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

OBJECTIVE: In 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. METHODS: To 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. RESULTS: We 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. CONCLUSIONS: These 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.

Lipid mediators of inhalation exposure-induced pulmonary toxicity and inflammation.

Many inhalation exposures induce pulmonary inflammation contributing to disease progression. Inflammatory processes are actively regulated via mediators including bioactive lipids. Bioactive lipids are potent signaling molecules involved in both pro-inflammatory and resolution processes through receptor interactions. The formation and clearance of lipid signaling mediators are controlled by multiple metabolic enzymes. An imbalance of these lipids can result in exacerbated and sustained inflammatory processes which may result in pulmonary damage and disease. Dysregulation of pulmonary bioactive lipids contribute to inflammation and pulmonary toxicity following exposures. For example, inhalation of cigarette smoke induces activation of pro-inflammatory bioactive lipids such as sphingolipids, and ceramides contributing to chronic obstructive pulmonary disease. Additionally, exposure to silver nanoparticles causes dysregulation of inflammatory resolution lipids. As inflammation is a common consequence resulting from inhaled exposures and a component of numerous diseases it represents a broadly applicable target for therapeutic intervention. With new appreciation for bioactive lipids, technological advances to reliably identify and quantify lipids have occurred. In this review, we will summarize, integrate, and discuss findings from recent studies investigating the impact of inhaled exposures on pro-inflammatory and resolution lipids within the lung and their contribution to disease. Throughout the review current knowledge gaps in our understanding of bioactive lipids and their contribution to pulmonary effects of inhaled exposures will be presented. New methods being employed to detect and quantify disruption of pulmonary lipid levels following inhalation exposures will be highlighted. Lastly, we will describe how lipid dysregulation could potentially be addressed by therapeutic strategies to address inflammation.

Glucagon-like Peptide-2 Acutely Enhances Chylomicron Secretion in Humans Without Mobilizing Cytoplasmic Lipid Droplets.

CONTEXT: A portion of ingested fats are retained in the intestine for many hours before they are mobilized and secreted in chylomicron (CM) particles. Factors such as glucagon-like peptide-2 (GLP-2) and glucose can mobilize these stored intestinal lipids and enhance CM secretion. We have recently demonstrated in rodents that GLP-2 acutely enhances CM secretion by mechanisms that do not involve the canonical CM synthetic assembly and secretory pathways. OBJECTIVE: To further investigate the mechanism of GLP-2's potent intestinal lipid mobilizing effect, we examined intracellular cytoplasmic lipid droplets (CLDs) in intestinal biopsies of humans administered GLP-2 or placebo. DESIGN, SETTING, PATIENTS, AND INTERVENTIONS: A single dose of placebo or GLP-2 was administered subcutaneously 5 hours after ingesting a high-fat bolus. In 1 subset of participants, plasma samples were collected to quantify lipid and lipoprotein concentrations for 3 hours after placebo or GLP-2. In another subset, a duodenal biopsy was obtained 1-hour after placebo or GLP-2 administration for transmission electron microscopy and proteomic analysis. RESULTS: GLP-2 significantly increased plasma triglycerides by 46% (P = 0.009), mainly in CM-sized particles by 133% (P = 0.003), without reducing duodenal CLD size or number. Several proteins of interest were identified that require further investigation to elucidate their potential role in GLP-2-mediated CM secretion. CONCLUSIONS: Unlike glucose that mobilizes enterocyte CLDs and enhances CM secretion, GLP-2 acutely increased plasma CMs without significant mobilization of CLDs, supporting our previous findings that GLP-2 does not act directly on enterocytes to enhance CM secretion and most likely mobilizes secreted CMs in the lamina propria and lymphatics.

Global landscape of protein complexes in postprandial-state livers from diet-induced obese and lean mice.

Obesity is associated with a spectrum of nonalcoholic fatty liver disease (NAFLD) which is characterized by steatosis. Prolonged fat deposition aggravates liver dysfunctions leading to an advanced form of NAFLD such as steatohepatitis and cirrhosis. As liver function in the postprandial state is critical for macronutrient metabolism and energy homeostasis, we sought to determine the differences in protein complex profiles in lean and fatty livers in the postprandial state. Protein complex profiling is of interest as proteins often do not function alone and the information on the interactions may reveal novel etiology of NAFLD, which is currently limited compared with proteome profiles or RNA-sequencing profiles. To this end, we fractionated liver lysates using size-exclusion chromatography (SEC) and analyzed each fraction using untargeted LC-MS/MS. We identified 1172 proteins that were discovered in lean and fatty livers, and their elution profiles were compared. We found that the majority of liver proteins were present as putative complexes. Also, the fatty liver protein elution profile showed great conservations as lean liver despite the metabolic disease state. Yet, we discovered a few proteins that showed different elution patterns in the fatty liver, including Acadm, Aldh1a7, Aldh1a1, Akr1a1, Eif3l, Fkbp2, G6pdx, Gm20441, Hao1, Pcna, Pkm, Ppif, Prdx4, Stmn1, Tagln, Tubb4b, Ubqln2, and Usp14, which may be involved in high fat diet-induced alterations of protein oligomerization and hepatic functions. Overall, our protein complex profiling could expand our understanding of hepatic abnormalities that cannot be uncovered by simple quantitation of protein expression.

Multi-Omics Approach Reveals Dysregulation of Protein Phosphorylation Correlated with Lipid Metabolism in Mouse Non-Alcoholic Fatty Liver.

Obesity caused by overnutrition is a major risk factor for non-alcoholic fatty liver disease (NAFLD). Several lipid intermediates such as fatty acids, glycerophospholipids and sphingolipids are implicated in NAFLD, but detailed characterization of lipids and their functional links to proteome and phosphoproteome remain to be elucidated. To characterize this complex molecular relationship, we used a multi-omics approach by conducting comparative proteomic, phoshoproteomic and lipidomic analyses of high fat (HFD) and low fat (LFD) diet fed mice livers. We quantified 2447 proteins and 1339 phosphoproteins containing 1650 class I phosphosites, of which 669 phosphosites were significantly different between HFD and LFD mice livers. We detected alterations of proteins associated with cellular metabolic processes such as small molecule catabolic process, monocarboxylic acid, long- and medium-chain fatty acid, and ketone body metabolic processes, and peroxisome organization. We observed a significant downregulation of protein phosphorylation in HFD fed mice liver in general. Untargeted lipidomics identified upregulation of triacylglycerols, glycerolipids and ether glycerophosphocholines and downregulation of glycerophospholipids, such as lysoglycerophospholipids, as well as ceramides and acylcarnitines. Analysis of differentially regulated phosphosites revealed phosphorylation dependent deregulation of insulin signaling as well as lipogenic and lipolytic pathways during HFD induced obesity. Thus, this study reveals a molecular connection between decreased protein phosphorylation and lipolysis, as well as lipid-mediated signaling in diet-induced obesity.

Albumin knockout mice exhibit reduced plasma free fatty acid concentration and enhanced insulin sensitivity.

Circulating albumin is expected to play a significant role in the trafficking of plasma free fatty acids (FFA) between tissues, such as FFA transfer from adipose tissue to the liver. However, it was not yet known how disrupting FFA binding to albumin in circulation would alter lipid metabolism and any resulting impacts upon control of glycemia. To improve understanding of metabolic control, we aimed to determine whether lack of serum albumin would decrease plasma FFA, hepatic lipid storage, whole body substrate oxidation, and glucose metabolism. Male and female homozygous albumin knockout mice and C57BL/6J wild type controls, each on a standard diet containing a moderate fat content, were studied at 6-8 weeks of age. Indirect calorimetry, glucose tolerance testing, insulin tolerance testing, exercise performance, plasma proteome, and tissue analyses were performed. In both sexes of albumin knockout mice compared to the wild type mice, significant reductions (p < 0.05) were observed for plasma FFA concentration, hepatic triacylglycerol and diacylglycerol content, blood glucose during the glucose tolerance test, and blood glucose during the insulin tolerance test. Albumin deficiency did not reduce whole body fat oxidation over a 24-h period and did not alter exercise performance in an incremental treadmill test. The system-level phenotypic changes in lipid and glucose metabolism were accompanied by reduced hepatic perilipin-2 expression (p < 0.05), as well as increased expression of adiponectin (p < 0.05) and glucose transporter-4 (p < 0.05) in adipose tissue. The results indicate an important role of albumin and plasma FFA concentration in lipid metabolism and glucoregulation.

The Roles of Cytoplasmic Lipid Droplets in Modulating Intestinal Uptake of Dietary Fat.

Dietary fat absorption is required for health but also contributes to hyperlipidemia and metabolic disease when dysregulated. One step in the process of dietary fat absorption is the formation of cytoplasmic lipid droplets (CLDs) in small intestinal enterocytes; these CLDs serve as dynamic triacylglycerol storage organelles that influence the rate at which dietary fat is absorbed. Recent studies have uncovered novel factors regulating enterocyte CLD metabolism that in turn influence the absorption of dietary fat. These include peroxisome proliferator-activated receptor α activation, compartmentalization of different lipid pools, the gut microbiome, liver X receptor and farnesoid X receptor activation, obesity, and physiological factors stimulating CLD mobilization. Understanding how enterocyte CLD metabolism is regulated is key in modulating the absorption of dietary fat in the prevention of hyperlipidemia and its associated metabolic disorders.

Proteomic Characterization of Cytoplasmic Lipid Droplets in Human Metastatic Breast Cancer Cells.

One of the characteristic features of metastatic breast cancer is increased cellular storage of neutral lipid in cytoplasmic lipid droplets (CLDs). CLD accumulation is associated with increased cancer aggressiveness, suggesting CLDs contribute to metastasis. However, how CLDs contribute to metastasis is not clear. CLDs are composed of a neutral lipid core, a phospholipid monolayer, and associated proteins. Proteins that associate with CLDs regulate both cellular and CLD metabolism; however, the proteome of CLDs in metastatic breast cancer and how these proteins may contribute to breast cancer progression is unknown. Therefore, the purpose of this study was to identify the proteome and assess the characteristics of CLDs in the MCF10CA1a human metastatic breast cancer cell line. Utilizing shotgun proteomics, we identified over 1500 proteins involved in a variety of cellular processes in the isolated CLD fraction. Interestingly, unlike other cell lines such as adipocytes or enterocytes, the most enriched protein categories were involved in cellular processes outside of lipid metabolism. For example, cell-cell adhesion was the most enriched category of proteins identified, and many of these proteins have been implicated in breast cancer metastasis. In addition, we characterized CLD size and area in MCF10CA1a cells using transmission electron microscopy. Our results provide a hypothesis-generating list of potential players in breast cancer progression and offers a new perspective on the role of CLDs in cancer.

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.

Proteome and phosphoproteome characterization of liver in the postprandial state from diet-induced obese and lean mice.

A metabolic consequence of obesity is hepatosteatosis, which can develop into more serious diseases in the non-alcoholic fatty liver disease (NAFLD) spectrum. The goal of this study was to identify the protein signature of liver in the postprandial state in obesity compared to leanness. The postprandial state is of interest due to the central role of the liver in regulating macronutrient and energy homeostasis during the fed-fast cycle and lack of previously reported controlled studies in the postprandial state. Therefore, we assessed the proteome and phosphoproteome of liver in the postprandial state from diet-induced obese (DIO) and lean mice using untargeted LC-MS/MS analysis. We identified significant alterations in the levels of proteins involved in fatty acid oxidation, activation, and transport, as well as proteins involved in energy metabolism including ketogenesis, tricarboxylic acid cycle, and electron transport chain in liver of DIO compared to lean mice. Additionally, phosphorylated proteins in liver of DIO and lean mice reflect possible regulatory mechanisms controlling fatty acid metabolism and gene expression that may contribute to hepatic metabolic alterations in obesity. Our data indicates PPARα-mediated transcriptional regulation of lipid metabolism and adaptation to hepatic lipid overload. The results of this study expand our knowledge of the molecular changes that occur in liver in the postprandial state in obesity compared to leanness. SIGNIFICANCE: Proteome and phosphoproteome studies of liver in a controlled postprandial state in obesity and leanness are lacking; however, this information is crucial to understanding how obesity-associated hepatosteatosis influences postprandial nutrient and energy metabolism. In this global shotgun proteome and phosphoproteome analysis, we identified unique protein signatures defining obesity and leanness in liver in the postprandial state and identified potential mechanisms contributing to hepatic metabolic alterations in obesity. The results of this study provide a foundation to focus future experiments on the contribution of altered protein and phosphorylation patterns to postprandial metabolism in obesity-associated hepatosteatosis.

High-fat-diet induced obesity increases the proportion of linoleic acyl residues in dam serum and milk and in suckling neonate circulation.

Maternal obesity increases the risk of offspring to become obese and develop related pathologies. Exposure to maternal high-fat diet (HFD) only during lactation increases the risk of obesity-related diseases, suggesting that factors in milk affect long-term health. We hypothesized that prepregnancy obesity induced by HFD alters milk lipidome, and in turn, alterations may affect neonate serum lipidome. The objective of this study was to determine the effect of prepregnancy obesity induced by HFD on circulating lipids in dams and neonates and in milk. Female mice were fed an HFD (60% kcal fat) or control diet (CON, 10% kcal fat) beginning 4 weeks before breeding. On postnatal day 2 (PND2), pups were cross-fostered to create pup groups exposed to HFD during pregnancy, lactation, or both or exposed to CON. On PND12, dams were milked and then euthanized along with pups to collect blood. Serum and milk were processed for multiple reaction monitoring (MRM) lipidomics profiling to quantify the relative expression of lipid classes. Lipidome of HFD dam serum and milk had increased proportion of C18:2 free fatty acid and fatty acyl residues in all lipid classes. Lipidome of serum from pups exposed to maternal HFD during lactation was similarly affected. Thus, maternal HFD induced redistribution of fatty acyl residues in the dam's circulation, which was associated with modification in milk and suckling neonate's lipidome. Further studies are needed to determine if increased circulating levels of C18:2 in neonate affects development and predisposes offspring to obesity and metabolic syndrome.

Maternal high-fat diet exposure during gestation, lactation, or gestation and lactation differentially affects intestinal morphology and proteome of neonatal mice.

Offspring nutrition depends on the mother during gestation and lactation; thus, maternal nutrition and metabolism can affect their development. We hypothesized that maternal exposure to high-fat (HF) diet affects neonate's gastrointestinal tract development. Our objective was to determine the effect of maternal HF diet during gestation and lactation on neonate's duodenum histomorphology and proteome. Female mice were fed either a control (C, 10% kcal fat) or an HF (60% kcal fat) diet for 4 weeks and bred. On postnatal day 2, half the pups were cross-fostered to dams fed on different diet, creating 4 treatments: C-C, C-HF, HF-C, and HF-HF, indicating maternal diet during gestation-lactation, respectively. On postnatal day 12, pups' duodenum was excised and prepared for histology and liquid chromatography-tandem mass spectrometry analysis of proteome. Villi were significantly longer in HF-HF pups, and crypt cell proliferation rate was not different among treatments. Between C-C and HF-HF, HF-C, or C-HF, 812, 601, or 894 proteins were differentially expressed (Tukey adjusted P < .05), respectively. Functional analysis clustered proteins upregulated in HF-HF vs C-C in fat digestion and absorption, extracellular matrix, cell adhesion, immune response, oxidation-reduction processes, phagocytosis, and transport categories. Proteins downregulated were classified as RNA splicing, translation, protein folding, endocytosis, and transport. There was evidence for a carryover effect of exposure to HF diet during gestation to the postnatal period. Alterations in proteome relative to HF exposure potentially reflect long-term changes in the functioning of the duodenum.

Diet Induced Obesity Alters Intestinal Cytoplasmic Lipid Droplet Morphology and Proteome in the Postprandial Response to Dietary Fat.

Dietary fat absorption by the small intestine is an efficient, multistep process that regulates the uptake and delivery of essential nutrients and energy. Fatty acids taken up by enterocytes, the absorptive cells of the small intestine, are resynthesized into triacylglycerol (TAG) and either secreted in chylomicrons or temporarily stored in cytoplasmic lipid droplets (CLDs). Proteins that associate with CLDs are thought to regulate the dynamics of TAG storage and mobilization. It is currently unclear what effect diet induced obesity (DIO) has on the balance between dietary fat storage and secretion. Specifically, there is limited knowledge of how DIO affects the level and diversity of proteins that associate with CLDs and regulate CLD dynamics. In the current study, we characterize CLDs from lean and DIO mice through histological and proteomic analyses. We demonstrate that DIO mice have larger intestinal CLDs compared to lean mice in response to dietary fat. Additionally, we identified 375 proteins in the CLD fraction isolated from enterocytes of lean and DIO mice. We identified a subgroup of lipid related proteins that are either increased or unique to the DIO CLD proteome. These proteins are involved in steroid synthesis, TAG synthesis, and lipolysis. This analysis expands our knowledge of the effect of DIO on the process of dietary fat absorption in the small intestine (D'Aquila, 2016).

Oral Glucose Mobilizes Triglyceride Stores From the Human Intestine.

BACKGROUND & AIMS: The small intestine regulates plasma triglyceride (TG) concentration. Within enterocytes, dietary TGs are packaged into chylomicrons (CMs) for secretion or stored temporarily in cytoplasmic lipid droplets (CLDs) until further mobilization. We and others have shown that oral and intravenous glucose enhances CM particle secretion in human beings, however, the mechanisms through which this occurs are incompletely understood. METHODS: Two separate cohorts of participants ingested a high-fat liquid meal and, 5 hours later, were assigned randomly to ingest either a glucose solution or an equivalent volume of water. In 1 group (N = 6), plasma and lipoprotein TG responses were assessed in a randomized cross-over study. In a separate group (N = 24), duodenal biopsy specimens were obtained 1 hour after ingestion of glucose or water. Ultrastructural and proteomic analyses were performed on duodenal biopsy specimens. RESULTS: Compared with water, glucose ingestion increased circulating TGs within 30 minutes, mainly in the CM fraction. It decreased the total number of CLDs and the proportion of large-sized CLDs within enterocytes. We identified 2919 proteins in human duodenal tissue, 270 of which are related to lipid metabolism and 134 of which were differentially present in response to glucose compared with water ingestion. CONCLUSIONS: Oral glucose mobilizes TGs stored within enterocyte CLDs to provide substrate for CM synthesis and secretion. Future studies elucidating the underlying signaling pathways may provide mechanistic insights that lead to the development of novel therapeutics for the treatment of hypertriglyceridemia.

DGAT1 deficiency disrupts lysosome function in enterocytes during dietary fat absorption.

Enterocytes, the absorptive cells of the small intestine, mediate the process of dietary fat absorption by secreting triacylglycerol (TAG) into circulation. When levels of dietary fat are high, TAG is stored in cytoplasmic lipid droplets (CLDs) and sequentially hydrolyzed for ultimate secretion. Mice with deficiency in acyl CoA: diacylglycerol acyltransferase 1 (Dgat1-/- mice) were previously reported to have a reduced rate of intestinal TAG secretion and abnormal TAG accumulation in enterocyte CLDs. This unique intestinal phenotype is critical to their resistance to diet-induced obesity; however, the underlying mechanism remains unclear. Emerging evidence shows that lysosomal TAG hydrolysis contributes to autophagy-mediated CLD mobilization termed lipophagy, and when disrupted results in CLD accumulation. In order to study how lipophagy contributes to the unique intestinal phenotype of Dgat1-/- mice, enterocytes from wild-type (WT) and Dgat1-/- mice were examined at 2 and 6 h after oral oil gavage. Through ultrastructural analysis we observed TAG present within autophagic vesicles (AVs) in mouse enterocytes, suggesting the role of lipophagy in intestinal CLD mobilization during dietary fat absorption. Furthermore, we found that Dgat1-/- mice had abnormal TAG accumulation within AVs and less acidic lysosomes compared to WT mice. Together these findings suggest that the delayed dietary fat absorption seen in Dgat1-/- mice is, in part, due to the dysregulated flux of autophagy-mediated CLD mobilization and impairment of lysosomal acidification in enterocytes. The present study highlights the critical role of lysosome in enterocyte CLD mobilization for proper dietary fat absorption.

Recent Advances in Triacylglycerol Mobilization by the Gut.

Dietary lipid absorption and lipoprotein secretion by the gut are important in maintaining whole-body energy homeostasis and have significant implications for health and disease. The processing of dietary lipids, including storage within and subsequent mobilization and transport from enterocyte cytoplasmic lipid droplets or other intestinal lipid storage pools (including the secretary pathway, lamina propria and lymphatics) and secretion of chylomicrons, involves coordinated steps that are subject to various controls. This review summarizes recent advances in our understanding of the mechanisms that underlie lipid storage and mobilization by small intestinal enterocytes and the intestinal lymphatic vasculature. Therapeutic targeting of lipid processing by the gut may provide opportunities for the treatment and prevention of dyslipidemia, and for improving health status.

Dgat1 and Dgat2 regulate enterocyte triacylglycerol distribution and alter proteins associated with cytoplasmic lipid droplets in response to dietary fat.

Enterocytes, the absorptive cells of the small intestine, mediate efficient absorption of dietary fat (triacylglycerol, TAG). The digestive products of dietary fat are taken up by enterocytes, re-esterified into TAG, and packaged on chylomicrons (CMs) for secretion into blood or temporarily stored within cytoplasmic lipid droplets (CLDs). Altered enterocyte TAG distribution impacts susceptibility to high fat diet associated diseases, but molecular mechanisms directing TAG toward these fates are unclear. Two enzymes, acyl CoA: diacylglycerol acyltransferase 1 (Dgat1) and Dgat2, catalyze the final, committed step of TAG synthesis within enterocytes. Mice with intestine-specific overexpression of Dgat1 (Dgat1Int) or Dgat2 (Dgat2Int), or lack of Dgat1 (Dgat1-/-), were previously found to have altered intestinal TAG secretion and storage. We hypothesized that varying intestinal Dgat1 and Dgat2 levels alters TAG distribution in subcellular pools for CM synthesis as well as the morphology and proteome of CLDs. To test this we used ultrastructural and proteomic methods to investigate intracellular TAG distribution and CLD-associated proteins in enterocytes from Dgat1Int, Dgat2Int, and Dgat1-/- mice 2h after a 200µl oral olive oil gavage. We found that varying levels of intestinal Dgat1 and Dgat2 altered TAG pools involved in CM assembly and secretion, the number or size of CLDs present in enterocytes, and the enterocyte CLD proteome. Overall, these results support a model where Dgat1 and Dgat2 function coordinately to regulate the process of dietary fat absorption by preferentially synthesizing TAG for incorporation into distinct subcellular TAG pools in enterocytes.

1α,25-dihydroxyvitamin D inhibits de novo fatty acid synthesis and lipid accumulation in metastatic breast cancer cells through down-regulation of pyruvate carboxylase.

Both increased de novo fatty acid synthesis and higher neutral lipid accumulation are a common phenotype observed in aggressive breast cancer cells, making lipid metabolism a promising target for breast cancer prevention. In the present studies, we demonstrate a novel effect of the active metabolite of vitamin D, 1α,25-dihydroxyvitamin D (1,25(OH)2D) on lipid metabolism in malignant breast epithelial cells. Treatment of MCF10CA1a breast epithelial cells with 1,25(OH)2D (10 nM) for 5 and 7 days decreased the level of triacylglycerol, the most abundant form of neutral lipids, by 20%(±3.9) and 50%(±5.9), respectively. In addition, 1,25(OH)2D treatment for 5 days decreased palmitate synthesis from glucose, the major fatty acid synthesized de novo (48%±5.5 relative to vehicle). We have further identified the anaplerotic enzyme pyruvate carboxylase (PC) as a target of 1,25(OH)2D-mediated regulation and hypothesized that 1,25(OH)2D regulates breast cancer cell lipid metabolism through inhibition of PC. PC mRNA expression was down-regulated with 1,25(OH)2D treatment at 2 (73%±6 relative to vehicle) and 5 (56%±8 relative to vehicle) days. Decrease in mRNA abundance corresponded with a decrease in PC protein expression at 5 days of treatment (54%±12 relative to vehicle). Constitutive overexpression of PC in MCF10CA1a cells using a pCMV6-PC plasmid inhibited the effect of 1,25(OH)2D on both TAG accumulation and de novo palmitate synthesis from glucose. Together, these studies demonstrate a novel mechanism through which 1,25(OH)2D regulates lipid metabolism in malignant breast epithelial cells.

Maternal high fructose and low protein consumption during pregnancy and lactation share some but not all effects on early-life growth and metabolic programming of rat offspring.

Maternal nutritional stress during pregnancy acts to program offspring metabolism. We hypothesized that the nutritional stress caused by maternal fructose or low protein intake during pregnancy would program the offspring to develop metabolic aberrations that would be exacerbated by a diet rich in fructose or fat during adult life. The objective of this study was to characterize and compare the fetal programming effects of maternal fructose with the established programming model of a low-protein diet on offspring. Male offspring from Sprague-Dawley dams fed a 60% starch control diet, a 60% fructose diet, or a low-protein diet throughout pregnancy and lactation were weaned onto either a 60% starch control diet, 60% fructose diet, or a 30% fat diet for 15 weeks. Offspring from low-protein and fructose-fed dam showed retarded growth (P<.05) at weaning (50.3, 29.6 vs 59.1±0.8 g) and at 18 weeks of age (420, 369 vs 464±10.9 g). At 18 weeks of age, offspring from fructose dams expressed greater quantities (P<.05) of intestinal Pgc1a messenger RNA compared with offspring from control or low-protein dams (1.31 vs 0.89, 0.85; confidence interval, 0.78-1.04). Similarly, maternal fructose (P=.09) and low-protein (P<.05) consumption increased expression of Pgc1a in offspring liver (7.24, 2.22 vs 1.22; confidence interval, 2.11-3.45). These data indicate that maternal fructose feeding is a programming model that shares some features of maternal protein restriction such as retarded growth, but is unique in programming of selected hepatic and intestinal transcripts.

1α,25-Dihydroxyvitamin D Inhibits the Metastatic Capability of MCF10CA1a and MDA-MB-231 Cells in an In Vitro Model of Breast to Bone Metastasis.

Breast cancer metastasis to the bone continues to be a major health problem, with approximately 80% of advanced breast cancer patients expected to develop bone metastasis. Although the problem of bone metastasis persists, current treatment options for metastatic cancer patients are limited. In this study, we investigated the preventive role of the active vitamin D metabolite, 1α,25-dihydroxyvitamin D (1,25(OH)2D), against the metastatic potential of breast cancer cells using a novel three-dimensional model (rMET) recapitulating multiple steps of the bone metastatic process. Treatment of MCF10CA1a and MDA-MB-231 cells inhibited metastasis in the rMET model by 70% (±5.7%) and 21% (±6%), respectively. In addition, 1,25(OH)2D treatment decreased invasiveness (20 ± 11% of vehicle) and decreased the capability of MCF10CA1a cells to survive in the reconstructed bone environment after successful invasion through the basement membrane (69 ± 5% of vehicle). An essential step in metastasis is epithelial-mesenchymal transition (EMT). Treatment of MCF10CA1a cells with 1,25(OH)2D increased gene (2.04 ± 0.28-fold increase) and protein (1.87 ± 0.20-fold increase) expression of E-cadherin. Additionally, 1,25(OH)2D treatment decreased N-cadherin gene expression (42 ± 8% decrease), a marker for EMT. Collectively, the present study suggests that 1,25(OH)2D inhibits breast cancer cell metastatic capability as well as inhibits EMT, an essential step in the metastatic process.