Peptide YY and neuropeptide Y induce villin expression, reduce adhesion, and enhance migration in small intestinal cells through the regulation of CD63, matrix metalloproteinase-3, and Cdc42 activity

Peptide YY (PYY) and neuropeptide Y (NPY) are regulatory peptides synthesized in the intestine and brain, respectively, that modify physiological functions affecting nutrient assimilation and feeding behavior. Because PYY and NPY also alter the expression of intestine-specific differentiation marker proteins and the tetraspanin CD63, which is involved in cell adhesion, we investigated whether intestinal cell differentiation could be linked to mucosal cell adhesion and migration through these peptides. PYY and NPY significantly decreased cell adhesion and increased cell migration in a dose-dependent manner prior to cell confluency in our model system, non-tumorigenic small intestinal hBRIE 380i cells. Both peptides reduced CD63 expression and CD63-dependent cell adhesion. CD63 overexpression increased and antisense CD63 cDNA decreased intestinal cell adhesion. In parallel, both PYY and NPY increased expression of matrix metalloproteinase-3 (MMP-3) to a level sufficient to induce cell migration by activating the Rho GTPase Cdc42. The effects of both peptides on cell migration were blocked in cells constitutively overexpressing dominant-negative Cdc42. PYY and NPY also significantly induced the expression of the differentiation marker villin, which could be eliminated by an MMP inhibitor at a concentration that inhibits cell migration. Increased MMP-3 activity, which enhanced cell migration, also induced villin mRNA levels. Therefore, these data indicate that the alteration of adhesion and migration by PYY and NPY occurs in part by synchronous modulation of three proteins that are involved in extracellular matrix-basolateral membrane interactions, CD63, MMP-3 and Cdc42, and that PYY/NPY regulation of expression of mucosal proteins such as villin is linked to the process of cell migration and adhesion.     

Cytokine production and DNA synthesis by Human peripheral Lymphocytes in response to Palmitic,Stearic, Oleic,and Linoleic acid

Effects of palmitic, stearic, oleic, and linoleic acid on mitogen-induced DNA synthesis, on production of IL-1β, IL-2, IFN-gamma, and TNF-α, and on IL-2R expression were determined in human peripheral lymphocytes. Free fatty acids (FFA) were added over a wide range of concentrations to cells cultured under serum free conditions with fatty acid free albumin. DNA synthesis was stimulated by low and inhibited by high FFA concentrations. Physiologica concentrations were stimulatory, except for linoleic acid. Cytokine production became affected by all FFA tested. Palmitic acid enhanced the release of IFN-gamma at concentrations that diminished TNF-α production. Saturated fatty acids were significantly more potent than unsaturated fatty acids in affecting cytokine production. IFN-gamma secretion was significantly more stimulated or inhibited by the various FFA compared with the other cytokines. IL-2R expression correlated with the production of IL-2. When tested in combination, stimulatory as well as inhibitory effects of the individual FFA became attenuated. It is suggested that palmitic, stearic, oleic, and linoleic acid are physiological regulators of DNA synthesis and cytokine release in human peripheral lymphocytes. Modulation of FFA ratios may be an effective means for the fine tuning of the immune system. As secretory mechanisms of cytokines appear to exhibit substrate specificity for FFA, the release of individual cytokines may be selectively influenced by FFA. © 1994 Wiley-Liss, Inc.     

Thiazolidinediones upregulate impaired fatty acid uptake in skeletal muscle of type 2 diabetic subjects

We examined the regulation of free fatty acid (FFA, palmitate) uptake into skeletal muscle cells of nondiabetic and type 2 diabetic subjects. Palmitate uptake included a protein-mediated component that was inhibited by phloretin. The protein-mediated component of uptake in muscle cells from type 2 diabetic subjects (78 +/- 13 nmol. mg protein-1. min-1) was reduced compared with that in nondiabetic muscle (150 +/- 17, P < 0.01). Acute insulin exposure caused a modest (16 +/- 5%, P < 0.025) but significant increase in protein-mediated uptake in nondiabetic muscle. There was no significant insulin effect in diabetic muscle (+19 +/- 19%, P = not significant). Chronic (4 day) treatment with a series of thiazolidinediones, troglitazone (Tgz), rosiglitazone (Rgz), and pioglitazone (Pio) increased FFA uptake. Only the phloretin-inhibitable component was increased by treatment, which normalized this activity in diabetic muscle cells. Under the same conditions, FFA oxidation was also increased by thiazolidinedione treatment. Increases in FFA uptake and oxidation were associated with upregulation of fatty acid translocase (FAT/CD36) expression. FAT/CD36 protein was increased by Tgz (90 +/- 22% over control), Rgz (146 +/- 42%), and Pio (111 +/- 37%, P < 0.05 for all 3) treatment. Tgz treatment had no effect on fatty acid transporter protein-1 and membrane-associated plasmalemmal fatty acid-binding protein mRNA expression. We conclude that FFA uptake into cultured muscle cells is, in part, protein mediated and acutely insulin responsive. The basal activity of FFA uptake is impaired in type 2 diabetes. In addition, chronic thiazolidinedione treatment increased FFA uptake and oxidation into cultured human skeletal muscle cells in concert with upregulation of FAT/CD36 expression. Increased FFA uptake and oxidation may contribute to lower circulating FFA levels and reduced insulin resistance in skeletal muscle of individuals with type 2 diabetes following thiazolidinedione treatment.     

Fat depot origin affects fatty acid handling in cultured rat and human preadipocytes

Regional differences in free fatty acid (FFA) handling contribute to diseases associated with particular fat distributions. As cultured rat preadipocytes became differentiated, FFA transfer into preadipocytes increased and was more rapid in single perirenal than in epididymal cells matched for lipid content. Uptake by human omental preadipocytes was greater than uptake by abdominal subcutaneous preadipocytes. Adipose-specific fatty acid binding protein (aP2) and keratinocyte lipid binding protein abundance was higher in differentiated rat perirenal than in epididymal preadipocytes. This interdepot difference in preadipocyte aP2 expression was reflected in fat tissue in older animals. Carnitine palmitoyltransferase 1 activity increased during differentiation and was higher in perirenal than in epididymal preadipocytes, particularly the muscle isoform. Long-chain acyl-CoA levels were higher in perirenal than in epididymal preadipocytes and isolated fat cells. These data are consistent with interdepot differences in fatty acid flux ensuing from differences in fatty acid binding proteins and enzymes of fat metabolism. Heterogeneity among depots results, in part, from distinct intrinsic characteristics of adipose cells. Different depots are effectively separate miniorgans.     

Y receptor-mediated induction of CD63 transcripts, a tetraspanin determined to be necessary for differentiation of the intestinal epithelial cell line, hBRIE 380i cells.

Peptide YY (PYY) and neuropeptide Y (NPY) are peptides that coordinate intestinal activities in response to luminal and neuronal signals. In this study, using the rat hybrid small intestinal epithelial cell line, hBRIE 380i cells, we demonstrated that PYY- and NPY-induced rearrangement of actin filaments may be in part through a Y1alpha and/or a nonneuronal Y2 receptor, which were cloned from both the intestinal mucosa and the hBRIE 380i cells. A number of PYY/NPY-responsive genes were also identified by subtractive hybridization of the hBRIE 380i cells in the presence or absence of a 6-h treatment with PYY. Several of these genes coded for proteins associated with the cell cytoskeleton or extracellular matrix. One of these proteins was the transmembrane-4 superfamily protein CD63, previously shown to associate with beta(1)-integrin and implicated in cell adhesion. CD63 immunoreactivity, using antibody to the extracellular domain, was highest in the differentiated cell clusters of the hBRIE 380i cells. The hBRIE 380i cells transfected with antisense CD63 cDNA lost these differentiated clusters. These studies suggest a new role for NPY and PYY in modulating differentiation through cytoskeletal associated proteins.     

Increased oral detection, but decreased intestinal signaling for fats in mice lacking gut microbiota

Germ-free (GF) mice lacking intestinal microbiota are significantly leaner than normal (NORM) control mice despite consuming more calories. The contribution of microbiota on the recognition and intake of fats is not known. Thus, we investigated the preference for, and acceptance of, fat emulsions in GF and NORM mice, and associated changes in lingual and intestinal fatty acid receptors, intestinal peptide content, and plasma levels of gut peptides. GF and NORM C57Bl/6J mice were given 48-h two-bottle access to water and increasing concentrations of intralipid emulsions. Gene expression of the lingual fatty acid translocase CD36 and protein expression of intestinal satiety peptides and fatty-acid receptors from isolated intestinal epithelial cells were determined. Differences in intestinal enteroendocrine cells along the length of the GI tract were quantified. Circulating plasma satiety peptides reflecting adiposity and biochemical parameters of fat metabolism were also examined. GF mice had an increased preference and intake of intralipid relative to NORM mice. This was associated with increased lingual CD36 (P<0.05) and decreased intestinal expression of fatty acid receptors GPR40 (P<0.0001), GPR41 (P<0.0001), GPR43 (P<0.05), and GPR120 (P<0.0001) and satiety peptides CCK (P<0.0001), PYY (P<0.001), and GLP-1 (P<0.001). GF mice had fewer enteroendocrine cells in the ileum (P<0.05), and more in the colon (P<0.05), relative to NORM controls. Finally, GF mice had lower levels of circulating leptin and ghrelin (P<0.001), and altered plasma lipid metabolic markers indicative of energy deficits. Increased preference and caloric intake from fats in GF mice are associated with increased oral receptors for fats coupled with broad and marked decreases in expression of intestinal satiety peptides and fatty-acid receptors.     

A new molecular form of PYY: structural characterization of human PYY(3-36) and PYY(1-36)

A radioimmunoassay was developed using an antibody raised in rabbits against synthetic porcine PYY. This radioimmunoassay was used to detect PYY immunoreactivity in human intestinal extracts. Human colonic mucosa was extracted with acid, centrifuged and the supernatant concentrated by low pressure preparative reverse phase chromatography. A subsequent C-18 reverse phase HPLC step separated two peaks of PYY immunoreactivity. Each peak was purified by sequential steps of ion-exchange FPLC and reverse phase HPLC. In the final purification step single absorbance peaks were associated with PYY immunoreactivity. Microsequence, amino acid, and mass spectral analysis of the intact and tryptic fragments of the two peptides were consistent with the structures: YPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY-amide [human PYY(1-36)] and--IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY-amide [human PYY(3-36)]. Human PYY(1-36) differs from porcine PYY only at position 3, with Ile instead of Ala, and position 18, with Asn instead of Ser. PYY(3-36) may differ in its biological activity from the intact peptide. Its high proportions in the colon suggest that it is released into the circulation where it could act as a partial antagonist of PYY(1-36).     

Effect of subcutaneous injections of PYY1-36 and PYY3-36 on appetite, ad libitum energy intake, and plasma free fatty acid concentration in obese males

Intraveneous (i.v.) PYY(3-36) infusions have been reported to reduce energy intake (EI) in humans, whereas few studies exist on effects of PYY(1-36). The aim of the present study was to examine effects of subcutaneous (sc) injections of PYY(1-36) and PYY(3-36) on appetite, ad libitum EI, plasma concentrations of PYY and free fatty acids (FFA) in obese males. Twenty-four males (BMI 27-40 kg/m(2)) were randomly assigned to two groups receiving sc injections of either PYY(1-36) or PYY(3-36) in a blinded, placebo-controlled, dose-escalating, cross-over study. Subjects were studied 5 days in succession, with escalating doses of PYY(1-36) [saline, 50, 100, 150, and 200 pmol PYY(1-36)/kg lean body mass (LBM)], or PYY(3-36) (saline, 25, 50, 75, and 100 pmol PYY(3-36)/kg LBM), respectively. PYY injections resulted in dose-dependent increases in plasma PYY levels but no effect on EI in either the PYY(1-36) or the PYY(3-36) group. However, increasing doses of PYY(3-36), but not PYY(1-36), resulted in increased ratings of satiety and decreased ratings of hunger, thirst, and prospective food consumption. Although not dose dependently, significant elevation of plasma FFA was seen after injection of PYY(3-36), but not PYY(1-36). Although sc administration of PYY was well tolerated, it remains to be determined whether high-dose PYY(3-36) is sufficient in reducing EI in long-term trials, and if so, whether the reduction in EI occurs without nausea. PYY(1-36) is unlikely to be important in regulating energy intake. The PYY(3-36) administrations caused a non-dose-dependent mobilization of FFA, likely through a direct effect.     

Free fatty acid release from endothelial cells

Cultured bovine aortic endothelial cells that have been previously enriched with fatty acid are able to release free fatty acid (FFA) into the extracellular fluid. No stimulus other than the presence of albumin in the medium is needed to elicit the FFA release. Intracellular triglycerides appear to be the source of most of the FFA that is released. The released FFA is composed of a mixture of fatty acids, with the fatty acid used to enrich the cells contributing about half of the total. Under certain conditions sufficient fatty acid can be released to increase the FFA concentration of the extracellular fluid. Cells enriched initially with arachidonic acid released 1.7- to 2.9-times more FFA as compared to cells enriched with corresponding amounts of oleic acid. Neither prostaglandins nor lipoxygenase products contributed appreciably to the amount of FFA released from cells enriched with arachidonic acid. Porcine pulmonary artery endothelial cells also can release net amounts of FFA. These findings indicate that endothelial cells have the capacity to release fatty acid in the form of FFA. This process could possibly play a role in the transfer of fatty acids, particularly arachidonic acid, across the endothelium.     

Lack of the intestinal Muc1 mucin impairs cholesterol uptake and absorption but not fatty acid uptake in Muc1-/- mice

Before cholesterol and fatty acid molecules in the small intestinal lumen can interact with their possible transporters for uptake and absorption, they must pass through a diffusion barrier, which may modify the kinetics of nutrient assimilation. This barrier includes an unstirred water layer and a surface mucous coat, which is located at the intestinal lumen-membrane interface. In the present study, we investigated whether disruption of the mucin gene (Muc)1 may influence intestinal uptake and absorption of cholesterol and fatty acid in male Muc1(-/-) mice. The wild-type mice displayed relatively high levels of Muc1, Muc2, Muc3, and Muc4 mRNAs and relatively low levels of Muc5ac and Muc5b mRNAs in the small intestine. The absence of Muc1 mRNA and protein in the small intestines of Muc1(-/-) mice confirmed complete knockout of the Muc1 gene, but the mRNA expression for other mucin genes remained unchanged. Intestinal uptake and absorption of cholesterol but not palmitic acid were significantly reduced in Muc1(-/-) mice compared with the wild-type mice. However, knockout of the Muc1 gene did not impair either expression levels of the genes that encode intestinal sterol efflux transporters Abcg5 and Abcg8 and fatty acid transporter Fatp4 or small intestinal transit rates. We conclude that physiological levels of the epithelial mucin produced by the Muc1 gene are necessary for normal intestinal uptake and absorption of cholesterol in mice. Our study implies that because cholesterol absorption efficiency is reduced by approximately 50% in Muc1-deficient mice, there may be one or more additional pathways for cholesterol absorption.     

Modulation of lipid-induced ER stress by fatty acid shape

Exposure of pancreatic β cells to long-chain saturated fatty acids (SFA) induces a so-called endoplasmic reticulum (ER) stress that can ultimately lead to cell death. This process is believed to participate in insulin deficiency associated with type 2 diabetes, via a decrease in β-cell mass. By contrast, some unsaturated fatty acid species appear less toxic to the cells and can even alleviate SFA-induced ER stress. In the present study, we took advantage of a simple yeast-based model, which brings together most of the trademarks of lipotoxicity in human cells, to screen fatty acids of various structures for their capacity to counter ER stress. Here we demonstrate that the tendency of a free fatty acid (FFA) to reduce SFA toxicity depends on a complex conjunction of parameters, including chain length, level of unsaturation, position of the double bonds and nature of the isomers (cis or trans). Interestingly, potent FFA act as building blocks for phospholipid synthesis and help to restore an optimal membrane organization, compatible with ER function and normal protein trafficking.     

Schlafen-3: A novel regulator of intestinal differentiation

Schlafen-3 (Slfn-3), a novel gene, has been shown to be a negative regulator of proliferation. The current investigation was undertaken to determine whether Slfn-3 might play a role in regulating cellular differentiation. Butyric acid, a short chain fatty acid, which induced differentiation of intestinal cells as evidenced by increased alkaline phosphatase (ALP) activity in the rat small intestinal IEC-6 cells, also produced a marked increase in Slfn-3 expression. Furthermore, overexpression of Slfn-3 caused stimulation of ALP activity in IEC-6 cells, which was exacerbated by butyrate. On the other hand, downregulation of Slfn-3 by slfn-3-si-RNA greatly attenuated the butyrate-mediated induction of differentiation of IEC-6 cells. Additionally, we observed that increased expression of Slfn-3 in colon cancer HCT-116 cells stimulated TGF-β expression and modulated expression of its downstream effectors as evidenced by increased expression of p27kip1 and downregulation of CDK-2. In addition, Slfn-3 increases E-cadherin expression but downregulates β-catenin. In conclusion, our data show that Slfn-3 plays a critical role in regulating intestinal mucosal differentiation. Furthermore our data also show that TGF-β signaling pathway plays an important role in mediating slfn-3 induced differentiation.     

Fatty acid (FFA) transport in cardiomyocytes revealed by imaging unbound FFA is mediated by an FFA pump modulated by the CD36 protein

Free fatty acid (FFA) transport across the cardiomyocyte plasma membrane is essential to proper cardiac function, but the role of membrane proteins and FFA metabolism in FFA transport remains unclear. Metabolism is thought to maintain intracellular FFA at low levels, providing the driving force for FFA transport, but intracellular FFA levels have not been measured directly. We report the first measurements of the intracellular unbound FFA concentrations (FFA(i)) in cardiomyocytes. The fluorescent indicator of FFA, ADIFAB (acrylodan-labeled rat intestinal fatty acid-binding protein), was microinjected into isolated cardiomyocytes from wild type (WT) and FAT/CD36 null C57B1/6 mice. Quantitative imaging of ADIFAB fluorescence revealed the time courses of FFA influx and efflux. For WT mice, rate constants for efflux (～0.02 s(-1)) were twice influx, and steady state FFA(i) were more than 3-fold larger than extracellular unbound FFA (FFA(o)). The concentration gradient and the initial rate of FFA influx saturated with increasing FFA(o). Similar characteristics were observed for oleate, palmitate, and arachidonate. FAT/CD36 null cells revealed similar characteristics, except that efflux was 2-3-fold slower than WT cells. Rate constants determined with intracellular ADIFAB were confirmed by measurements of intracellular pH. FFA uptake by suspensions of cardiomyocytes determined by monitoring FFA(o) using extracellular ADIFAB confirmed the influx rate constants determined from FFA(i) measurements and demonstrated that rates of FFA transport and etomoxir-sensitive metabolism are regulated independently. We conclude that FFA influx in cardiac myocytes is mediated by a membrane pump whose transport rate constants may be modulated by FAT/CD36.     

Fatty acid changes during the induction of differentiation of human promyelocytic leukemia (HL-60) cells by phorbolmyristate acetate.

We studied fatty acid changes that are likely to occur during phorbolmyristate acetate (PMA)-induced differentiation of HL-60 cells. It was observed that PMA-induced differentiation is associated with increased uptake, but not synthesis, of fatty acids. Fatty acid analysis revealed that arachidonic acid (AA), 20:5 n-3 and 22:6 n-3 levels are reduced with a concomitant increase in 22:5 n-6 in the phospholipid fraction. In the FFA fraction there are increases in free AA, free 20:5 n-3, 22:5 n-3 and 22:6 n-3, and a fall in free 22:5 n-6 in PMA-treated cells. PMA-induced differentiation and nitroblue tetrazolium reduction by PMA-treated cells was only partially inhibited (about 20-30%) by indomethacin and nordihydroguiaretic acid (cyclooxygenase and lipoxygenase inhibitors respectively), but not by superoxide dismutase, catalase or mannitol. These results indicate that PMA-induced differentiation of HL-60 cells is accompanied by specific changes in the fatty acid composition of the cells.     

Phytanic acid and docosahexaenoic acid increase the metabolism of all-trans-retinoic acid and CYP26 gene expression in intestinal cells.

Retinoids are essential for growth and cell differentiation of epithelial tissues. The effects of the food compounds phytol, the phytol metabolite phytanic acid, and the fatty acid docosahexaenoic acid (DHA) on the retinoid signaling pathway in intestinal cells were studied. Phytol inhibited the formation of all-trans-retinoic acid (RA) from dietary retinol in intestinal cells. Phytanic acid, a known retinoic X receptor (RXRalpha) and peroxisome proliferator activating receptor (PPARalpha) activator, also activated PPARdelta, and to a lesser degree PPARgamma, in a transactivation assay. Phytanic acid had no effect on intestinal RA hydroxylase CYP26 (also named P450RAI) gene expression and metabolism of all-trans-RA in intestinal Caco-2 cells. However, in combination with retinoic acid receptor (RAR)-ligands (all-trans-RA or synthetic Am580) phytanic acid enhanced the induction of CYP26 and RA-metabolism in comparison to treatments with all-trans-RA or Am580 alone. Also treatment with DHA did not affect CYP26 gene expression and RA-metabolism but cotreatment of the cells with DHA and all-trans-RA or Am580 enhanced the induction of CYP26, in comparison to the induction caused by all-trans-RA or Am580 alone. This study indicates that food compounds such as phytanic acid and DHA that are RXR-agonists and have an impact on intestinal CYP26 gene expression and metabolism of all-trans-RA in intestinal cells.     

Effect of maternal triglycerides and free fatty acids on placental LPL in cultured primary trophoblast cells and in a case of maternal LPL deficiency

Maternal hypertriglyceridemia is a normal condition in late gestation and is an adaptation to ensure an adequate nutrient supply to the fetus. Placental lipoprotein lipase (LPL) is involved in the initial step in transplacental fatty acid transport as it hydrolyzes maternal triglycerides (TG) to release free fatty acids (FFA). We investigated LPL activity and protein (Western blot) and mRNA expression (real-time RT-PCR) in the placenta of an LPL-deficient mother with marked hypertriglyceridemia. The LPL activity was fourfold lower, LPL protein expression 50% lower, and mRNA expression threefold higher than that of normal, healthy placentas at term (n = 4-7). To further investigate the role of maternal lipids in placental LPL regulation, we isolated placental cytotrophoblasts from term placentas and studied LPL activity and protein and mRNA expression after incubation in Intralipid (as a source of TG) and oleic, linoleic, and a combination of oleic, linoleic, and arachidonic acids as well as insulin. Intralipid (40 and 400 mg/dl) decreased LPL activity by approximately 30% (n = 10-14, P < 0.05) and 400 microM linoleic and linoleic-oleic-arachidonic acid (n = 10) decreased LPL activity by 37 and 34%, respectively. No major changes were observed in LPL protein or mRNA expression. We found no effect of insulin on LPL activity or protein expression in the cultured trophoblasts. To conclude, the activity of placental LPL is reduced by high levels of maternal TG and/or FFA. This regulatory mechanism may serve to counteract an excessive delivery of FFA to the fetus in conditions where maternal TG levels are markedly increased.     

Peptide YY as a growth factor for intestinal epithelium

Peptide YY is an abundant distal gut hormone that may play a significant role in intestinal epithelial proliferation. Gut epithelial cells express specific receptors for PYY, PYY induces proliferation in intestinal cells in vivo and in vitro, and the Y1 receptor subtype couples to mitogenic signaling pathways. In addition to proposed physiologic effects on gut mucosal maintenance, PYY proliferative effects may be hypothesized to contribute to pathophysiologic consequences of stimulated growth.     

Mangiferin decreases plasma free fatty acids through promoting its catabolism in liver by activation of AMPK

Mangiferin has been shown to have the effect of improving dyslipidemia. Plasma free fatty acids (FFA) are closely associated with blood lipid metabolism as well as many diseases including metabolic syndrome. This study is to investigate whether mangiferin has effects on FFA metabolism in hyperlipidemic rats. Wistar rats were fed a high-fat diet and administered mangiferin simultaneously for 6 weeks. Mangiferin (50, 100, 150 mg/kg BW) decreased dose-dependently FFA and triglycerides (TG) levels in plasma, and their accumulations in liver, but increased the β-hydroxybutyrate levels in both plasma and liver of hyperlipidemic rats. HepG2 cells were treated with oleic acid (OA, 0.2 mmol/L) to simulate the condition of high level of plasma FFA in vitro, and were treated with different concentrations of mangiferin simultaneously for 24 h. We found that mangiferin significantly increased FFA uptake, significantly decreased intracellular FFA and TG accumulations in HepG2 cells. Mangiferin significantly increased AMP-activated protein kinase (AMPK) phosphorylation and its downstream proteins involved in fatty acid translocase (CD36) and carnitine palmitoyltransferase 1 (CPT1), but significantly decreased acyl-CoA: diacylgycerol acyltransferase 2 (DGAT2) expression and acetyl-CoA carboxylase (ACC) activity by increasing its phosphorylation level in both in vivo and in vitro studies. Furthermore, these effects were reversed by Compound C, an AMPK inhibitor in HepG2 cells. For upstream of AMPK, mangiferin increased AMP/ATP ratio, but had no effect on LKB1 phosphorylation. In conclusion, mangiferin decreased plasma FFA levels through promoting FFA uptake and oxidation, inhibiting FFA and TG accumulations by regulating the key enzymes expression in liver through AMPK pathway. Therefore, mangiferin is a possible beneficial natural compound for metabolic syndrome by improving FFA metabolism.