexaenoic acid in regio- and enantiopure triacylglycerols on gut metabolome and microbiota in rats

Docosahexaenoic acid [22:6(n-3), DHA] plays an important role in human physiology including gut health. This research aimed to investigate the impact of positional distribution of DHA in dietary triacylglycerols (TAG) on gut metabolomic profile and microbiota. In the 4-week feeding trial, the Sprague-Dawley rats were fed on an n-3 deficient feed supplemented with TAG containing DHA at the sn-1, 2, or 3 position and palmitic acid at the remaining positions. Three groups receiving standard n-3 adequate feed, n-3 deficient feed, or n-3 deficient feed supplemented with tripalmitin were included as controls. The gut metabolome was studied using LC–MS-based non-targeted metabolomics, and microbiota profiles were investigated by 16S rRNA sequencing. Compared to the n-3 adequate diet, four-week feeding on the n-3 deficient diet affected the fecal pyrimidine metabolism, steroid biosynthesis, and arginine and proline metabolism. Feeding with DHA-containing TAGs, especially TAG with DHA at sn-3 position, increased the level of N5-Carboxy aminoimidazole ribonucleotide related to purine biosynthesis and dimethylbenzimidazole involved in vitamin B2 biosynthesis. N-3 deficient diet lowered the abundance of the genus Alistipes and the species Bacteroides massiliensis in the gut microbiota. Compared to the n-3 deficient groups, feeding with DHA-containing TAGs decreased the abundance of species Prevotella sp. CAG: 1031, and feeding with sn-2 DHA resulted in an increase in the abundance of Bacteroides fragilis and a decrease in the abundance of Faecalibacterium sp. CAP 74. This is the first study showing that dietary DHA from different positions of TAG may affect gut metabolites and microbiota differently.

Intestinal Acyl-CoA synthetase 5 (ACSL5) deficiency potentiates postprandial GLP-1 & PYY secretion, reduces food intake, and protects against diet-induced obesity

ObjectiveIn the small intestine, the products of digestion of dietary triacylglycerol (TAG), fatty acids (FA) and monoacylglycerol, are taken up by absorptive cells, enterocytes, for systemic energy delivery. These digestion products can also bind receptors on endocrine cells to stimulate the release of hormones capable of influencing systemic energy metabolism. The initial phase of intestinal FA absorption involves the acylation of FAs to acyl-CoA by the acyl-CoA long chain synthetase (ACSL) enzymes. ACSL5 is abundantly expressed in the small intestinal epithelium where it is the major ACSL isoform, contributing approximately 80% of total ACSL activity. In mice with whole body deficiency of ACSL5, the rate of dietary fat absorption is reduced and energy expenditure is increased. However, the mechanisms by which intestinal ACSL5 contributes to intestinal FA metabolism, enteroendocrine signaling, and regulation of energy expenditure remain undefined. Here, we test the hypothesis that intestinal ACSL5 regulates energy metabolism by influencing dietary fat absorption and enteroendocrine signaling. MethodsTo explore the role of intestinal ACSL5 in energy balance and intestinal dietary fat absorption, a novel mouse model of intestine specific ACSL5 deficiency (ACSL5IKO) was generated by breeding ACSL5 floxed (ACSL5loxP/loxP) to mice harboring the tamoxifen inducible, villin-Cre recombinase. ACSL5IKO and control, ACSL5loxP/loxP mice were fed chow (low in fat) or a 60% high fat diet (HFD), and metabolic phenotyping was performed including, body weight, body composition, insulin and glucose tolerance tests, energy expenditure, physical activity, and food intake studies. Pair-feeding studies were performed to determine the role of food intake in regulating development of obesity. Studies of dietary fat absorption, fecal lipid excretion, intestinal mucosal FA content, and circulating levels of glucagon like peptide 1 (GLP-1) and peptide YY (PYY) in response to a TAG challenge were performed. Treatment with a GLP-1 receptor antagonist was performed to determine the contribution of GLP-1 to acute regulation of food intake. ResultsWe found that ACSL5IKO mice experienced rapid and sustained protection from body weight and fat mass accumulation during HFD feeding. While intestine specific deficiency of ACSL5 delayed gastric emptying and reduced dietary fat secretion, it did not result in increased excretion of dietary lipid in feces. Energy expenditure and physical activity were not increased in ACSL5IKO mice. Mice deficient in intestinal ACSL5 display significantly reduced energy intake during HFD, but not chow feeding. When HFD intake of control mice was matched to ACSL5IKO during pair-feeding studies, no differences in body weight or fat mass gain were observed between groups. Postprandial GLP-1 and PYY were significantly elevated in ACSL5IKO mice secondary to increased FA content in the distal small intestine. Blockade of GLP-1 signaling by administration of a long-acting GLP-1 receptor antagonist partially restored HFD intake of ACSL5IKO. ConclusionsThese data indicate that intestinal ACSL5 serves as a critical regulator of energy balance, protecting mice from diet-induced obesity exclusively by increasing satiety and reducing food intake during HFD feeding. The reduction in food intake observed in ACSL5IKO mice is driven, in part, by increased postprandial GLP-1 and PYY secretion. These effects are only observed during HFD feeding, suggesting that altered processing of dietary fat following intestinal ACSL5 ablation contributes to GLP-1 and PYY mediated increases in satiety.

Metabolomic Investigation of Brain and Liver in Rats Fed Docosahexaenoic Acid in Regio- and Enantiopure Triacylglycerols.

N-3 polyunsaturated fatty acids (n-3 PUFAs) play important roles in cognitive functions. However, there is a lack of knowledge on the metabolic impact of regio- and stereo-specific positioning of n-3 PUFAs in dietary triacylglycerols. Rats in a state of mild n-3 PUFA deficiency are fed daily with 360mg triacylglycerols containing DHA (docosahexaenoic acid) at sn (stereospecific numbering)-1, 2, or 3 positions and 18:0 at remaining positions, or an equal amount of tristearin for 5 days. Groups fed with n-3 deficient diet and normal n-3 adequate diet are included as controls. The metabolic profiles of the brain and liver are studied using NMR (nuclear magnetic resonance)-based metabolomics. Several metabolites of significance in membrane integrity and neurotransmission, and glutamate, in particular, are significantly lower in the brain of the groups fed with sn-1 and sn-3 DHA compared to the sn-2 DHA group. Further, the tristearin and DHA groups show a lower lactate level compared to the groups fed on normal or n-3 deficient diet, suggesting a prominent role of C18:0 in regulating energy metabolism. This study sheds light on the impact of stereospecific positioning of DHA in triacylglycerols and the role of dietary stearic acid on metabolism in the brain and liver.

Influence of Dietary Triacylglycerol Structure on the Accumulation of Docosahexaenoic Acid [22:6(n-3)] in Organs in a Short-Term Feeding Trial with Mildly Omega-3 Deficient Rats.

To study the effect of positional distribution of docosahexaenoic acid (DHA) in dietary triacylglycerols (TAG) on the tissue fatty acid content and composition of mildly (n-3) deficient rats. In a 5-day feeding trial, mildly (n-3) deficient rats received 360mg daily structured TAGs: sn-22:6(n-3)-18:0-18:0, sn-18:0-18:0-22:6(n-3), sn-18:0-22:6(n-3)-18:0, or tristearin. A fifth group receives standard (n-3) adequate feed AIN-93G from birth till the end of the trial. The DHA-fed groups show significantly higher DHA levels in the liver and visceral fat compared to the tristearin or normal feed groups showing that the dose and the short feeding period of DHA were sufficient to restore the DHA content in the organs of (n-3) deficient rats. Feeding sn-1 DHA resulted in higher levels of DHA in the liver TAG compared to sn-3 DHA feeding, although the difference did not reach statistical significance. These findings indicated a possible difference in the tissue accumulation and/or metabolic fate of DHA from the sn-1 and sn-3 positions of TAG.

Identifying edible plants with high anti-obesity potential: In vitro inhibitory effect against pancreatic lipase followed by bioactive compound metabolomic screening

The ever-growing number of individuals facing obesity related health problems represents an alarming situation. Within the available obesity control strategies, there is evidence that some natural compounds can inhibit pancreatic lipase (PNLIP, EC 3.1.1.3), a digestive enzyme with pivotal role in the hydrolysis of dietary triacylglycerols, resulting in reduced fat metabolism. In this study, to identify sources of bioactive compounds with anti-obesity potential, 278 extracts originating by 126 food species were prepared in water and dichloromethane (DCM) and their in vitro inhibitory potential against PNLIP was measured. Importantly, 12 extracts exceeded the set cut-off level of significant inhibitory effect (inhibition> 70%) and for them dose-response curves were prepared and metabolomic target screening was performed. By using ultra-high-performance liquid chromatography hybrid quadrupole time-of-flight mass spectrometry (UHPLC-q-TOF-MS), it was possible to tentatively identify 28 and 22 secondary metabolites in the aqueous and DCM extracts, respectively. Several species-specific compounds were detected, e.g., hesperidin and eriocitrin in lemon and lime fruit aqueous extracts or curcuminoids in DCM turmeric extracts. Additionally, the highest percentage of the total analytical signal detected in dill, walnut, caper and bell pepper DCM extracts corresponded to various fatty acids. All in all, the edible plant extracts with strong anti-obesity potential contained various bioactive phytochemicals that may be used in nutraceutical applications.

Metabolic fate of DHA from regio- and stereospecific positions of triacylglycerols in a long-term feeding trial in rats

This study investigated the impact of regio- and stereospecific position of docosahexaenoic acid (DHA) in dietary triacylglycerols (TAGs) on the fatty acid composition of tissues and organs in rats. Four-week feeding with TAGs containing DHA in sn-1, 2, or 3 position and palmitic acid in the remaining positions at a daily dosage of 500 mg TAG/kg body weight significantly increased the DHA content in all organs and tissues in rats, except in the brain, where the change in DHA level was not statistically significant. The group fed sn-1 DHA showed a significantly higher content of DHA in the plasma TAG than the group fed sn-3 DHA. The sn-3 DHA group had higher levels of DHA in the visceral fat compared to the sn-1, sn-2, as well as all other groups. This is the first study showing that DHA from sn-1 and sn-3 positions of dietary TAGs have differential accumulation in tissues. The new findings improved the current knowledge on the significance of TAG isomeric structure for the bioavailability and metabolic fate of DHA.

Solvent-Free Enzymatic Synthesis of Dietary Triacylglycerols from Cottonseed Oil in a Fluidized Bed Reactor.

The synthesis of structured lipids with nutraceutical applications, such as medium-long-medium (MLM) triacylglycerols, via modification of oils and fats represents a challenge for the food industry. This study aimed to synthesize MLM-type dietary triacylglycerols by enzymatic acidolysis of cottonseed oil and capric acid (C10) catalyzed by Lipozyme RM IM (lipase from Rhizomucor miehei) in a fluidized bed reactor (FBR). After chemical characterization of the feedstock and hydrodynamic characterization of the reactor, a 22 central composite rotatable design was used to optimize capric acid incorporation. The independent variables were cycle number (20-70) and cottonseed oil/capric acid molar ratio (1:2-1:4). The temperature was set at 45 °C. The best conditions, namely a 1:4 oil/acid molar ratio and 80 cycles (17.34 h), provided a degree of incorporation of about 40 mol%, as shown by compositional analysis of the modified oil. Lipozyme RM IM showed good operational stability (kd = 2.72 × 10-4 h-1, t1/2 = 2545.78 h), confirming the good reuse capacity of the enzyme in the acidolysis of cottonseed oil with capric acid. It is concluded that an FBR configuration is a promising alternative for the enzymatic synthesis of MLM triacylglycerols.

Petroselinic Acid from Dietary Triacylglycerols Reduces the Concentration of Arachidonic Acid in Tissue Lipids of Rats

Studies in vitro have revealed that triacylglycerols containing petroselinoyl [18:1(n-12)] moieties are hydrolyzed by pancreatic lipase at much lower rates than other triacylglycerols. To assess the lipolysis and absorption in vivo of such unusual triacylglycerols, diets containing 120 g seed oil triacylglycerols of coriander (Coriandrum sativum) per kg diet at a level of 72 g 18:1(n-12) moieties/100 g oil were fed to a group of weaned male Wistar rats without restriction for a period of 10 wk. For comparison, groups of rats were fed similar isocaloric diets containing plant oil triacylglycerols with various levels of oleoyl [18:1(n-9)] moieties, e.g., high oleic sunflower seed oil [75 g 18:1(n-9)/100 g oil], olive oil [(66 g 18:1(n-9)/100 g oil], medium oleic rapeseed oil [54 g 18:1(n-9)/100 g oil] and conventional high linoleic sunflower seed oil [25 g 18:1(n-9)/100 g oil]. All diets were supplemented with 20 g corn oil/kg diet. Consumption of coriander oil, compared with the other oils, led to significantly greater liver weights. No significant differences were observed among the groups fed various levels of oleic acid in body weight, the weights of heart, liver, kidneys, spleen or testes, lipid content of heart, or total cholesterol, HDL cholesterol and triacylglycerol concentrations of blood plasma. Ingestion of coriander oil led to incorporation of 18:1(n-12) into heart, liver and blood lipids and to a significant reduction in the concentration of arachidonic acid in the lipids of heart, liver and blood with a concomitant increase in the concentration of linoleic acid compared with results for the other groups. Our data show that petroselinic acid from dietary triacylglycerols is absorbed by rats as readily as oleic acid, but the former reduces the concentration of arachidonic acid in tissue lipids.

Probing dietary triacylglycerol metabolism and meibogenesis in mice: A stable isotope-labeled tracer liquid chromatography–tandem mass spectrometry study

Exocrine Meibomian glands (MGs) play a central role in the ocular physiology and biochemistry by producing in situ and, mostly, de novo, a secretion (meibum), which is composed of a complex mixture of homologous lipids of various classes, in a metabolic pathway termed meibogenesis. Recent in vivo experiments with a number of mouse models demonstrated that inactivation of any of the major genes of meibogenesis led to alterations in the lipid composition of meibum and severe ocular and MG abnormalities that replicated various human ocular pathologies. However, the role of dietary lipids in meibogenesis, and in the onset and/or alleviation of these diseases, remains controversial. To uncover the role of dietary lipids, the metabolic transformations of a dietary lipid tracer - stable isotope-labeled glyceryl tri(oleate-1,2,3,7,8-13C5) (13C15-TO) - were investigated using LC-high-resolution TOF-MS/MS. We demonstrated that major metabolic transformations of the tracer occurred in the stomach and small intestines where 13C15-TO underwent immediate and extensive transesterification into 13C5- and 13C10-substituted triacylglycerols of various lengths, giving a mixture of 13C-labeled compounds that remain virtually unchanged in the mouse plasma, liver, and white adipose tissue, but were almost undetectable in the feces. Importantly, the tracer and its metabolites were virtually undetectable in MGs, even after 4 weeks of daily supplementation. Notably, unbiased Principal Component Analysis of the data revealed no measurable changes in the overall chemical composition of meibum after the treatment, which implies no direct effect of dietary triacylglycerols on meibogenesis, and left their systemic effects as the most likely mechanism.

Dietary triacylglycerol hydroperoxide is not absorbed, yet it induces the formation of other triacylglycerol hydroperoxides in the gastrointestinal tract

The in vivo presence of triacylglycerol hydroperoxide (TGOOH), a primary oxidation product of triacylglycerol (TG), has been speculated to be involved in various diseases. Thus, considerable attention has been paid to whether dietary TGOOH is absorbed from the intestine. In this study, we performed the lymph duct-cannulation study in rats and analyzed the level of TGOOH in lymph following administration of a TG emulsion containing TGOOH. As we successfully detected TGOOH from the lymph, we hypothesized that this might be originated from the intestinal absorption of dietary TGOOH [hypothesis I] and/or the in situ formation of TGOOH [hypothesis II]. To determine the validity of these hypotheses, we then performed another cannulation study using a TG emulsion containing a deuterium-labeled TGOOH (D2-TGOOH) that is traceable in vivo. After administration of this emulsion to rats, we clearly detected unlabeled TGOOH instead of D2-TGOOH from the lymph, indicating that TGOOH is not absorbed from the intestine but is more likely to be produced in situ. By discriminating the isomeric structures of TGOOH present in lymph, we predicted the mechanism by which the intake of dietary TGOOH triggers oxidative stress (e.g., via generation of singlet oxygen) and induces in situ formation of TGOOH. The results of this study hereby provide a foothold to better understand the physiological significance of TGOOH on human health.

Mechanisms of intestinal triacylglycerol synthesis

Triacylglycerols are a major source of stored energy that are obtained either from the diet or can be synthesized to some extent by most tissues. Alterations in pathways of triacylglycerol metabolism can result in their excessive accumulation leading to obesity, insulin resistance, cardiovascular disease and nonalcoholic fatty liver disease. Most tissues in mammals synthesize triacylglycerols via the glycerol 3-phosphate pathway. However, in the small intestine the monoacylglycerol acyltransferase pathway is the predominant pathway for triacylglycerol biosynthesis where it participates in the absorption of dietary triacylglycerol. In this review, the enzymes that are part of both the glycerol 3-phosphate and monoacylglycerol acyltransferase pathways and their contributions to intestinal triacylglycerol metabolism are reviewed. The potential of some of the enzymes involved in triacylglycerol synthesis in the small intestine as possible therapeutic targets for treating metabolic disorders associated with elevated triacylglycerol is briefly discussed.

2-Arachidonoyl glycerol potently induces cholecystokinin secretion in murine enteroendocrine STC-1 cells via cannabinoid receptor CB1.

Cholecystokinin (CCK) is a peptide hormone secreted from enteroendocrine cells and regulates the exocrine pancreas, gastric motility, and appetite. Dietary triacylglycerols are hydrolyzed to fatty acids (FA) and 2-monoacylglycerols (2-MAG) in the small intestine. Although it is well known that FA stimulate CCK secretion, whether 2-MAG have the CCK-releasing activity remains unclear. We examined the CCK-releasing activity of four commercially available 2-MAG in a murine CCK-producing cell line, STC-1, and the molecular mechanism underlying 2-MAG-induced CCK secretion. CCK released from the cells was measured using ELISA. Among four 2-MAG (2-palmitoyl, 2-oleoyl, 2-linoleoyl, and 2-arachidonoyl monoacylglycerols) examined, 2-arachidonoyl glycerol (2-AG) potently stimulated CCK secretion in a dose-dependent manner. Structurally related compounds, such as 2-arachidonoyl glycerol ether and 1-arachidonoyl glycerol, did not stimulate CCK secretion. Both arachidonic acid and 2-AG stimulated CCK secretion at 100 μM, but only 2-AG did at 50 μM. 2-AG-induced CCK secretion but not arachidonic acid-induced CCK secretion was attenuated by treatment with a cannabinoid receptor 1 (CB1) antagonist. These results indicate that a specific 2-MAG, 2-AG, directly stimulates CCK secretion via CB1.

Characterization of cytoplasmic lipid droplets in each region of the small intestine of lean and diet-induced obese mice in response to dietary fat.

The absorptive cells of the small intestine, namely, enterocytes, contribute to postprandial blood lipid levels by secreting dietary triacylglycerol in chylomicrons. The rate and amount of dietary triacylglycerol absorbed vary along the length of the small intestine. Excess dietary triacylglycerol not immediately secreted in chylomicrons can be temporarily stored in cytoplasmic lipid droplets (CLDs) and repackaged in chylomicrons at later times. The characteristics of CLDs, including their size, number per cell, and associated proteins, may influence CLD metabolism and reflect differences in lipid processing or storage in each intestinal region. However, it is unknown whether the characteristics or proteomes of CLDs differ in enterocytes of each intestine region in response to dietary fat. Furthermore, it is unclear if obesity influences the characteristics or proteomes of CLDs in each intestine region. To address this, we used transmission electron microscopy and shotgun liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis to assess the characteristics and proteome of CLDs in the proximal, middle, and distal regions of the small intestine of lean and diet-induced obese (DIO) mice 2 h after an oil gavage. We identified differences in lipid storage along the length of the small intestine and between lean and DIO mice, as well as distinct CLD proteomes reflecting potentially unique roles of CLDs in each region. This study reveals differences in lipid processing along the length of the small intestine in response to dietary fat in lean and DIO mice and reflects distinct features of the proximal, middle, distal region of the small intestine.NEW & NOTEWORTHY This study reflects the dynamics of fat absorption along the length of the small intestine in lean and obese mice in the physiological response to dietary fat. We identified unique features of cytoplasmic lipid droplets (CLDs) in the proximal, middle, and distal regions of the small intestine of lean and obese mice that may contribute to regional differences in dietary fat processing, absorption, or CLD metabolism.

Single cell oil production by Trichosporon sp.: Effects of fermentation conditions on fatty acid composition and applications in synthesis of structured triacylglycerols

Effects of various fermentation conditions on fatty acid composition of single cell oil (SCO) from Trichosporon sp. F1-2 were investigated. Ten carbon sources clustered into two groups using hierarchical clustering algorithm and principal component analysis. Xylose and soluble starch from each group were further studied. SCO using xylose had a high oleic acid content, while the linoleic acid and α-linolenic acid content increased significantly in SCO using soluble starch especially at the sn-2 position. 20 and 31 triacylglycerol (TAG) types were detected, respectively. The increased types were rich in linoleic acid and α-linolenic acid. The effect of C/N ratios was the weakest (distance threshold 3.05), followed by inoculation amounts (3.08), fermentation time (4.30) and nitrogen sources (5.64). The optimum nitrogen source, C/N ratio, inoculation amount and fermentation time for SCO accumulation were ammonium sulfate, 60, 6% and 120 h, respectively. Two different SCOs were produced using xylose and soluble starch under optimum conditions, which were demonstrated to be potential substrates for synthesis of 1,3-distearoyl-2-oleoyl-glycerol (a cocoa butter improver) and MLM-type TAG (the dietary TAG with low caloric value) respectively. Therefore, changing the carbon source can help broaden application fields of SCOs and increase feedstock sources of structured TAG synthesis.

Is dietary phosphatidylcholine essential for juvenile slipper lobster (Thenus australiensis)?

Phospholipids, especially phosphatidylcholine (PC), are considered essential nutrients for larval and juvenile stages of many decapod crustacean species due to low rates of endogenous biosynthesis and potential nutritional benefits in assisting lipid digestion and transport. This study was conducted to test the hypothesis that dietary PC is essential and would subsequently have beneficial effects on lipid utilisation and digestibility of fish oil triacylglycerol (TAG) in the slipper lobster, Thenus australiensis. Juvenile slipper lobsters were supplied six formulated experimental feeds with actual PC contents of 0.05, 0.48, 0.65, 0.98, 1.15 and 1.43% (dry matter) for 12 weeks. Manipulating dietary PC content had no effects on survival, growth, chemical composition, whole body or tissue lipid distribution, or apparent digestibility (AD) of gross energy (GE), total lipid (TL) or PC. Juvenile slipper lobsters appeared to possess fully developed and efficient mechanisms for GE, TAG and PC digestion (>95% AD). In vivo lipid class mass balance calculations demonstrated net intake of TL and TAG was considerably greater than was accumulated in the whole-body during growth. Thus, a significant proportion of dietary TL and TAG was utilised for energy and other metabolic processes. In contrast, particularly for low PL dietary treatments (≤ 0.5%), net gain of PC was greater than net intake, indicating a reliance on biosynthesis of PC. Thus, it is concluded that there is no or a very low requirement for dietary PC and thus may not be essential for the examined size class of juvenile T. australiensis when supplied adequate dietary PC biosynthetic precursors and other potentially related dietary nutrients. Further targeted research is recommended to verify if these findings are applicable to larval and early juvenile stages, and to enhance overall understanding of lipid nutritional requirements for T. australiensis.

Novel Cell-Based Assay to Investigate Monoacylglycerol Acyltransferase 2 Inhibitory Activity Using HIEC-6 Cell Line.

The dietary triacylglycerol (TAG) gets absorbed and accumulated in the body through the monoacylglycerol (MAG) pathway, which plays a major role in obesity and related disorders. The main enzyme of this pathway, monoacylglycerol acyltransferase 2 (MGAT2), is considered as a potential target for developing antiobesity compounds. Hence, there is a need for in vitro cell-based assays for screening the potential leads for MGAT2 inhibitors. Because of synthetic inhibitor’s side effects, there is an increased interest in natural extracts as potential leads. Hence, we have optimized a 2-MAG-induced TAG accumulation inhibitory cell-based assay to screen natural extracts using the HIEC-6 cell line. A concentration-dependent TAG accumulation was observed when the HIEC-6 cells were fed with exogenous 2-MAG. The TAG accumulation was confirmed by in situ BODIPY staining and was quantified. However, no TAG accumulation was seen when the cells were fed with exogenous DAG or TAG, suggesting MGAT2-mediated MAG uptake and its conversion to TAG. We demonstrated the utility of this assay by screening five different plant-based aqueous extracts. These extracts showed various inhibition levels (25% to 30%) of 2-MAG-induced TAG accumulation in the HIEC-6. The MGAT2 inhibitory potential of these extracts was confirmed by an in vitro MGAT2 assay. This cell-based assay adds a new methodology for screening, developing, and evaluating MGAT2 inhibitors for addressing obesity and related disorders.

Spatially Resolved Activity-based Proteomic Profiles of the Murine Small Intestinal Lipases

Despite the crucial function of the small intestine in nutrient uptake our understanding of the molecular events underlying the digestive function is still rudimentary. Recent studies demonstrated that enterocytes do not direct the entire dietary triacylglycerol toward immediate chylomicron synthesis. Especially after high-fat challenges, parts of the resynthesized triacylglycerol are packaged into cytosolic lipid droplets for transient storage in the endothelial layer of the small intestine. The reason for this temporary storage of triacylglycerol is not completely understood. To utilize lipids from cytosolic lipid droplets for chylomicron synthesis in the endoplasmic reticulum, stored triacylglycerol has to be hydrolyzed either by cytosolic lipolysis or lipophagy. Interestingly, triacylglycerol storage and chylomicron secretion rates are unevenly distributed along the small intestine, with the proximal jejunum exhibiting the highest intermittent storage capacity. We hypothesize that correlating hydrolytic enzyme activities with the reported distribution of triacylglycerol storage and chylomicron secretion in different sections of the small intestine is a promising strategy to determine key enzymes in triacylglycerol remobilization. We employed a serine hydrolase specific activity-based labeling approach in combination with quantitative proteomics to identify and rank hydrolases based on their relative activity in 11 sections of the small intestine. Moreover, we identified several clusters of enzymes showing similar activity distribution along the small intestine. Merging our activity-based results with substrate specificity and subcellular localization known from previous studies, carboxylesterase 2e and arylacetamide deacetylase emerge as promising candidates for triacylglycerol mobilization from cytosolic lipid droplets in enterocytes.

Diacylglycerol acyltransferase 1/2 inhibition induces dysregulation of fatty acid metabolism and leads to intestinal barrier failure and diarrhea in mice.

The intestinal metabolism and transport of triacylglycerol (TAG) play a critical role in dietary TAG absorption, and defects in the process are associated with congenital diarrhea. The final reaction in TAG synthesis is catalyzed by diacylglycerol acyltransferase (DGAT1 and DGAT2), which uses activated fatty acids (FA) as substrates. Loss‐of‐function mutations in DGAT1 cause watery diarrhea in humans, but mechanisms underlying the relationship between altered DGAT activity and diarrhea remain largely unclear. Here, the effects of DGAT1 and DGAT2 inhibition, alone or in combination, on dietary TAG absorption and diarrhea in mice were investigated by using a selective DGAT1 inhibitor (PF‐04620110) and DGAT2 inhibitor (PF‐06424439). Simultaneous administration of a single dosing of these inhibitors drastically decreased intestinal TAG secretion into the blood circulatory system and TAG accumulation in the duodenum at 60 min after lipid gavage. Under 60% high‐fat diet (HFD) feeding, their repeated simultaneous administration for 2 days induced severe watery diarrhea and occasionally led to death. The diarrhea was accompanied by enhanced fecal FA excretion, intestinal injury and barrier failure. DGAT1 or DGAT2 inhibition alone did not induce the phenotypic changes observed in DGAT1/2 inhibitor‐treated mice. The results demonstrate that DGAT1/2 inhibition alters TAG absorption and results in watery diarrhea in mice. DGAT1/2 inhibition‐induced diarrhea may be caused by intestinal barrier dysfunction due to dysregulation of the cytotoxic FA metabolism. These findings suggest that DGAT‐mediated intestinal TAG synthesis is a vital step for maintaining intestinal barrier integrity under HFD feeding.

Intestinal sensing and handling of dietary lipids in gastric bypass–operated patients and matched controls

Altered meal-related gut hormone secretion seems important for weight loss and diabetes remission after Roux-en-Y gastric bypass (RYGB). Elucidating the responsible meal components and receptors could aid discovery of new treatments of obesity and diabetes. Enteroendocrine cells respond to digestion products of dietary triacylglycerol, especially long-chain fatty acids (LCFAs) and 2-oleoyl-glycerol (2-OG), but not medium-chain fatty acids (MCFAs). We examined the impact of olive oil (20 mL) and its derivates, LCFAs and 2-OG, on enteroendocrine secretions [glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), cholecystokinin (CCK), peptide YY (PYY), and neurotensin (NT)] and on glucose, lipid, and bile acid metabolism in RYGB-operated and unoperated individuals. In an exploratory randomized crossover design, 10 RYGB-operated patients and 10 matched controls ingested 3 equimolar triacylglycerol formulations on separate days: olive oil (digested to 2-OG+LCFAs), C8-dietary oil (2-OG+MCFAs), and tricaprylin (MCFAs; negative control). Hormone responses were calculated as area under the curve (AUC). Independent of group status, olive oil had greater effects than C8-dietary oil on AUCs of plasma GLP-1 (+32%; 95% CI: 23%, 43%; P<0.01), CCK (+53%, P<0.01), and NT (+71%, P<0.01), whereas the effect on GIP differed between groups (+90% in controls, P<0.01; +24% in RYGB, P=0.10). Independent of group status, C8-dietary oil had greater effects than tricaprylin on AUCs of plasma CCK (+40%, P<0.01) and NT (+32%, P<0.01), but not GLP-1 (+5%; 95% CI: -2.9%, 13%; P=0.22), whereas the effect on GIP again differed between groups (+78% in controls, P<0.01; +39% in RYGB, P=0.01). Distal (GLP-1/PYY/NT), but not proximal (CCK/GIP), enteroendocrine responses were generally greater in RYGB patients than in controls. The combination of LCFAs plus 2-OG was substantially more effective than 2-OG plus MCFAs in stimulating enteroendocrine secretion in RYGB-operated and matched control individuals. Distal lipid-induced gut hormone release was greater after RYGB.This trial was registered at clinicaltrials.gov as NCT03223389.

Intestinal basolateral lipid substrate transport is linked to chylomicron secretion and is regulated by apoC-III

Chylomicron metabolism is critical for determining plasma levels of triacylglycerols (TAGs) and cholesterol, both of which are risk factors for CVD. The rates of chylomicron secretion and remnant clearance are controlled by intracellular and extracellular factors, including apoC-III. We have previously shown that human apoC-III overexpression in mice (apoC-IIITg mice) decreases the rate of chylomicron secretion into lymph, as well as the TAG composition in chylomicrons. We now find that this decrease in chylomicron secretion is not due to the intracellular effects of apoC-III, but instead that primary murine enteroids are capable of taking up TAG from TAG-rich lipoproteins (TRLs) on their basolateral surface; and via Seahorse analyses, we find that mitochondrial respiration is induced by basolateral TRLs. Furthermore, TAG uptake into the enterocyte is inhibited when excess apoC-III is present on TRLs. In vivo, we find that dietary TAG is diverted from the cytosolic lipid droplets and driven toward mitochondrial FA oxidation when plasma apoC-III is high (or when basolateral substrates are absent). We propose that this pathway of basolateral lipid substrate transport (BLST) plays a physiologically relevant role in the maintenance of dietary lipid absorption and chylomicron secretion. Further, when apoC-III is in excess, it inhibits BLST and chylomicron secretion.