Aziz Hichami

The gustatory pathway is involved in CD36-mediated orosensory perception of long-chain fatty acids in the mouse

The sense of taste informs the body about the quality of ingested foods. Tastant‐mediated signals are generated by a rise in free intracellular calcium levels ([Ca2+]i) in the taste bud cells and then are transferred to the gustatory area of brain via connections between the gustatory nerves (chorda tym‐ pani and glossopharyngeal nerves) and the nucleus of solitary tract in the brain stem. We have recently shown that lingual CD36 contributes to fat preference and early digestive secretions in the mouse. We show here that 1) the induction of an increase in [Ca2+]i by linoleic acid is CD36‐dependent in taste receptor cells, 2) the spontaneous preference for or conversely con ditioned aversion to linoleic acid requires intact gusta tory nerves, and 3) the activation of gustatory neurons in the nucleus of the solitary tract elicited by a linoleic acid deposition on the tongue in wild‐type mice cannot be reproduced in CD36‐null animals. We conclude that the CD36‐mediated perception of long‐chain fatty acids involves the gustatory pathway, suggesting that the mouse may have a “taste“ for fatty foods. This system would constitute a potential physiological advantage under conditions of food scarcity by leading the mouse to select and absorb fatty foods. However, it might also lead to a risk of obesity and associated diseases in a context of constantly abundant food.—Gaillard, D., Laugerette, F., Darcel, N., El‐Yassimi, A., Passilly‐De grace, P., Hichami, A., Khan, N. A., Montmayeur, J.‐P., Besnard, P. The gustatory pathway is involved in CD36‐ mediated orosensory perception of long‐chain fatty acids in the mouse. FASEB J. 22, 1458–1468 (2008)

Stat3 and Gfi-1 Transcription Factors Control Th17 Cell Immunosuppressive Activity via the Regulation of Ectonucleotidase Expression

Although Th17 cells are known to promote tissue inflammation and autoimmunity, their role during cancer progression remains elusive. Here, we showed that in vitro Th17 cells generated with the cytokines IL-6 and TGF-β expressed CD39 and CD73 ectonucleotidases, leading to adenosine release and the subsequent suppression of CD4(+) and CD8(+) T cell effector functions. The IL-6-mediated activation of the transcription factor Stat3 and the TGF-β-driven downregulation of Gfi-1 transcription factor were both essential for the expression of ectonucleotidases during Th17 cell differentiation. Stat3 supported whereas Gfi-1 repressed CD39 and CD73 expression by binding to their promoters. Accordingly, Th17 cells differentiated with IL-1β, IL-6, and IL-23 but without TGF-β did not express ectonucleotidases and were not immunosuppressive. Finally, adoptive transfer of Th17 cells induced by TGF-β and IL-6 promoted tumor growth in a CD39-dependent manner. Thus, ectonucleotidase expression supports the immunosuppressive fate of Th17 cells in cancer.

Linoleic Acid Induces Calcium Signaling, Src Kinase Phosphorylation, and Neurotransmitter Release in Mouse CD36-positive Gustatory Cells

We have recently demonstrated that the cells expressing CD36, localized apically on the taste buds of mouse lingual circumvallate papillae, act as gustatory cells. In the present study we isolated these CD36-positive cells from mouse circumvallate papillae and investigated intracellular signaling events, triggered by a long-chain polyunsaturated fatty acid, i.e. linoleic acid (LA). LA induced increases in free intracellular calcium concentrations, [Ca2+]i, by recruiting calcium from endoplasmic reticulum pool via inositol 1,4,5-triphosphate production followed by calcium influx via opening of store-operated calcium (SOC) channels. LA also induced phosphorylation of Src-protein-tyrosine kinases (Src-PTKs), particularly of Fyn59 and Yes62. LA-evoked phosphorylation of Fyn59 and Yes62 was implicated in the activation of SOC channels. Reverse transcription-quantitative PCR revealed that the CD36-positive gustatory cells possessed mRNA of enzymes like tryptophan hydroxylase-1, l-aromatic amino acid decarboxylase, tyrosine hydroxylase, and dopamine β-hydroxylase, involved in the synthesis of monoamine neurotransmitters. Interestingly, the addition of LA to these cells induced the release of 5-hydroxytryptamine and noradrenalin to the extracellular environment. The LA-induced release of these neurotransmitters was curtailed by SOC channel blockers and Src-PTK inhibitors. These results altogether demonstrate that LA binds to mouse CD36-positive gustatory cells, induces Src-PTKs phosphorylation, triggers calcium signaling, and evokes the release of 5-hydroxytryptamine and noradrenalin, which in turn may be implicated in the downstream signaling to the afferent nerve fibers, thus transmitting the output signal from taste buds to the central nervous system.

Implication of acyl chain of diacylglycerols in activation of different isoforms of protein kinase C

We synthesized diacylglycerols (DAGs) containing ω‐6 or ω‐3 polyunsaturated fatty acids [i.e., 1‐stearoyl‐2‐arachidonoyl‐sn‐glycerol (SAG), 1‐stearoyl‐2‐docosahexaenoyL‐sn‐glycerol (SDG), and 1‐stearoyl‐2‐eicosapentaenoyl‐sn‐glycerol (SEG)] and assessed their efficiency on activation of conventional (α, βI, γ) and novel (ε, δ) protein kinase C (PKC). SAG exerted significantly higher stimulatory effects than SDG and SEG on activation of PKCα and PKCδ. Activation of PKCβI by SEG and SDG was higher than that by SAG. Activation of PKCγ did not differ significantly among DAG molecular species. Addition of SAG to assays containing SEG and SDG exerted additive effects on activation of α and ε, but not on βI and γ, isoforms of PKC. SDG‐ and SEG‐induced activation of PKCδ was significantly curtailed by the addition of SAG. Three DAG species significantly curtailed the PMA‐induced activation of βl, γ, and δ, but not of α and ε, isoforms of PKC. Our study demonstrates for the first time that in vitro activation of different PKC isoenzymes vary in response to different DAG species, and one can envisage that this differential regulation may be responsible for their in vivo effects on target organs.—Madani, S., Hichami, A., Legrand, A., Belleville, J., Khan, N. A. Implication of acyl chain of diacylglycerols in activation of different isoforms of protein kinase C. FASEB J. 15, 2595–2601 (2001)

Endocytosis of resveratrol via lipid rafts and activation of downstream signaling pathways in cancer cells.

trans-Resveratrol has been proposed to prevent tumor growth and to sensitize cancer cells to anticancer agents. Polyphenol entry into the cells has remained poorly understood. Here, we show that [3H]-resveratrol enters colon cancer cells (SW480, SW620, HT29) and leukemia U937 cells through a monensin (5–20 μmol/L) -sensitive process that suggests clathrin-independent endocytosis. Uptake of the molecule can be prevented by methyl-β-cyclodextrin (2–12 mg/mL), nystatin (12 ng/mL), and filipin (1 μg/mL), which all disrupt plasma membrane lipid rafts. Accordingly, radiolabeled resveratrol accumulates in sphingomyelin- and cholesterol-enriched cell fractions. Interestingly, extracellular signal–regulated kinases (ERK), c-Jun NH2-terminal kinases (JNK), and Akt also accumulate in lipid rafts on resveratrol exposure (IC50 at 48 h ≈ 30 μmol/L in SW480 and U937 cells). In these rafts also, resveratrol promotes the recruitment, by the integrin αVβ3 (revealed by coimmunoprecipitation with an anti-integrin αVβ3 antibody), of signaling molecules that include the FAK (focal adhesion kinase), Fyn, Grb2, Ras, and SOS proteins. Resveratrol-induced activation of downstream signaling pathways and caspase-dependent apoptosis is prevented by endocytosis inhibitors, lipid raft–disrupting molecules, and the integrin antagonist peptide arginine-glycine-aspartate (500 nmol/L). Altogether, these data show the role played by lipid rafts in resveratrol endocytosis and activation of downstream pathways leading to cell death. Cancer Prev Res; 4(7); 1095–106. ©2011 AACR.

Docosahexaenoic acid reduces suppressive and migratory functions of CD4 + CD25 + regulatory T-cells

Immunological tolerance is one of the fundamental aspects of the immune system. The CD4+CD25+ regulatory T (Treg) cells have emerged as key players in the development of tolerance to self and foreign antigens. However, little is known about the endogenous factors and mechanisms controlling their suppressive capacity on immune response. In this study, we observed that docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid, diminished, in a dose-dependent manner, the capacity of Treg cells to inhibit the CD4+CD25− effector T-cell proliferation. DHA not only reduced the migration of Treg cells toward chemokines but also downregulated the mRNA expression of CCR-4 and CXCR-4 in Treg cells. DHA also curtailed ERK1/2 and Akt phosphorylation and downregulated the Smad7 levels in these cells. Contradictorily, DHA upregulated the mRNA expression of Foxp3, CTLA-4, TGF-β, and IL-10; nonetheless, this fatty acid increased the expression of p27KIP1 mRNA, known to be involved in Treg cell unresponsiveness. In Foxp3-immunoprepitated nuclear proteins, DHA upregulated histone desacetylase 7 levels that would again participate in the unresposnsiveness of these cells. Finally, a DHA-enriched diet also diminished, ex vivo, the suppressive capacity of Treg cells. Altogether, these results suggest that DHA, by diminishing Treg cell functions, may play a key role in health and disease.

STIM1 regulates calcium signaling in taste bud cells and preference for fat in mice.

Understanding the mechanisms underlying oro-gustatory detection of dietary fat is critical for the prevention and treatment of obesity. The lipid-binding glycoprotein CD36, which is expressed by circumvallate papillae (CVP) of the mouse tongue, has been implicated in oro-gustatory perception of dietary lipids. Here, we demonstrate that stromal interaction molecule 1 (STIM1), a sensor of Ca(2+) depletion in the endoplasmic reticulum, mediates fatty acid-induced Ca(2+) signaling in the mouse tongue and fat preference. We showed that linoleic acid (LA) induced the production of arachidonic acid (AA) and lysophosphatidylcholine (Lyso-PC) by activating multiple phospholipase A2 isoforms via CD36. This activation triggered Ca(2+) influx in CD36-positive taste bud cells (TBCs) purified from mouse CVP. LA also induced the production of Ca(2+) influx factor (CIF). STIM1 was found to regulate LA-induced CIF production and the opening of multiple store-operated Ca(2+) (SOC) channels. Furthermore, CD36-positive TBCs from Stim1-/- mice failed to release serotonin, and Stim1-/- mice lost the spontaneous preference for fat that was observed in wild-type animals. Our results suggest that fatty acid-induced Ca(2+) signaling, regulated by STIM1 via CD36, might be implicated in oro-gustatory perception of dietary lipids and the spontaneous preference for fat.

Modulation of lipid metabolism by n-3 polyunsaturated fatty acids in gestational diabetic rats and their macrosomic offspring.

The time course of changes in lipid metabolism by dietary n-3 PUFAs (polyunsaturated fatty acids) in streptozotocin-induced diabetic rats during pregnancy (days 12 and 21) and their macrosomic offspring at birth (day 0) and through adulthood (days 60 and 90) was studied with respect to adipose tissue, liver and serum lipid concentrations, and fatty acid composition. Glucose and insulin levels were also assessed in order to characterize the diabetic state of macrosomic offspring. Pregnant diabetic and control rats were fed either an Isio-4 or EPAX diet (enriched with n-3 PUFA). The same diets were also consumed by pups at weaning. Compared with control rats, during pregnancy diabetic rats had a significant elevation in liver and serum triacylglycerol (triglyceride) and cholesterol concentrations. At birth, macrosomic pups had higher serum insulin and glucose levels than control pups. The macrosomic rats maintained accelerated postnatal growth combined with high adipose tissue weight and lipid content through the first 12 weeks of age. The macrosomic pups from diabetic rats fed the Isio-4 diet also showed a significant enhancement in liver and serum triacylglycerol and cholesterol levels at birth and during adulthood. Feeding the EPAX diet to diabetic mothers as well as their macrosomic pups increased serum and liver levels of EPA (eicospentaenoic acid) and DHA (docosahexaenoic acid) with a reduction in arachidonic acid. The EPAX diet induced a significant decrease in liver and serum triacylglycerol and cholesterol concentrations in mothers during pregnancy and in their macrosomic pups during adulthood. Since the EPAX diet improves lipid anomalies considerably in diabetic mothers and their macrosomic offspring, it may prevent long-term metabolic abnormalities associated with macrosomia.

Immunomodulation and Anti-inflammatory Roles of Polyphenols as Anticancer Agents

Cancers are the largest cause of mortality and morbidity in industrialized countries. Several new concepts have emerged in relation to mechanisms that contribute to the regulation of carcinogenesis processes and associated inflammatory effects such as the modulation of innate immune cells and adaptive immune cells that could infiltrate the tumor. In the tumor microenvironment, there is a delicate balance between antitumor immunity and tumor-originated proinflammatory activity, which weaken antitumor immunity. Consequently; modulation of immune cells and inflammatory processes represent attractive targets for therapeutic intervention in malignant diseases with the goal to restore the sensitivity of cancer cells to chemotherapies and to overcome resistance to current cytotoxic therapies. Numerous studies have reported interesting properties of dietary polyphenols in anticancer strategies notably by their pleiotropic properties on cancer cells, immune cells and inflammation. This review is dedicated to the current knowledge of the mechanisms of polyphenols (resveratrol, curcumin, genistein and epigallocatechin) against cancers through a modulation of the immune system and the pro-inflammatory mediator production. We describe the effects of polyphenols on the adaptative and innate immune cells that could infiltrate the tumor. Reduction of chronic inflammation or its downstream consequences may represent a key mechanism in the fight of cancer development and polyphenols could reduce various pro-inflammatory substance productions through targeting signal transduction or through antioxidant effects. Lastly, we analyze key molecular links between inflammation and tumor progression through nuclear factors such as NFκB or microRNAs.

Eicosapentaenoic acid and docosahexaenoic acid modulate MAP kinase enzyme activity in human T-cells

In order to investigate the implication of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in T signalling, we assessed their effects on the activation of two mitogen activated protein (MAP) kinases, i.e. extracellularly-regulated kinases 1 and 2 (ERK1/ERK2) in Jurkat T-cells. The n-3 polyunsaturated fatty acids (PUFAs) alone failed to induce MAP kinase (MAPK) enzyme activity. To elucidate whether DHA and EPA act via protein kinase C (PKC) dependent and independent pathways, we employed their respective activators, i.e. phorbol 12-myristate 13-acetate (PMA) and antiCD3 antibodies. We observed that U0126, an inhibitor of MAPK kinase-ERK kinase 1/2 (MEK1/2), abolished the actions of these two agents on MAPK activation, suggesting that they act upstream of MEK1/2. Further EPA and DHA diminished both the PMA- and antiCD3 antibodies-induced enzyme activity of ERK1/ERK2 in Jurkat T-cells. Interestingly, okadaic acid (OA), a phosphatase inhibitor seems to act downstream of MEK1/2 as U0126 failed to inhibit the OA-induced MAPK activation. It is noteworthy that EPA and DHA not only failed to curtail the OA-induced MAPK activity but also these n-3 PUFAs at 20 μM potentiated the action of OA. Therefore, EPA and DHA seem to modulate MAPK activation upstream and downstream of MEK1/2. On the hand, arachidonic acid, an n-6 PUFA potentiated the MAPK enzyme activity. In conclusion, our study shows that EPA and DHA may regulate T-cells functions by modulating MAPK enzyme activity.