Expression Of Fatty Acid Transport Protein Research Articles

Abstract 5129: Neutrophil extracellular traps induced by surgical stress regulate cancer metabolism leading to tumor growth

Abstract Introduction: Surgery is a crucial intervention to cure malignancies. However, evidence shows that the surgical insult and its accompanying immune response can enhance tumor growth and metastasis. Neutrophil Extracellular Traps (NETs) released systemically during surgical stress were previously shown to contribute to this process. In fact, to generate increased energy demands required for growth and metastasis, cancer cells in a stressful microenvironment need an adaptive mechanism where metabolic reprogramming to fatty acid (FA) oxidation has been emerging as an important process. Here, we aim to explore the direct effects of surgical NETs in regulating cancer cell metabolism leading to tumor growth and metastasis. Methods: For the indolent tumor model, C57BL/6 mice were injected with a small number of LLC-1 (Lewis Lung Cancer-1) cells subcutaneously (SQ) and a group was subjected to laparotomy for 30 mins, with or without intraperitoneal DNAse (NET inhibitor) injection perioperatively. Tumor growth was then followed for 4 weeks. For the tumor metastasis model, LLC-1 cells were injected SQ and allowed to grow for 3 weeks before resection with or without perioperative DNAse. Tumor metastasis to the lungs was assessed after 3 weeks. In vitro experiments on LLC-1 and MC-38 (murine colon adenocarcinoma) cancer cells were performed using NETs released from neutrophils freshly isolated from mice bone marrow and stimulated with Phorbol myristate acetate (PMA). Results: For the indolent tumor model, injection of cancer cells did not lead to any significant tumor growth after 4 weeks unless mice underwent surgical stress via laparotomy. Treatment with DNAse significantly reversed their growth. For the tumor metastasis model, tumor resection alone led to significant lung metastasis, but DNAse treatment during resection or resection in PAD-4 KO mice (unable to form NETs) led to significantly less metastatic burden. Furthermore, in vitro treatment of cancer cells with NETs lead to significant cell proliferation, relatively more under hypoxic and serum conditions. This was paralleled with a significant increase in fluorescently labeled FA uptake into cancer cells as detected by flow cytometry, as well as a significant upregulation of gene expression of FA transport proteins and oxidation enzymes. Conclusion: Surgical stress can promote tumor growth and metastasis through cancer cell metabolic reprogramming induced by systemically released NETs. We hope that elucidating the molecular mechanism behind this process can help in finding therapeutics to prevent the protumorigenic effects of surgical stress. Citation Format: Tony Haykal, Ruiqi Yang, Celine Tohme, Ronghua Wang, David Geller, Christof Kaltenmeier, Hamza Yazdani, Samer Tohme. Neutrophil extracellular traps induced by surgical stress regulate cancer metabolism leading to tumor growth. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5129.

Vitamin D and Swimming Exercise Prevent Obesity in Rats under a High-Fat Diet via Targeting FATP4 and TLR4 in the Liver and Adipose Tissue.

The prevalence of obesity has risen in the last decades, and it has caused massive health burdens on people's health, especially metabolic and cardiovascular issues. The risk of vitamin D insufficiency is increased by obesity, because adipose tissue alters both the requirements for and bioavailability of vitamin D. Exercise training is acknowledged as having a significant and long-term influence on body weight control; the favorable impact of exercise on obesity and obesity-related co-morbidities has been demonstrated via various mechanisms. The current work illustrated the effects of vitamin D supplementation and exercise on obesity induced by a high-fat diet (HFD) and hepatic steatosis in rats and explored how fatty acid transport protein-4 (FATP4) and Toll-like receptor-4 antibodies (TLR4) might be contributing factors to obesity and related hepatic steatosis. Thirty male albino rats were divided into five groups: group 1 was fed a normal-fat diet, group 2 was fed an HFD, group 3 was fed an HFD and given vitamin D supplementation, group 4 was fed an HFD and kept on exercise, and group 5 was fed an HFD, given vitamin D, and kept on exercise. The serum lipid profile adipokines, interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) were analyzed, and the pathological changes in adipose and liver tissues were examined. In addition, the messenger-ribonucleic acid (mRNA) expression of FATP4 and immunohistochemical expression of TLR4 in adipose and liver tissues were evaluated. Vitamin D supplementation and exercise improved HFD-induced weight gain and attenuated hepatic steatosis, along with improving the serum lipid profile, degree of inflammation, and serum adipokine levels. The expression of FATP4 and TLR4 in both adipose tissue and the liver was downregulated; it was noteworthy that the group that received vitamin D and was kept on exercise showed also improvement in the histopathological picture of this group. According to the findings of this research, the protective effect of vitamin D and exercise against obesity and HFD-induced hepatic steatosis is associated with the downregulation of FATP4 and TLR4, as well as a reduction in inflammation.

The effect of bile salt diet supplementation on genes related to fat metabolism in yellow-feathered broilers.

Background and Aim:As a new feed additive, bile acid (BA) can promote the absorption and transport of lipids and fat-soluble vitamins. In recent years, BAs have been widely used in animal feed to promote fat absorption. Therefore, this study aimed to investigate the effect of bile salt supplementation in the diet of yellow-feathered broilers on messenger RNA (mRNA) expression of sterol regulatory element-binding protein 1 (SREBF1), fatty acid synthase (FAS), acetyl-coenzyme A carboxylase (ACC), and fatty acid transport protein 4 (FATP4).Materials and Methods:Four hundred and twenty commercial male chicks were randomly divided into seven groups (with four replicates per group and 15 chickens per replicate). They were fed diets supplemented with bile salts at 0, 1.5, 2.5, 3.5, 4.5, 5.5 mg/kg, and 2 mg/kg tylosin for 30 days. Changes in SREBF1, fatty acid transporter 4, FAS, and acetyl-CoA carboxylase genes in intestinal mucosa and liver of yellow-feathered broilers were determined using a quantitative fluorescence polymerase chain reaction.Results:mRNA expression of SREBF1, FAS, ACC, and FATP4 in the small intestine decreased in chicks fed diets supplemented with 3.5 and 4.5 mg/kg bile salts (p<0.05) compared with the control group on 7 days and 14 d. The mRNA expressions of SREBF1, FAS, ACC, and FATP4 in liver tissue decreased in chicks fed diets supplemented with 4.5 and 5.5 mg/kg bile salts (p<0.05) compared to the control group on 7 days. The mRNA expression of SREBF1, FAS, ACC, and FATP4 in the liver at 14 days and the small intestine on 21 days also decreased in chicks fed diets supplemented with 4.5 mg/kg bile salts (p<0.05) compared to the control group. When contrasted with the control group on day 21, the mRNA expression of SRWBF1, FAS, ACC, and FATP4 detected in the liver was lower in chicks fed diets supplemented with bile salts (p<0.05).Conclusion:The dietary supplementation of bile salts at 4.5 mg/kg effectively regulates the expression of fat metabolism genes, such as SREBF1, FAS, ACC, and FATP4 mRNA. At this concentration, bile salts promote fat catabolism, inhibit fat synthesis, and play an essential role in improving the fat deposition of broilers.

Significance of Altered Fatty Acid Transporter Expressions in Uterine Cervical Cancer and Its Precursor Lesions.

High-risk human papilloma virus (HR HPV) infection is a major factor leading to the development of uterine cervical cancer. Data suggest that alterations in lipid metabolism are related to the pathogenesis of cervical cancer. Specifically, the uptake of exogenous fatty acids and their intracellular storage in lipid droplets enables cancer cells to survive and adapt to the changing tumor environment. We compared the immunohistochemical expression of fatty acid transport protein 4 (FATP4), and cluster of differentiation 36/fatty acid translocase (CD36/FAT) in normal cervical epithelium, low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL), and squamous cell carcinoma (SCC) tissues of the uterine cervix. We also investigated the clinicopathological implications of these fatty acid transporters in SCC. Compared with that in normal cervical tissues, the expression of FATP4 was lower in LSIL (p=0.002), HSIL (p=0.006), and SCC (p=0.001). In contrast, CD36 expression was higher in SCC tissues than in normal cervical tissues (p<0.001). In normal cervical tissues, HR HPV-infected lesions exhibited a decrease in FATP4 (p<0.001) and an increase of CD36 (p=0.134), compared to those that were not infected with HR HPV. High CD36 expression was associated with a shorter recurrence-free survival (p<0.001). However, high FATP4 levels showed no significant correlation with the clinicopathological parameters of SCC. Altered expression levels of FATP4 and CD36 are unique features that might be related to HR HPV infection and promote tumorigenesis and progression of cervical cancer.

Association of placental toll-like receptor 4 and fatty acid transport protein expression with neonatal weight

Association of placental toll-like receptor 4 and fatty acid transport protein expression with neonatal weight

Enterotoxigenic Escherichia coli Interferes FATP4-Dependent Long-Chain Fatty Acid Uptake of Intestinal Epithelial Enterocytes via Phosphorylation of ERK1/2-PPARγ Pathway

Sufficient fatty acid (FA) uptake from jejunal lumen is closely associated with pediatric growth. Enterotoxigenic Escherichia coli (ETEC), which poses a big threat to young mammals’ health, is also targeted on the jejunum, however, the effects on FA uptake is not understood yet. To explore the impacts of ETEC on the FA uptake ability of jejunum epithelial enterocytes during early life, we orally gavaged weaning piglets with ETEC K88 and found intestinal inflammation combined with compromised uptake of LCFA (C16:0, C18:0, C20:3, C20:4) except for C14:0 whose chain length is similar to medium chain fatty acid (MCFA). Furthermore, we observed reduced protein expression of TJs, fatty acid transport protein 4 (FATP4), peroxisome proliferator-activated receptor γ (PPARγ), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), and upregulated expression of p-PPARγ. In the in vitro study, we challenged polarized porcine intestine jejunum cell line IPEC-J2 with ETEC K88 and discovered similar results on intestinal barrier and expression of associated genes combined with morphological changes. Based on the constructed cellular model, we then determined lower uptake of BODIPY-labeled C16:0 without any difference in the uptake of BODIPY-labeled C12:0. The content of intracellular triglyceride which was mainly synthesized by LCFA concomitantly lowered down. Using gene knock down and overexpression, FATP4 was confirmed to be responsible for LCFA uptake. Moreover, ERK1/2 inhibitor U0126 and PPARγ antagonist T0070907 revealed ETEC could initiate cascaded phosphorylation of ERK1/2 and PPARγ resulting in hindered expression of FATP4. These results indicate ETEC challenge will cause dysfunction in FATP4-dependent LCFA uptake by phosphorylation of ERK1/2 and PPARγ. Furthermore, intestinal uptake of MCFA is in a FATP4-independent manner which is not easily disturbed by ETEC.

High Membranous Expression of Fatty Acid Transport Protein 4 Is Associated with Tumorigenesis and Tumor Progression in Clear Cell Renal Cell Carcinoma.

The increased requirement of fatty acids forces cancer cells to enhance uptake of fatty acids from the extracellular milieu, in addition to de novo lipogenesis. Coexpression of cluster of differentiation 36 (CD36) with fatty acid transport protein 4 (FATP4) or long-chain acyl CoA synthetase 1 (ACSL1) synergistically activated fatty acid uptake in experimental models. In this study, we investigated the immunohistochemical expression of CD36, FATP4, and ACSL1 in 180 cases of clear cell renal cell carcinoma (RCC) in comparison with 80 specimens of the normal kidney. We also examined the clinical implication of these three fatty acid transporters in RCC, which was validated by an open-access The Cancer Genome Atlas data analysis. Both CD36 and FATP4 revealed higher membranous expressions in RCC tumor cells than in normal cells. In contrast, ACSL1 expression was remarkably reduced in RCC tumor cells compared to normal cells. CD36, FATP4, and ACSL1 showed high expressions in 74 (41.1%), 85 (47.2%), and 72 (40.0%) out of 180 RCC cases, respectively. Clinically, high FATP4 in tumor cells was associated with female gender (p = 0.05), high TNM stage (p = 0.039), tumor necrosis (p = 0.009), and tumor recurrence (p = 0.037), while high ACSL1 was only related to female gender (p = 0.023). CD36 expression revealed no correlation with the clinicopathologic parameters of RCC. Increased FATP4 expression displayed an association with short recurrence-free survival (p = 0.003). In conclusion, the high FATP4 expression was clinically associated with poor prognostic factors of RCC. Overexpression of membranous FATP4 and CD36 combined with reduced cytoplasmic expression of ACSL1 might be a tumor-specific feature of RCC, contributing to the tumorigenesis and tumor progression.

Maternal BMI and gestational diabetes alter placental lipid transporters and fatty acid composition

IntroductionPlacental fatty acid (FA) uptake and metabolism depend on maternal supply which may be altered when women have a high pre-pregnancy body mass index (BMI) or develop gestational diabetes (GDM). Consequently, an impaired FA transport to the fetus may negatively affect fetal development. While placental adaptation of maternal-fetal glucose transfer in mild GDM has been described, knowledge on placental FA acid metabolism and possible adaptations in response to maternal obesity or GDM is lacking.We aimed to analyze the FA composition and the expression of key genes involved in FA uptake and metabolism in placentas from women with pre-pregnancy normal weight (18.5 ≤ BMI<25 kg/m2), overweight (25 ≤ BMI<30 kg/m2), obesity (BMI ≥ 30 kg/m2), and lean pregnant women with GDM. MethodsPlacental FA content was determined by gas liquid chromatography. Placental mRNA expression of FA transport proteins (FATP1, FATP4, FATP6), FA binding proteins (FABP3, FABP4, FABP7), FA translocase (FAT/CD36) and enzymes (Endothelial lipase (EL) and lipoprotein lipase (LPL)) were quantified by qRT-PCR. ResultsHigh pre-pregnancy BMI and GDM were associated with decreased placental FATP1, FATP4, EL and increased FAT/CD36 and FATP6 expressions. LPL mRNA levels and placental total FA content were similar among groups. Specific FA, including some long-chain polyunsaturated FA, were altered. DiscussionOur results demonstrate that high pre-pregnancy BMI or GDM independently alter mRNA expression levels of genes involved in FA uptake and metabolism and the placental FA profile, which could affect fetal development and long-term health.

Ruminant and industrial trans-fatty acid uptake in the heart

Dietary trans-fats are strongly associated with heart disease. However, the capacity for the tissues of the body, and specifically the heart, to take up trans-fats is unknown. It is also unknown if different trans-fats have different uptake capacities in the heart and other tissues of the body. Diets of low-density lipoprotein receptor-deficient mice were supplemented for 14weeks with foods that contained 1.5% of the trans-fat elaidic acid or vaccenic acid. Tissues were extracted and frozen in liquid nitrogen, and then lipids were analyzed by gas chromatography for fatty acid content. Isolated cardiomyocytes were also exposed to elaidic or vaccenic acid in cell culture media for 24h. Dietary supplementation with vaccenic or elaidic acid resulted in a 20-fold higher accumulation of these TFAs in fat deposits in the body in comparison to liver. Liver tissue accumulated about twice as much per gram tissue as heart. Similar quantities of both elaidic acid and vaccenic acid were taken up by the tissues. Isolated cardiomyocytes exhibited an unusually large uptake of trans-fat, and this was dependent upon both the concentration and duration of exposure to the trans-fats but not upon the type of trans-fat. Expression levels of CD36 and FATP4 were not significantly changed during dietary interventions or exposure of cells to trans-fats. We conclude that fat, liver and heart (including cardiomyocytes) are all capable of accumulating trans-fat in response to dietary supplementation without changes in fatty acid transport protein expression.

Glucagon-like peptide-1 analogue prevents nonalcoholic steatohepatitis in non-obese mice.

To investigate whether a glucagon-like peptide-1 (GLP-1) analogue inhibits nonalcoholic steatohepatitis (NASH), which is being increasingly recognized in Asia, in non-obese mice. A methionine-choline-deficient diet (MCD) along with exendin-4 (20 μg/kg per day, ip), a GLP-1 analogue, or saline was administered to male db/db mice (non-obese NASH model). Four or eight weeks after commencement of the diet, the mice were sacrificed and their livers were excised. The excised livers were examined by histochemistry for evidence of hepatic steatosis and inflammation. Hepatic triglyceride (TG) and free fatty acid (FFA) content was measured, and the expression of hepatic fat metabolism- and inflammation-related genes was evaluated. Oxidative stress-related parameters and macrophage recruitment were also examined using immunohistochemistry. Four weeks of MCD feeding induced hepatic steatosis and inflammation and increased the hepatic TG and FFA content. The expression of fatty acid transport protein 4 (FATP4), a hepatic FFA influx-related gene; macrophage recruitment; and the level of malondialdehyde (MDA), an oxidative stress marker, were significantly augmented by a 4-wk MCD. The levels of hepatic sterol regulatory element-binding protein-1c (SREBP-1c) mRNA (lipogenesis-related gene) and acyl-coenzyme A oxidase 1 (ACOX1) mRNA (β-oxidation-related gene) had decreased at 4 wk and further decreased at 8 wk. However, the level of microsomal triglyceride transfer protein mRNA (a lipid excretion-related gene) remained unchanged. The administration of exendin-4 significantly attenuated the MCD-induced increase in hepatic steatosis, hepatic TG and FFA content, and FATP4 expression as well as the MCD-induced augmentation of hepatic inflammation, macrophage recruitment, and MDA levels. Additionally, it further decreased the hepatic SREBP-1c level and alleviated the MCD-mediated inhibition of the ACOX1 mRNA level. These results suggest that GLP-1 inhibits hepatic steatosis and inflammation through the inhibition of hepatic FFA influx and oxidative stress in a non-obese NASH model.

New evidence for the role of ceramide in the development of hepatic insulin resistance.

AimThere are few and contradictory data on the role of excessive accumulation of intracellular sphingolipids, particularly ceramides, in the development of hepatic insulin resistance. In our study we assessed accumulated sphingolipid fractions and clarify the mechanisms of hepatic insulin resistance development as well as involvement of fatty acid and ceramide transporters in this process.MethodsIn culture of primary rat hepatocytes, exposed to high concentration of palmitic acid (0.75mM) during short and prolonged incubation, high performance liquid chromatography was used to assess intra- and extracellular sphingolipid fractions content. Degree of palmitate-induced insulin resistance was estimated by measuring changes in phosphorylation of insulin pathway proteins by western blotting as well as changes in expression of different type of transporters.ResultsIn our study short and prolonged exposure of primary hepatocytes to palmitic acid resulted in increased intracellular accumulation of ceramide which inhibited insulin signaling pathway. We observed a significant increase in the expression of fatty-acid transport protein (FATP2) and ceramide transfer protein (CERT) what is consistent with enhanced intracellular ceramide content. The content of extracellular ceramide was increased nearly threefold after short and twofold after long incubation period. Expression of microsomal triglyceride transfer protein (MTP) and ATP-binding cassette transporter (ABCA1) was increased significantly mainly after short palmitate incubation.ConclusionOur data showed that increase in intarcellular ceramide content contributes to the development of hepatic insulin resistance. We suggest pivotal role of transporters in facilitating fatty acid influx (FATP2), accumulation of ceramides (CERT) and export to the media (MTP and ABCA1).

Seasonal upregulation of catabolic enzymes and fatty acid transporters in the flight muscle of migrating hoary bats, Lasiurus cinereus

The high energy density of fat, and limited capacity for carbohydrate storage suggest that migrating bats should fuel endurance flights with fat, as observed in migrating birds. Yet, cursorial mammals are unable to support high intensity exercise with fat stores. We hypothesized that migratory bats and birds have converged on similar physiological mechanisms to fuel endurance flight with fat. We predicted bats would seasonally upregulate fatty acid transport and oxidation pathways when migration demands were high. We studied seasonal variation in mitochondrial oxidative enzyme activities and fatty acid transport protein expression in the flight muscle of hoary bats (Lasiurus cinereus). Carnitine palmitoyl transferase, 3-hydroxyacyl-CoA dehydrogenase and citrate synthase activity increased during migration. There were no changes in expression of fatty acid translocase or plasma membrane fatty acid binding protein. Heart-type fatty acid binding protein expression increased 5-fold in migrating females, but did not vary seasonally in males. An aerial insectivore lifestyle, and the coincidence of migration and pregnancy may explain differences in transporter expression compared to previously studied birds. Overall, our results are consistent with seasonal upregulation of lipid metabolism and aerobic capacity, and confirm that migration poses distinct physiological challenges for bats.

Requirement of Fatty Acid Transport Protein 4 for Development, Maturation, and Function of Sebaceous Glands in a Mouse Model of Ichthyosis Prematurity Syndrome

Fatty acid transport protein 4 (FATP4) is one of a family of six transmembrane proteins that facilitate long- and very long-chain fatty acid uptake. FATP4 is expressed in several tissues, including skin. Mutations in human SLC27A4, which encodes FATP4, cause ichthyosis prematurity syndrome, characterized by a thick desquamating epidermis and premature birth. Mice lacking FATP4, which genetically model the human disease, are born with tight, thick skin and a defective skin barrier; they die neonatally due to dehydration and restricted movements. Both the skin phenotype and the lethality are rescued by transgene expression of FATP4 in suprabasal keratinocytes. Sebaceous glands in Fatp4 null skin grafted onto nude mice were found to be dystrophic and enwrapped by thick layers of epithelial cells. Consistent with these results, transgene-rescued Fatp4 null mice showed a subnormal level of FATP4 expression in sebocytes and exhibited abnormal development of both sebaceous glands and meibomian glands, specialized sebaceous glands of the eyelids. Sebum from these mice contained a reduced level of type II diester wax, a major mouse sebum lipid species, and showed perturbations in mass spectrometric profiles of diester wax and cholesteryl ester species. In addition, these mice showed an impaired ability to repel water and regulate body temperature after water immersion. Taken together, our results suggest that FATP4 plays crucial roles in the development and maturation of both sebaceous and meibomian glands, as well as in the formation and composition of sebum, likely by regulating the trafficking of fatty acids necessary for proper synthesis of sebum lipids.

Effect of rumen-protected choline supplementation on liver and adipose gene expression during the transition period in dairy cattle

We previously reported that supplementation of rumen-protected choline (RPC) reduces the hepatic triacylglycerol concentration in periparturient dairy cows during early lactation. Here, we investigated the effect of RPC on the transcript levels of lipid metabolism-related genes in liver and adipose tissue biopsies, taken at wk −3, 1, 3, and 6 after calving, to elucidate the mechanisms underlying this RPC-induced reduction of hepatic lipidosis. Sixteen multiparous cows were blocked into 8 pairs and randomly allocated to either 1 of 2 treatments, with or without RPC. Treatments were applied from 3wk before to 6wk after calving. Both groups received a basal diet and concentrate mixture. One group received RPC supplementation, resulting in an intake of 14.4g of choline per day, whereas controls received an isoenergetic mixture of palm oil and additional soybean meal. Liver and adipose tissue biopsies were taken at wk −3, 1, 3, and 6 to determine the mRNA abundance of 18 key genes involved in liver and adipose tissue lipid and energy metabolism. Milk samples were collected in wk 1, 2, 3, and 6 postpartum for analysis of milk fatty acid (FA) composition. The RPC-induced reduction in hepatic lipidosis could not be attributed to altered lipolysis in adipose tissue, as no treatment effect was observed on the expression of peroxisome proliferator-activated receptor γ, lipoprotein lipase, or FA synthase in adipose tissue, or on the milk FA composition. Rumen-protected choline supplementation increased the expression of FA transport protein 5 and carnitine transporter SLC22A5 in the liver, suggesting an increase in the capacity of FA uptake and intracellular transport, but no treatment effect was observed on carnitine palmitoyl transferase 1A, transporting long-chain FA into mitochondria. In the same organ, RPC appeared to promote apolipoprotein B-containing lipoprotein assembly, as shown by elevated microsomal triglyceride transfer protein expression and apolipoprotein B100 expression. Cows supplemented with RPC displayed elevated levels of glucose transporter 2 mRNA and a reduced peak in pyruvate carboxylase mRNA immediately after calving, showing that supplementation also resulted in improved carbohydrate metabolism. The results from this study suggest that RPC supplementation reduces liver triacylglycerol by improved FA processing and very-low-density lipoprotein synthesis, and RPC also benefits hepatic carbohydrate metabolism.

FATP4 contributes as an enzyme to the basal and insulin-mediated fatty acid uptake of C2C12muscle cells

The function of membrane proteins in long-chain fatty acid transport is controversial. The acyl-CoA synthetase fatty acid transport protein-4 (FATP4) has been suggested to facilitate fatty acid uptake indirectly by its enzymatic activity, or directly by transport across the plasma membrane. Here, we investigated the function of FATP4 in basal and insulin mediated fatty acid uptake in C(2)C(12) muscle cells, a model system relevant for fatty acid metabolism. Stable expression of exogenous FATP4 resulted in a twofold higher fatty acyl-CoA synthetase activity, and cellular uptake of oleate was enhanced similarly. Kinetic analysis demonstrated that FATP4 allowed the cells to reach apparent saturation of fatty acid uptake at a twofold higher level compared with control. Short-term treatment with insulin increased fatty acid uptake in line with previous reports. Surprisingly, insulin increased the acyl-CoA synthetase activity of C(2)C(12) cells within minutes. This effect was sensitive to inhibition of insulin signaling by wortmannin. Affinity purified FATP4 prepared from insulin-treated cells showed an enhanced enzyme activity, suggesting it constitutes a novel target of short-term metabolic regulation by insulin. This offers a new mechanistic explanation for the concomitantly observed enhanced fatty acid uptake. FATP4 was colocalized to the endoplasmic reticulum by double immunofluorescence and subcellular fractionation, clearly distinct from the plasma membrane. Importantly, neither differentiation into myotubes nor insulin treatment changed the localization of FATP4. We conclude that FATP4 functions by its intrinsic enzymatic activity. This is in line with the concept that intracellular metabolism plays a significant role in cellular fatty acid uptake.

Fatty acid transport protein expression in human brain and potential role in fatty acid transport across human brain microvessel endothelial cells

The blood-brain barrier (BBB), formed by the brain capillary endothelial cells, provides a protective barrier between the systemic blood and the extracellular environment of the CNS. Passage of fatty acids from the blood to the brain may occur either by diffusion or by proteins that facilitate their transport. Currently several protein families have been implicated in fatty acid transport. The focus of the present study was to identify the fatty acid transport proteins (FATPs) expressed in the brain microvessel endothelial cells and characterize their involvement in fatty acid transport across an in vitro BBB model. The major fatty acid transport proteins expressed in human brain microvessel endothelial cells (HBMEC), mouse capillaries and human grey matter were FATP-1, -4 and fatty acid binding protein 5 and fatty acid translocase/CD36. The passage of various radiolabeled fatty acids across confluent HBMEC monolayers was examined over a 30-min period in the presence of fatty acid free albumin in a 1 : 1 molar ratio. The apical to basolateral permeability of radiolabeled fatty acids was dependent upon both saturation and chain length of the fatty acid. Knockdown of various fatty acid transport proteins using siRNA significantly decreased radiolabeled fatty acid transport across the HBMEC monolayer. Our findings indicate that FATP-1 and FATP-4 are the predominant fatty acid transport proteins expressed in the BBB based on human and mouse expression studies. While transport studies in HBMEC monolayers support their involvement in fatty acid permeability, fatty acid translocase/CD36 also appears to play a prominent role in transport of fatty acids across HBMEC.

Epidermal hyperproliferation in mice lacking fatty acid transport protein 4 (FATP4) involves ectopic EGF receptor and STAT3 signaling

Fatty acid transport protein (FATP) 4 is one of a family of six FATPs that facilitate long- and very long-chain fatty acid uptake. Mice lacking FATP4 are born with tight, thick skin and a defective epidermal barrier; they die neonatally due to dehydration and restricted movements. Both the skin phenotype and the lethality are rescued by transgene-driven expression of FATP4 solely in suprabasal keratinocytes. Here we show that Fatp4 mutants exhibit epidermal hyperplasia resulting from an increased number of proliferating suprabasal cells. In addition, barrier formation initiates precociously but never progresses to completion. To investigate possible mechanisms whereby Fatp4 influences skin development, we identified misregulated genes in Fatp4 mutants. Remarkably, three members of the epidermal growth factor (EGF) family ( Ereg, Areg, and Epgn) showed increased expression that was associated with elevated epidermal activation of the EGF receptor (EGFR) and STAT3, a downstream effector of EGFR signaling. Both Tyrphostin AG1478, an EGFR tyrosine kinase inhibitor, and curcumin, an inhibitor of both STAT3 and EGFR, attenuated STAT3 activation/nuclear translocation, reduced skin thickening, and partially suppressed the barrier abnormalities. These data identify FATP4 activity as negatively influencing EGFR activation and the resulting STAT3 signaling during normal skin development. These findings have important implications for understanding the pathogenesis of ichthyosis prematurity syndrome, a disease recently shown to be caused by FATP4 mutations.

Increased expression of FABP1 and cFLIPL by free fatty acid treatment is associated with reduced death-receptor mediated apoptosis in human primary hepatocytes

Aims: The non-alcoholic fatty liver disease (NAFLD) is associated with excessive accumulation of lipid-droplets in hepatocytes and causes increased death-receptor expression, altered apoptosis-induction, liver damage and fibrosis. Investigation of fatty acid transport protein expression and lipid accumulation in steatotic hepatocytes may elucidate the correlation of FFA transport mechanisms, death-receptor expression and apoptosis. Methods: Human primary hepatocytes were treated with 0.5mM and 1mM free fatty acids (FFA; oleate: palmitate=2: 1) for 24–96h. Expression of the FFA transport proteins FABP-1, FATP-5, CD36/FAT and Caveolin-1 as well as the death-receptors DR4, DR5, CD95/Fas and TNFR-1 and the apoptosis-related proteins PUMA, NOXA, cFLIPL was investigated. Activation of the stress-responsive kinase ERK was determined by Western-blot. Results: Expression of CD36 (0.5±0.1,p<0.01;means±SEM) and Caveolin-1 (0.5±0.1,p<0.01) was reduced in hepatocytes after treatment with FFA for 96h while FABP-1 (2.2±0.2,p<0.01) expression-levels were elevated. FATP-5-expression was not changed significantly under FFA-treatment. FFA-incubation for 96h slightly decreased the expression of DR4 (0.7±0.1,p=0.014) and DR5 (0.6±0.1,p=0.011). Expression-levels of CD95/Fas (2.7±0.1, p=0.04) and TNFR-1 (2.1±0.1,p<0.01) were increased after incubation with 0.5mM FFA. Furthermore the expression of the pro-apoptotic molecule NOXA (0.4±0.1,p<0.01) was repressed. FFA-treatment did not affect apoptosis-induction (M30) significantly but activated the expression of the anti-apoptotic protein cFLIPL (2.9±0.5,p=0.017). Western-blot analyses showed increased phosphorylation of the stress-responsive kinase ERK after FFA-treatment. Conclusions: FFA-treatment induces FABP-1-expression consequently FFA-uptake seems to be an active mechanism. FFA-incubation activates the expression of CD95/Fas and TNFR-1 but does not activate apoptosis, which may be due to increased cFLIPL expression and ERK activation.

Clay ingestion enhances intestinal triacylglycerol hydrolysis and non-esterified fatty acid absorption

Consumption by animals and humans of earthy materials such as clay is often related to gut pathologies. Our aim was to determine the impact of kaolinite ingestion on glucose and NEFA transport through the intestinal mucosa. The expression of hexose transporters (Na/glucose co-transporter 1 (SGLT1), GLUT2, GLUT5) and of proteins involved in NEFA absorption (fatty acid transporter/cluster of differentiation 36 (FAT/CD36), fatty acid transport protein 4 (FATP4) and liver fatty acid binding protein (L-FABP)) was measured (1) in rats whose jejunum was perfused with a solution of kaolinite, and (2) in rats who ate spontaneously kaolinite pellets during 7 and 28 d. Also, we determined TAG and glucose absorption in the kaolinite-perfused group, and pancreatic lipase activity, gastric emptying and intestinal transit in rats orally administered with kaolinite. Glucose absorption was not affected by kaolinite perfusion or ingestion. However, kaolinite induced a significant increase in intestinal TAG hydrolysis and NEFA absorption. The cytoplasmic expression of L-FABP and FATP4 also increased due to kaolinite ingestion. NEFA may enter the enterocytes via endocytosis mainly since expression of NEFA transporters in the brush-border membrane was not affected by kaolinite. After uptake, rapid binding of NEFA by L-FABP and FATP4 could act as an intracellular NEFA buffer to prevent NEFA efflux. Increased TAG hydrolysis and NEFA absorption may be due to the adsorption properties of clay and also because kaolinite ingestion caused a slowing down of gastric emptying and intestinal transit.

Dairy protein and leucine alter GLP-1 release and mRNA of genes involved in intestinal lipid metabolism in vitro

Objective A growing body of evidence supports an antiobesity effect of dairy products; however, the mechanisms remain unclear. The objective of this study was to explore possible intestinal mechanisms by which dairy delivers an antiobesity effect. The human intestinal cell line, NCI-H716, was used to test the hypothesis that branched-chain amino acids and dairy proteins regulate satiety hormone secretion and modulate genes involved in fatty acid and cholesterol metabolism. Methods In dose–response (0.5%, 1.0%, 2.0%, and 3.0%) studies, the effect of leucine, isoleucine, valine, skim milk, casein, and whey on glucagon-like peptide-1 release and the expression of selected genes were tested. Results Leucine, isoleucine, skim milk, and casein stimulated glucagon-like peptide-1 release ( P < 0.05). Isoleucine and whey downregulated the expression of intestinal-type fatty acid binding protein ( i-FABP), fatty acid transport protein 4 ( FATP4), Niemann-Pick C-1–like-1 protein ( NPC1L1), acetyl-coenzyme A carboxylase ( ACC), fatty acid synthase ( FAS), sterol regulatory element-binding protein-2 ( SREBP-2), and 3-hydroxy-3-methylglutaryl-CoA reductase ( HMGCR; P < 0.05). Leucine and valine downregulated the expression of NPC1L1, ACC, FAS, SREBP-2, and HMGCR ( P < 0.05). Casein downregulated the expression of i-FABP, FATP4, ACC, FAS, SREBP-2, and HMGCR ( P < 0.05). Skim milk downregulated the expression of ACC, FAS, and SREBP-2, but not i-FABP, FATP4, and NPC1L1. Conclusion This work suggests that the antiobesity effect of dairy may be mediated, at least in part, by integration of events that promote glucagon-like peptide-1 secretion and inhibit expression of genes involved in intestinal fatty acid and cholesterol absorption and synthesis.