Western Human Nutrition Research Center

Studies in animals have documented that, compared with glucose, dietary fructose induces dyslipidemia and insulin resistance. To assess the relative effects of these dietary sugars during sustained consumption in humans, overweight and obese subjects consumed glucose- or fructose-sweetened beverages providing 25% of energy requirements for 10 weeks. Although both groups exhibited similar weight gain during the intervention, visceral adipose volume was significantly increased only in subjects consuming fructose. Fasting plasma triglyceride concentrations increased by approximately 10% during 10 weeks of glucose consumption but not after fructose consumption. In contrast, hepatic de novo lipogenesis (DNL) and the 23-hour postprandial triglyceride AUC were increased specifically during fructose consumption. Similarly, markers of altered lipid metabolism and lipoprotein remodeling, including fasting apoB, LDL, small dense LDL, oxidized LDL, and postprandial concentrations of remnant-like particle-triglyceride and -cholesterol significantly increased during fructose but not glucose consumption. In addition, fasting plasma glucose and insulin levels increased and insulin sensitivity decreased in subjects consuming fructose but not in those consuming glucose. These data suggest that dietary fructose specifically increases DNL, promotes dyslipidemia, decreases insulin sensitivity, and increases visceral adiposity in overweight/obese adults.

Continuing improvements in analytical technology along with an increased interest in performing comprehensive, quantitative metabolic profiling, is leading to increased interest pressures within the metabolomics community to develop centralized metabolite reference resources for certain clinically important biofluids, such as cerebrospinal fluid, urine and blood. As part of an ongoing effort to systematically characterize the human metabolome through the Human Metabolome Project, we have undertaken the task of characterizing the human serum metabolome. In doing so, we have combined targeted and non-targeted NMR, GC-MS and LC-MS methods with computer-aided literature mining to identify and quantify a comprehensive, if not absolutely complete, set of metabolites commonly detected and quantified (with today's technology) in the human serum metabolome. Our use of multiple metabolomics platforms and technologies allowed us to substantially enhance the level of metabolome coverage while critically assessing the relative strengths and weaknesses of these platforms or technologies. Tables containing the complete set of 4229 confirmed and highly probable human serum compounds, their concentrations, related literature references and links to their known disease associations are freely available at http://www.serummetabolome.ca.

Methods are described for the extraction and analysis of hydrophilic and lipophilic antioxidants, using modifications of the oxygen radical absorbing capacity (ORAC(FL)) procedure. These methods provide, for the first time, the ability to obtain a measure of "total antioxidant capacity" in the protein free plasma, using the same peroxyl radical generator for both lipophilic and hydrophilic antioxidants. Separation of the lipophilic and hydrophilic antioxidant fractions from plasma was accomplished by extracting with hexane after adding water and ethanol to the plasma (hexane/plasma/ethanol/water, 4:1:2:1, v/v). Lipophilic and hydrophilic antioxidants were efficiently partitioned between hexane and aqueous solvents. Conditions for controlling temperature effects and decreasing assay variability using fluorescein as the fluorescent probe were validated in different laboratories. Incubation (37 degrees C for at least 30 min) of the buffer to which AAPH was dissolved was critical in decreasing assay variability. Lipophilic antioxidants represented 33.1 +/- 1.5 and 38.2 +/- 1.9% of the total antioxidant capacity of the protein free plasma in two independent studies of 6 and 10 subjects, respectively. Methods are described for application of the assay techniques to other types of biological and food samples.

Damage to the DNA of germ cells can lead to mutation, which may result in birth defects, genetic diseases, and cancer. The very high endogenous rate of oxidative DNA damage and the importance of dietary ascorbic acid (AA) in preventing this damage has prompted an examination of these factors in human sperm DNA. The oxidized nucleoside 8-hydroxy-2'-deoxyguanosine (8-oxo-7,8-dihydro-2'-deoxyguanosine; oxo8dG), 1 of approximately 20 major products of oxidative damage to DNA, was measured in DNA isolated from human sperm provided by healthy subjects and compared to the seminal fluid AA levels. This relationship was studied in two groups. In a group of 24 free-living individuals 20-50 years old high levels of oxo8dG were correlated with low seminal plasma AA. The endogenous level of oxo8dG in this group was 13 fmol per microgram of DNA or approximately 25,000 adducts per sperm cell. The second group of individuals was maintained on a controlled diet that varied only in AA content. When dietary AA was decreased from 250 to 5 mg/day, the seminal fluid AA decreased by half and the level of oxo8dG in sperm DNA increased 91%. Repletion of dietary AA for 28 days (from 5 mg/day to 250 or 60 mg/day) caused a doubling in seminal fluid AA and reduced oxo8dG by 36%. These results indicate that dietary AA protects human sperm from endogenous oxidative DNA damage that could affect sperm quality and increase risk of genetic defects, particularly in populations with low AA such as smokers.

In populations where vitamin A availability from food is low, infectious diseases can precipitate vitamin A deficiency by decreasing intake, decreasing absorption, and increasing excretion. Infectious diseases that induce the acute-phase response also impair the assessment of vitamin A status by transiently depressing serum retinol concentrations. Vitamin A deficiency impairs innate immunity by impeding normal regeneration of mucosal barriers damaged by infection, and by diminishing the function of neutrophils, macrophages, and natural killer cells. Vitamin A is also required for adaptive immunity and plays a role in the development of T both-helper (Th) cells and B-cells. In particular, vitamin A deficiency diminishes antibody-mediated responses directed by Th2 cells, although some aspects of Th1-mediated immunity are also diminished. These changes in mucosal epithelial regeneration and immune function presumably account for the increased mortality seen in vitamin A-deficient infants, young children, and pregnant women in many areas of the world today.

Previous studies indicate that leptin secretion is regulated by insulin-mediated glucose metabolism. Because fructose, unlike glucose, does not stimulate insulin secretion, we hypothesized that meals high in fructose would result in lower leptin concentrations than meals containing the same amount of glucose. Blood samples were collected every 30-60 min for 24 h from 12 normal-weight women on 2 randomized days during which the subjects consumed three meals containing 55, 30, and 15% of total kilocalories as carbohydrate, fat, and protein, respectively, with 30% of kilocalories as either a fructose-sweetened [high fructose (HFr)] or glucose-sweetened [high glucose (HGl)] beverage. Meals were isocaloric in the two treatments. Postprandial glycemic excursions were reduced by 66 +/- 12%, and insulin responses were 65 +/- 5% lower (both P < 0.001) during HFr consumption. The area under the curve for leptin during the first 12 h (-33 +/- 7%; P < 0.005), the entire 24 h (-21 +/- 8%; P < 0.02), and the diurnal amplitude (peak - nadir) (24 +/- 6%; P < 0.0025) were reduced on the HFr day compared with the HGl day. In addition, circulating levels of the orexigenic gastroenteric hormone, ghrelin, were suppressed by approximately 30% 1-2 h after ingestion of each HGl meal (P < 0.01), but postprandial suppression of ghrelin was significantly less pronounced after HFr meals (P < 0.05 vs. HGl). Consumption of HFr meals produced a rapid and prolonged elevation of plasma triglycerides compared with the HGl day (P < 0.005). Because insulin and leptin, and possibly ghrelin, function as key signals to the central nervous system in the long-term regulation of energy balance, decreases of circulating insulin and leptin and increased ghrelin concentrations, as demonstrated in this study, could lead to increased caloric intake and ultimately contribute to weight gain and obesity during chronic consumption of diets high in fructose.

Toll-like receptor 4 (TLR4) and TLR2 were shown to be activated by saturated fatty acids (SFAs) but inhibited by docosahexaenoic acid (DHA). However, one report suggested that SFA-induced TLR activation in cell culture systems is due to contaminants in BSA used for solubilizing fatty acids. This report raised doubt about proinflammatory effects of SFAs. Our studies herein demonstrate that sodium palmitate (C16:0) or laurate (C12:0) without BSA solubilization induced phosphorylation of inhibitor of nuclear factor-κB α, c-Jun N-terminal kinase (JNK), p44/42 mitogen-activated-kinase (ERK), and nuclear factor-κB subunit p65, and TLR target gene expression in THP1 monocytes or RAW264.7 macrophages, respectively, when cultured in low FBS (0.25%) medium. C12:0 induced NFκB activation through TLR2 dimerized with TLR1 or TLR6, and through TLR4. Because BSA was not used in these experiments, contaminants in BSA have no relevance. Unlike in suspension cells (THP-1), BSA-solubilized C16:0 instead of sodium C16:0 is required to induce TLR target gene expression in adherent cells (RAW264.7). C16:0-BSA transactivated TLR2 dimerized with TLR1 or TLR6 and through TLR4 as seen with C12:0. These results and additional studies with the LPS sequester polymixin B and in MyD88−/− macrophages indicated that SFA-induced activation of TLR2 or TLR4 is a fatty acid-specific effect, but not due to contaminants in BSA or fatty acid preparations. Toll-like receptor 4 (TLR4) and TLR2 were shown to be activated by saturated fatty acids (SFAs) but inhibited by docosahexaenoic acid (DHA). However, one report suggested that SFA-induced TLR activation in cell culture systems is due to contaminants in BSA used for solubilizing fatty acids. This report raised doubt about proinflammatory effects of SFAs. Our studies herein demonstrate that sodium palmitate (C16:0) or laurate (C12:0) without BSA solubilization induced phosphorylation of inhibitor of nuclear factor-κB α, c-Jun N-terminal kinase (JNK), p44/42 mitogen-activated-kinase (ERK), and nuclear factor-κB subunit p65, and TLR target gene expression in THP1 monocytes or RAW264.7 macrophages, respectively, when cultured in low FBS (0.25%) medium. C12:0 induced NFκB activation through TLR2 dimerized with TLR1 or TLR6, and through TLR4. Because BSA was not used in these experiments, contaminants in BSA have no relevance. Unlike in suspension cells (THP-1), BSA-solubilized C16:0 instead of sodium C16:0 is required to induce TLR target gene expression in adherent cells (RAW264.7). C16:0-BSA transactivated TLR2 dimerized with TLR1 or TLR6 and through TLR4 as seen with C12:0. These results and additional studies with the LPS sequester polymixin B and in MyD88−/− macrophages indicated that SFA-induced activation of TLR2 or TLR4 is a fatty acid-specific effect, but not due to contaminants in BSA or fatty acid preparations. endogenous damage associated molecular pattern docosahexaenoic acid p44/42 mitogen-activated-kinase inhibitor of nuclear factor-κB interleukin c-Jun N-terminal kinase Limulus Amebocyte Lysate lipopolysaccharide muramyldipeptide MurNAc-L-Ala-D-isoGln nuclear factor-κB nucleotide-binding oligomerization domain family pathogen-associated molecular pattern pattern recognition receptor reactive oxygen species saturated fatty acid Toll-like receptor Pattern recognition receptors (PRRs), including Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain protein (NOD) like receptors detect invading pathogens by recognizing conserved pathogen-associated molecular patterns (PAMPs) and activate innate immune responses for host defense. However, PRRs can be activated by a wide variety of endogenous damage associated molecular patterns (DAMPs) derived from tissue injury or stress and induce sterile inflammation to initiate wound healing processes. Emerging evidence suggests that PRRs can also sense metabolic disturbance and link immune responses to metabolic homeostasis (1Hotamisligil G.S. Erbay E. Nutrient sensing and inflammation in metabolic diseases.Nat. Rev. Immunol. 2008; 8: 923-934Crossref PubMed Scopus (778) Google Scholar, 2Schroder K. Zhou R. Tschopp J. The NLRP3 inflammasome: a sensor for metabolic danger?.Science. 2010; 327: 296-300Crossref PubMed Scopus (847) Google Scholar). Such a functional diversity of PRRs may be achieved by their ability to recognize a wide variety of structurally unrelated molecules. However, such a broad specificity of PRRs in recognizing agonists can make them susceptible to dysregulation leading to development of chronic inflammation, which in turn can promote development and progression of many chronic diseases including atherosclerosis, insulin resistance, Alzheimer's disease, and cancer. Recent studies revealed that dietary components and metabolic intermediates can alter the activity and expression of PRRs, suggesting that PRR-mediated inflammation and its functional consequence are dynamically modulated by what we eat (3Lee J.Y. Zhao L. Hwang D.H. Modulation of pattern recognition receptor-mediated inflammation and risk of chronic diseases by dietary fatty acids.Nutr. Rev. 2010; 68: 38-61Crossref PubMed Scopus (117) Google Scholar, 4Zhao L. Lee J.Y. Hwang D.H. Inhibition of pattern recognition receptor-mediated inflammation by bioactive phytochemicals.Nutr. Rev. 2011; 69: 310-320Crossref PubMed Scopus (85) Google Scholar). High saturated fat diets have been used for diet-induced obesity and insulin resistance in many animal studies. Both in vitro and in vivo studies suggest that saturated fatty acids (SFAs) can activate proinflammatory signaling pathways leading to insulin resistance (5Glass C.K. Olefsky J.M. Inflammation and lipid signaling in the etiology of insulin resistance.Cell Metab. 2012; 15: 635-645Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar). The molecular mechanism by which SFAs activate proinflammatory signaling pathways remains obscure. Our previous studies revealed that SFAs activate but n-3 PUFA docosahexaenoic acid (DHA) inhibits TLR4- and TLR2-mediated signaling pathways leading to expression of proinflammatory marker gene products (6Lee J.Y. Sohn K.H. Rhee S.H. Hwang D. Saturated fatty acids, but not unsaturated fatty acids, induce the expression of cyclooxygenase-2 mediated through Toll-like receptor 4.J. Biol. Chem. 2001; 276: 16683-16689Abstract Full Text Full Text PDF PubMed Scopus (990) Google Scholar–9Lee J.Y. Plakidas A. Lee W.H. Heikkinen A. Chanmugam P. Bray G. Hwang D.H. Differential modulation of Toll-like receptors by fatty acids: preferential inhibition by n-3 polyunsaturated fatty acids.J. Lipid Res. 2003; 44: 479-486Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar). Numerous studies with cells in culture and in animal models of mutated or deleted TLR4 or TLR2 subsequently demonstrated that SFAs indeed can activate TLR4- and TLR2-mediated proinflammatory signaling pathways and consequently, increase risk of insulin resistance (10Shi H. Kokoeva M.V. Inouye K. Tzameli I. Yin H. Flier J.S. TLR4 links innate immunity and fatty acid-induced insulin resistance.J. Clin. Invest. 2006; 116: 3015-3025Crossref PubMed Scopus (2681) Google Scholar–19Davis J.E. Braucher D.R. Walker-Daniels J. Spurlock M.E. Absence of Tlr2 protects against high-fat diet-induced inflammation and results in greater insulin-stimulated glucose transport in cultured adipocytes.J. Nutr. Biochem. 2011; 22: 136-141Crossref PubMed Scopus (79) Google Scholar). However, one report (20Erridge C. Samani N.J. Saturated fatty acids do not directly stimulate Toll-like receptor signaling.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 1944-1949Crossref PubMed Scopus (215) Google Scholar) suggested that SFA-induced TLR activation is due to contaminants in BSA used for solubilizing fatty acids. This report casted doubt upon the proinflammatory effects of SFAs. TLRs are activated by various microbial components (i.e., endotoxins) that are ubiquitously present in our envi­ronment. Therefore, potential contamination of microbial components in reagents used for in vitro and in vivo studies is not a trivial technical issue for investigations focusing on the of endogenous in TLRs and Such is the are in This issue was in the the activation of TLR4 by as a protein is a of or due to contaminants K. H. protein is a endogenous of the Immunol. PubMed Scopus Google Scholar, protein not induce the of from Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). is not for the of can also be used to detect and agonists for Therefore, we used to potential contamination of TLR agonists in of evidence are that the activation of proinflammatory signaling pathways by SFAs is a fatty acid-specific and not due to contaminants in the of cells to fatty acids revealed that the of cells to TLR agonists is on the of reactive oxygen species in cell culture and were from and of polymixin and for were from for c-Jun N-terminal kinase p44/42 mitogen-activated-kinase of nuclear B B nuclear B and B were from B was from immune was a as were as FBS and and FBS from FBS from FBS from and FBS from BSA was from laurate was from acid sodium palmitate sodium and BSA and were from was from TLR4 inhibitor was from cell from macrophages by of and by were cultured in FBS and cells cell were cultured in and The macrophages were macrophages from with A. H. E. and activate the through Immunol. 2008; PubMed Scopus Google Scholar). cell were in a and were from of was by was by was from Lee of The from these expression was in for the were to the cells were of and the with of and or and or and in to and expression of was used in to the of and expression for as the cells were in for by with fatty acids in the low medium. The cells were and were from the the and to the activity was by activity to in of the was acid (C16:0) is and to be with BSA to The solubilization was as G. for but not in the of insulin by saturated fatty acids.J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) with C16:0 was in or with BSA in a in or medium. The was in a for by on a in for The C16:0 was through a of sodium was in of sodium was in in a and to sodium was to and cells were to indicated in of sodium was in with in and and cells were of The the cells were in for and with fatty acids in the medium. cells were in and in for The cells were with fatty acids in the low medium. the the were The cells were with and by in cell sodium and The were by and to by of the to The was in and The was with for or by with for The were by the reagents by to were for and interleukin in the cell culture a and respectively, and a the RAW264.7 cells were of in medium. The the cells were in for by with or BSA-solubilized C16:0 for The cells were with and with for The cells were with and by in The of were from the cells by with a a The of were also by cells were a in a of in The cells were for and with in for with the cells were with or BSA-solubilized C16:0 for in The cells were with in for and with The were on was with a with The Amebocyte were a the was with reagents and for The was The were also a the of were with and for of was to of the and for or low The were by of the The were by a the The of the were from the the by the of the was from the and as were by a Our previous studies demonstrated that sodium laurate (C12:0) BSA PRRs J.Y. Zhao L. R. L. Lee W.H. Hwang D.H. Saturated fatty acid but polyunsaturated fatty acid inhibits Toll-like receptor dimerized with Toll-like receptor or Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L. Lee J.Y. K. Hwang D.H. Differential modulation of signaling pathways by fatty acids in Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, potential contamination of agonists in BSA was not with our previous of cells with sodium laurate (C12:0) to of and expression However, acid (C16:0) is in and to be with BSA to expression of target is to contaminants in we cells with BSA or BSA-solubilized C16:0 to sodium C16:0-BSA induced and expression BSA used to C16:0 no we C12:0 or expression of or is due to contamination of LPS in BSA or fatty acid preparations. The LPS polymixin B not C12:0 or and expression that the effects of the SFAs are not due to LPS contamination in BSA or fatty acid B not sodium laurate or acid expression that the of expression by these fatty acids is not due to LPS contamination in the fatty acid or BSA preparations. cells were in for and with polymixin B for by with or of BSA in the for The protein were with or by were for by polymixin from no B and Samani (20Erridge C. Samani N.J. Saturated fatty acids do not directly stimulate Toll-like receptor signaling.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 1944-1949Crossref PubMed Scopus (215) Google Scholar) suggested that SFA-induced expression in cells is due to contaminants present in BSA or fatty acid that can activate that is the or or expression be in macrophages TLR2 signaling Therefore, we or C16:0-BSA induced or expression is in The results that C12:0 or C16:0-BSA induced and expression in macrophages the that the of or expression by C12:0 or C16:0-BSA is due to contaminants in BSA or fatty acid that can activate TLR2 or TLRs or studies have shown that SFAs can activate and in macrophages (6Lee J.Y. Sohn K.H. Rhee S.H. Hwang D. Saturated fatty acids, but not unsaturated fatty acids, induce the expression of cyclooxygenase-2 mediated through Toll-like receptor 4.J. Biol. Chem. 2001; 276: 16683-16689Abstract Full Text Full Text PDF PubMed Scopus (990) Google Scholar, J.Y. Zhao L. R. L. Lee W.H. Hwang D.H. Saturated fatty acid but polyunsaturated fatty acid inhibits Toll-like receptor dimerized with Toll-like receptor or Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, H. Kokoeva M.V. Inouye K. Tzameli I. Yin H. Flier J.S. TLR4 links innate immunity and fatty acid-induced insulin resistance.J. Clin. Invest. 2006; 116: 3015-3025Crossref PubMed Scopus (2681) Google Scholar, D. A. R. C.K. Olefsky J.M. of macrophages tissue and is activated by fatty acids Toll-like receptors and 4 and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L. Lee J.Y. K. Hwang D.H. Differential modulation of signaling pathways by fatty acids in Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). TLR4 and and is not the of SFAs induce the expression of or in cells as shown in evidence that proinflammatory effects of SFAs are not due to the potential contaminants in the reagents in in vitro systems is that SFAs activate proinflammatory signaling n-3 fatty acid SFA-induced activation of proinflammatory signaling pathways (3Lee J.Y. Zhao L. Hwang D.H. Modulation of pattern recognition receptor-mediated inflammation and risk of chronic diseases by dietary fatty acids.Nutr. Rev. 2010; 68: 38-61Crossref PubMed Scopus (117) Google Scholar, J.Y. Sohn K.H. Rhee S.H. Hwang D. Saturated fatty acids, but not unsaturated fatty acids, induce the expression of cyclooxygenase-2 mediated through Toll-like receptor 4.J. Biol. Chem. 2001; 276: 16683-16689Abstract Full Text Full Text PDF PubMed Scopus (990) Google K. Hwang D.H. acids Toll-like receptor 4 activation through of receptor and lipid in a reactive oxygen Biol. Chem. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). C12:0 induced phosphorylation of and but inhibited phosphorylation and expression cells were with fatty acids in the of polymixin B C12:0 or C16:0-BSA induced and expression effects of LPS and contaminants that can activate TLRs are be that we have BSA for the of potential contaminants that can induce the expression of TLR or target gene products in RAW264.7 BSA induced the expression of TLR target gene suggesting the of contaminants that can activate TLR or PRRs in these BSA preparations. and which induced expression in RAW264.7 cells in the of polymixin B not induce expression in cells suggesting that the contaminants in these BSA are that can activate TLR2 or proinflammatory these results demonstrate that fatty acid-induced or expression is not due to LPS or contaminants that can activate TLR2 or TLRs in BSA or fatty acid preparations. that these BSA of these BSA the one from not of and expression also that BSA of Therefore, BSA was used the studies. the of RAW264.7 cells to SFAs with the of FBS This in the of RAW264.7 cells to C12:0 was not with the fatty acid of in FBS not laurate (C12:0) without BSA solubilization activated the signaling pathways of TLRs in adherent leading to phosphorylation of and phosphorylation was by leading to of expression of cells suspension cell with sodium palmitate (C16:0) without BSA solubilization activated the signaling pathways of leading to phosphorylation of B and B subunit The activation of the TLR pathways by C16:0 to expression of to expression was also by polymixin B no C16:0-BSA but not BSA also induced phosphorylation and expression in cells expression induced by C16:0-BSA was inhibited by but was not by polymixin B Because BSA was not used in studies with sodium laurate and sodium potential contaminants in BSA that can activate PRRs were not C12:0 transactivated B mediated through TLR2 dimerized with TLR1 or TLR6, or through TLR4 C16:0-BSA also induced B activation mediated through these TLRs these results that SFA-induced activation of signaling pathways of TLR4 and TLR2 is the of SFAs and not due to potential contaminants in fatty acid or BSA laurate (C12:0) or acid induced B activation through TLR2 dimerized with TLR1 or TLR6 and through TLR4. cells were cultured in and with TLR2 and TLR1 TLR2 and TLR6 TLR4 and in to and expression the cells were in for and with C12:0 or C16:0-BSA for The cell were for and are as LPS was used as from or from BSA our previous studies that SFAs activate TLR2 and cells were cultured in the medium. we the of cells to C12:0 or TLR agonists is by in the culture The of cells to or TLR agonists a TLR4 a TLR2 was when the cells were cultured in the (0.25%) with the cells cultured in FBS The of expression by C12:0 was in the cells cultured in the with FBS results were with C16:0-BSA The activation of expression by or C16:0-BSA was also in cells cultured in the (0.25%) with cells cultured in the be that the results by and Samani (20Erridge C. Samani N.J. Saturated fatty acids do not directly stimulate Toll-like receptor signaling.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 1944-1949Crossref PubMed Scopus (215) Google Scholar) that sodium laurate not induce expression or B were in cells cultured in the with laurate or TLR and reactive oxygen species and phosphorylation of and were in the culture with low FBS (0.25%) with with FBS cells were in for and with C12:0 or C16:0-BSA for in the or cultured in and with C12:0 or C16:0-BSA for The protein were for and by The culture from and C16:0-BSA were for by cells were cultured in or FBS for and with or C16:0-BSA for by for were by as in of the by the in as of from the of FBS for and C16:0-BSA cells were in for and with C12:0 for indicated or cultured in and with C12:0 for the The protein were for and by what to the of cells to fatty acids in the medium. The of been shown to be by in or cells in culture H. of reactive oxygen species induced by in Res. PubMed Scopus Google I. H. J. of Chem. 2011; PubMed Scopus Google Scholar). activation of TLR4 of the receptor lipid in K. A. stress by Toll-like receptor 4 to the in 2006; PubMed Scopus Google Scholar, K. A. inhibits TLR signaling pathways by of TLRs to lipid 2006; PubMed Scopus Google Scholar). Our previous studies demonstrated that C12:0 and of TLR4 lipid in K. Hwang D.H. acids Toll-like receptor 4 activation through of receptor and lipid in a reactive oxygen Biol. Chem. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). of TLR2 was also shown to stimulate the activity of K.H. Lee Lee with TLR2 is required for innate immune responses to Immunol. 2009; PubMed Scopus Google Scholar, Lee acid expression the and in Immunol. 2008; PubMed Scopus Google Scholar). these results suggest that the of cells to TLR4 agonists in on of cells in Therefore, we the in the cells cultured in the with or low (0.25%) FBS shown in and in or cells was by the culture with FBS with that with suggesting that the effects of SFAs. phosphorylation of and was the cells were cultured in low FBS (0.25%) with FBS These results suggest that low in culture in the of cells to LPS or SFAs in signaling with of RAW264.7 cells with sodium C12:0 or C16:0-BSA in the to inhibitor of the phosphorylation of and and the expression of and induced by C12:0 or C16:0-BSA in the these results demonstrate that in cells in culture is one of the that can the of the cells to SFAs. Our results demonstrate that SFAs without to BSA can activate proinflammatory signaling pathways in macrophages and monocytes in a low culture medium. However, inhibits saturated fatty acid-induced activation of signaling These results that SFA-induced activation of signaling pathways and target gene expression is not due to contaminants in BSA or fatty acid preparations. The results from also that the fatty acids and reagents used in these studies or of that sodium palmitate and acid used in these of and which is to LPS and respectively, as from the LPS However, in sodium palmitate and acid as by not a pattern suggesting that for acid may by be that a of the for or the to M.E. with the and to of 22: Scholar). Therefore, we have to and contaminants in our reagents that can activate TLR2 or TLR4. cells in the of polymixin effects of and potential contaminants that can activate TLR2 and TLRs are Therefore, the activation of TLR2 or TLR4 by SFAs is due to contaminants that can activate TLR2 or the SFAs not be to induce the expression of TLR target gene products in cells in the of polymixin The that the SFAs induce the expression of the TLR target gene products that the activation of TLR2 or TLR4 by the SFAs is not due to the of potential contaminants that can activate TLR2 or TLR4. we the of in palmitate as by as these of in palmitate for the expression of or induced by inhibited or SFA-induced TLR target gene expression C12:0 or C16:0 effects on TLR activation in our studies these fatty acids are in in the is that SFAs are with that can stimulate TLR is with that can TLR these results the that the effects of SFAs may be due to contaminants in fatty acid preparations. a inhibitor of TLR4 and and suggesting that activation of and expression is in mediated through TLR4 signaling Our previous studies also that C12:0 and of TLR4 lipid K. Hwang D.H. acids Toll-like receptor 4 activation through of receptor and lipid in a reactive oxygen Biol. Chem. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). The of cells to SFAs is in the culture with low FBS (0.25%) with the culture with which is with by the cells cultured in the medium. These results about the activation of signaling pathways induced by SFAs and suggest that that can the of cells to SFAs in signaling pathways and its functional SFAs can activate TLR2 and TLR4 is receptor of the receptor lipid and of the signaling are the potential through which fatty acids can activation of these The Lipid in LPS is by SFAs of Rev. Biochem. PubMed Scopus Google Scholar). of these SFAs from Lipid results in of activity of by a PubMed Scopus Google Scholar). Lipid by unsaturated fatty acids instead of SFAs is and as against the responses to LPS in a cell without LPS by a PubMed Scopus Google K. R. lipid from the lipopolysaccharide of is in PubMed Google Scholar). that activate TLR2 are also by SFAs. These results suggest that the fatty acids in lipid or a in receptor activation for TLR2 and TLR4. The for revealed that of fatty in LPS are the in D.H. Lee H. Lee The of lipopolysaccharide recognition by the 2009; PubMed Scopus Google Scholar). of LPS to with TLR4 through and mediated by the fatty and in Lipid is fatty acids the of Lipid the SFAs in Lipid can with the of and of TLR4 with or The for revealed that fatty of are the in the fatty is a in TLR1 J.Y. Lee M.E. Lee H. Lee of the induced by of a Full Text Full Text PDF PubMed Scopus Google Scholar). of fatty of TLR2 of is saturated fatty acids the fatty acids in the can with the in TLR2 or TLR1 and promote the of the receptors or the of the receptors with the through TLR2 and TLR1 or TLR6 can in a when are A. L. A. The for pattern recognition of pathogens by the innate immune is by PubMed Scopus Google Scholar). was shown that acid can activate TLR4 and TLR2 dimerized with TLR1 or TLR6 but no TLRs (6Lee J.Y. Sohn K.H. Rhee S.H. Hwang D. Saturated fatty acids, but not unsaturated fatty acids, induce the expression of cyclooxygenase-2 mediated through Toll-like receptor 4.J. Biol. Chem. 2001; 276: 16683-16689Abstract Full Text Full Text PDF PubMed Scopus (990) Google Scholar, J.Y. Zhao L. R. L. Lee W.H. Hwang D.H. Saturated fatty acid but polyunsaturated fatty acid inhibits Toll-like receptor dimerized with Toll-like receptor or Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The and indicated the of the lipid or in or of and TLR6 but not in TLRs J.Y. Lee of the Toll-like receptor Rev. Biochem. 2011; PubMed Scopus Google Scholar). for TLRs TLR4 and TLR2 are to be by SFAs. the molecular LPS or and TLR4 or respectively, been revealed receptors without potential activity of various endogenous fatty remains to be of TLR4 of the receptor lipid signaling such as and for the activation of signaling pathways K. Hwang D.H. acids Toll-like receptor 4 activation through of receptor and lipid in a reactive oxygen Biol. Chem. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar, K. A. stress by Toll-like receptor 4 to the in 2006; PubMed Scopus Google Scholar, K. A. inhibits TLR signaling pathways by of TLRs to lipid 2006; PubMed Scopus Google Scholar). Lipid are of which as signaling to and signaling to promote the of to signaling Lipid have a lipid that is in and The in lipid are by SFAs in which are by suggesting that saturated fatty lipid Both TLR4 and TLR2 LPS and respectively, are by SFAs. for the of lipid suggests that of with for lipid can increase the and of lipid Lipid and 2006; PubMed Scopus Google Scholar). was shown that acid but n-3 PUFA inhibits and of TLR4 lipid in macrophages K. Hwang D.H. acids Toll-like receptor 4 activation through of receptor and lipid in a reactive oxygen Biol. Chem. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). mechanism by which SFAs activate TLR2 and TLR4 of endogenous signaling or of that can with However, no evidence been to The results demonstrate that SFA-induced activation of TLR2 and TLR4 is fatty acid effects but not due to contaminants in TLRs are not in host but also wound healing and metabolic disturbance to immune Such of TLRs can be by recognizing a variety of endogenous molecules. the molecular TLR4 or TLR2 and their (i.e., LPS and been revealed by studies receptors without the potential activity of a variety of endogenous fatty remains to be of the mechanism by which such endogenous activate signaling pathways our of sterile inflammation is associated with the development and progression of many chronic

The saturated fatty acids acylated on Lipid A of lipopolysaccharide (LPS) or bacterial lipoproteins play critical roles in ligand recognition and receptor activation for Toll-like Receptor 4 (TLR4) and TLR2. The results from our previous studies demonstrated that saturated and polyunsaturated fatty acids reciprocally modulate the activation of TLR4. However, the underlying mechanism has not been understood. Here, we report for the first time that the saturated fatty acid lauric acid induced dimerization and recruitment of TLR4 into lipid rafts, however, dimerization was not observed in non-lipid raft fractions. Similarly, LPS and lauric acid enhanced the association of TLR4 with MD-2 and downstream adaptor molecules, TRIF and MyD88, into lipid rafts leading to the activation of downstream signaling pathways and target gene expression. However, docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid, inhibited LPS- or lauric acid-induced dimerization and recruitment of TLR4 into lipid raft fractions. Together, these results demonstrate that lauric acid and DHA reciprocally modulate TLR4 activation by regulation of the dimerization and recruitment of TLR4 into lipid rafts. In addition, we showed that TLR4 recruitment to lipid rafts and dimerization were coupled events mediated at least in part by NADPH oxidase-dependent reactive oxygen species generation. These results provide a new insight in understanding the mechanism by which fatty acids differentially modulate TLR4-mediated signaling pathway and consequent inflammatory responses which are implicated in the development and progression of many chronic diseases. The saturated fatty acids acylated on Lipid A of lipopolysaccharide (LPS) or bacterial lipoproteins play critical roles in ligand recognition and receptor activation for Toll-like Receptor 4 (TLR4) and TLR2. The results from our previous studies demonstrated that saturated and polyunsaturated fatty acids reciprocally modulate the activation of TLR4. However, the underlying mechanism has not been understood. Here, we report for the first time that the saturated fatty acid lauric acid induced dimerization and recruitment of TLR4 into lipid rafts, however, dimerization was not observed in non-lipid raft fractions. Similarly, LPS and lauric acid enhanced the association of TLR4 with MD-2 and downstream adaptor molecules, TRIF and MyD88, into lipid rafts leading to the activation of downstream signaling pathways and target gene expression. However, docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid, inhibited LPS- or lauric acid-induced dimerization and recruitment of TLR4 into lipid raft fractions. Together, these results demonstrate that lauric acid and DHA reciprocally modulate TLR4 activation by regulation of the dimerization and recruitment of TLR4 into lipid rafts. In addition, we showed that TLR4 recruitment to lipid rafts and dimerization were coupled events mediated at least in part by NADPH oxidase-dependent reactive oxygen species generation. These results provide a new insight in understanding the mechanism by which fatty acids differentially modulate TLR4-mediated signaling pathway and consequent inflammatory responses which are implicated in the development and progression of many chronic diseases. Toll-like receptors (TLRs) 3The abbreviations used are: TLRToll-like receptorLPSlipopolysaccharidePUFApolyunsaturated fatty acidDHAdocosahexaenoic acidROSreactive oxygen speciesGFPgreen fluorescent proteinFBSfetal bovine serumDMEMDulbecco's modified Eagle's mediumBSAbovine serum albuminDPIdiphenyleneiodonium chlorideNAcN-acetyl-l-cysteineMyD88myeloid differentiation factor 88TIRToll/IL-1 receptorTRIFTIR domain-containing adaptor inducing interferon-βCM-H2DCFDA5-(and 6-)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetylester. 3The abbreviations used are: TLRToll-like receptorLPSlipopolysaccharidePUFApolyunsaturated fatty acidDHAdocosahexaenoic acidROSreactive oxygen speciesGFPgreen fluorescent proteinFBSfetal bovine serumDMEMDulbecco's modified Eagle's mediumBSAbovine serum albuminDPIdiphenyleneiodonium chlorideNAcN-acetyl-l-cysteineMyD88myeloid differentiation factor 88TIRToll/IL-1 receptorTRIFTIR domain-containing adaptor inducing interferon-βCM-H2DCFDA5-(and 6-)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetylester. are one of the key pattern recognition receptor families that play a critical role in inducing innate and adaptive immune responses in mammals by recognizing conserved pathogen-associated molecular pattern of invading microbes. So far, at least thirteen TLRs have been identified in mammalian species (1Uematsu S. Akira S. J. Mol. Med. 2006; 84: 712-725Crossref PubMed Scopus (346) Google Scholar, 2Kawai T. Akira S. Trends Mol. Med. 2007; 13: 460-469Abstract PubMed Scopus Google (LPS) from the ligand for the TLR4 PubMed Scopus Google of and (1Uematsu S. Akira S. J. Mol. Med. 2006; 84: 712-725Crossref PubMed Scopus (346) Google Scholar, 2Kawai T. Akira S. Trends Mol. Med. 2007; 13: 460-469Abstract PubMed Scopus Google LPS a with in serum leading to the of of LPS to which are to LPS to S. 2006; PubMed Scopus Google Lipid which of the of acylated with saturated fatty The of these saturated fatty acids are by saturated fatty of these saturated fatty acids from Lipid A not results in of Lipid A an the Lipid A PubMed Scopus Google Scholar, J. Med. PubMed Scopus Google or Lipid fatty acids are to and an J. J. PubMed Scopus Google Scholar, PubMed Google In addition, lipoproteins are to and to in PubMed Scopus Google Together, these results that saturated fatty acids acylated on Lipid A or bacterial lipoproteins play critical roles in ligand recognition and receptor activation for TLR4 and TLR2. that the of bacterial with of of acylated saturated fatty acids by and S. J. PubMed Scopus Google Scholar, S. J. Google a with or which the molecular of or T. Akira S. 13: PubMed Scopus Google Scholar, S. T. Akira S. J. PubMed Scopus Google Scholar, J. Med. PubMed Scopus Google So that for TLRs are acylated by saturated fatty from our previous studies demonstrated that saturated fatty acids TLR4 and polyunsaturated fatty acids saturated fatty and activation of TLR4 J. J. PubMed Scopus Google Scholar, J. Lipid PubMed Scopus Google In addition, the saturated fatty acid lauric acid the n-3 docosahexaenoic acid activation J. PubMed Scopus Google Together, these results that and TLR4 signaling pathways and target gene are reciprocally by saturated and polyunsaturated fatty However, the mechanism for by fatty acids not to lipid raft are with LPS and in J. PubMed Google in an of NADPH TLR4 recruitment to lipid rafts T. J. Med. 2006; PubMed Scopus Google a lipid raft the of T. J. Med. 2006; PubMed Scopus Google that lipid rafts are for TLR4-mediated and target gene expression. Lipid rafts are a of lipid by in Lipid rafts a Mol. PubMed Scopus Google Lipid rafts have a lipid that in and PubMed Scopus Google The in saturated fatty with J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google that saturated fatty lipid raft the n-3 signaling from lipid rafts by lipid and the to the of signaling J. J. PubMed Scopus Google that TLRs or (1Uematsu S. Akira S. J. Mol. Med. 2006; 84: 712-725Crossref PubMed Scopus (346) Google Scholar, 2Kawai T. Akira S. Trends Mol. Med. 2007; 13: 460-469Abstract PubMed Scopus Google TLR4 the of the receptor from our previous studies that the molecular target by which saturated fatty acids and n-3 reciprocally modulate TLR4 activation the receptor or the leading to the receptor activation of the downstream signaling J. J. PubMed Scopus Google Scholar, J. Lipid PubMed Scopus Google we the of TLR4 activation mediated by regulation of the dimerization and recruitment of TLR4 into lipid rafts, and these in an that chronic one of the key for the development and progression of many chronic and that the of the of development of diseases. induced by Toll-like receptors by recognizing invading and downstream signaling pathways that to the of of gene (1Uematsu S. Akira S. J. Mol. Med. 2006; 84: 712-725Crossref PubMed Scopus (346) Google Scholar, 2Kawai T. Akira S. Trends Mol. Med. 2007; 13: 460-469Abstract PubMed Scopus Google However, not and from a that TLRs by from and saturated fatty acids J. PubMed Scopus Google Scholar, J. PubMed Scopus Google that TLRs not induced by that TLRs and downstream signaling pathways provide for and the chronic diseases. from and studies TLRs to the development and progression of many chronic and J. PubMed Scopus Google Scholar, J. J. 2007; PubMed Scopus Google Scholar, J. Akira S. PubMed Scopus Google Scholar, J. 2006; PubMed Scopus Google studies demonstrated for the first time that saturated fatty acids and n-3 polyunsaturated fatty acids reciprocally modulate TLR4 activation J. Med. PubMed Scopus Google Scholar, J. J. PubMed Scopus Google Scholar, J. Lipid PubMed Scopus Google Scholar, J. PubMed Scopus Google The of that saturated fatty acid-induced and or are mediated by the activation of TLR4 J. J. 2007; PubMed Scopus Google Scholar, J. 2006; PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, S. J. 2007; PubMed Scopus Google Scholar, T. J. S. S. S. 2007; PubMed Scopus Google However, the molecular mechanism by which these fatty acids modulate the activation of TLRs not from our previous studies that the molecular target of the n-3 polyunsaturated fatty acid DHA in TLR4 activation the receptor or the events of downstream leading to the activation of TLR4 J. Med. PubMed Scopus Google Scholar, J. J. PubMed Scopus Google Scholar, J. Lipid PubMed Scopus Google Scholar, J. PubMed Scopus Google was demonstrated that TLR4 to lipid raft are with the LPS J. PubMed Google The activation of to recruitment of downstream adaptor PubMed Scopus Google Scholar, J. PubMed Scopus Google TLRs are to lipid rafts, activation by enhanced recruitment of into lipid which by of and TLRs or PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, T. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, PubMed Scopus Google TLR4 with or The and T. Akira S. 13: PubMed Scopus Google Scholar, S. T. Akira S. J. PubMed Scopus Google Scholar, J. Med. PubMed Scopus Google receptor dimerization and recruitment of TLR4 into lipid rafts to of the events leading to the activation of TLR4 signaling results demonstrate that saturated fatty acid lauric acid the n-3 polyunsaturated fatty acid DHA the dimerization and recruitment of TLR4 into lipid rafts. In addition, lauric acid DHA the association of TLR4 with MD-2 and downstream adaptor molecules, and the activation of and target gene expression. The of with the lipid raft of the of TLR4 in and lipid rafts. inhibited LPS- or lauric acid-induced activation of and the of TLR4 target acid, NADPH and DHA inhibited NADPH NADPH with and TLR4 dimerization induced by LPS or lauric Together, these results that the recruitment and dimerization of TLR4 into lipid rafts are the for the activation of the downstream signaling pathways and that lauric acid and DHA reciprocally modulate these TLR4 was not in non-lipid raft with LPS or lauric acid, that dimerization of TLR4 in the lipid rafts the non-lipid raft These results that the recruitment of TLR4 into lipid rafts coupled with the dimerization of TLR4. The results from our previous studies showed that saturated fatty acids and n-3 reciprocally modulate the dimerization of pattern recognition receptor that bacterial S. J. 2007; PubMed Scopus Google studies showed that LPS recruitment of TLR4 to lipid rafts NADPH oxidase-dependent T. J. Med. 2006; PubMed Scopus Google was that by TLR4 to lipid rafts in J. Med. 2006; PubMed Scopus Google In our lauric acid, DHA inhibited NADPH The NADPH or the TLR4 dimerization induced by LPS or lauric These results that lauric acid, DHA dimerization and recruitment of TLR4 to lipid rafts in a was that NADPH 4 with TLR4 in with and J. J. PubMed Scopus Google However, was that MyD88, the downstream of NADPH in J. PubMed Scopus Google not NADPH in the of TLR4 signaling and dimerization and recruitment of TLR4 to lipid rafts induced by LPS or lauric acid, that these one or a part of the signaling with TLR4 in lipid the saturated fatty acid lauric acid DHA dimerization and recruitment of TLR4 to lipid rafts, and lauric acid and DHA reciprocally modulate NADPH are The that Lipid A acylated with saturated fatty that of saturated fatty acids from Lipid A results in of J. J. PubMed Scopus Google Scholar, J. Lipid PubMed Scopus Google and that Lipid fatty acids are or an J. PubMed Scopus Google Scholar, J. PubMed Google that saturated fatty not fatty play the key role in LPS in in lipid rafts are acylated with saturated fatty acids that the of lipid rafts. results that LPS and lauric acid dimerization and recruitment of TLR4 to lipid rafts the that the saturated fatty acids acylated in Lipid A the of lipid rafts, and TLR4 to lipid rafts TLR4 with signaling to the downstream signaling was demonstrated that of with n-3 acid of fatty acid into in lipid rafts, which are with saturated fatty acids J. J. PubMed Scopus Google an of the fatty acid of in lipid rafts by n-3 to of signaling from lipid rafts, and downstream signaling pathways J. J. PubMed Scopus Google Scholar, J. PubMed Scopus Google results demonstrate that DHA LPS- or saturated fatty acid-induced dimerization and recruitment of TLR4 to lipid rafts, which the of TLR4 signaling a that n-3 the of lipid rafts by the fatty acid of that are to acylated with saturated fatty acid for the of lipid rafts. the of the of lipid rafts by DHA to of signaling to lipid results provide a new in understanding the mechanism by which saturated fatty acids and n-3 polyunsaturated fatty acids differentially modulate inflammatory responses the regulation of dimerization and recruitment of TLR4 to lipid rafts. Toll-like receptors (TLRs) 3The abbreviations used are: TLRToll-like receptorLPSlipopolysaccharidePUFApolyunsaturated fatty acidDHAdocosahexaenoic acidROSreactive oxygen speciesGFPgreen fluorescent proteinFBSfetal bovine serumDMEMDulbecco's modified Eagle's mediumBSAbovine serum albuminDPIdiphenyleneiodonium chlorideNAcN-acetyl-l-cysteineMyD88myeloid differentiation factor 88TIRToll/IL-1 receptorTRIFTIR domain-containing adaptor inducing interferon-βCM-H2DCFDA5-(and 6-)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetylester. 3The abbreviations used are: TLRToll-like receptorLPSlipopolysaccharidePUFApolyunsaturated fatty acidDHAdocosahexaenoic acidROSreactive oxygen speciesGFPgreen fluorescent proteinFBSfetal bovine serumDMEMDulbecco's modified Eagle's mediumBSAbovine serum albuminDPIdiphenyleneiodonium chlorideNAcN-acetyl-l-cysteineMyD88myeloid differentiation factor 88TIRToll/IL-1 receptorTRIFTIR domain-containing adaptor inducing interferon-βCM-H2DCFDA5-(and 6-)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetylester. are one of the key pattern recognition receptor families that play a critical role in inducing innate and adaptive immune responses in mammals by recognizing conserved pathogen-associated molecular pattern of invading microbes. So far, at least thirteen TLRs have been identified in mammalian species (1Uematsu S. Akira S. J. Mol. Med. 2006; 84: 712-725Crossref PubMed Scopus (346) Google Scholar, 2Kawai T. Akira S. Trends Mol. Med. 2007; 13: 460-469Abstract PubMed Scopus Google Toll-like receptor lipopolysaccharide polyunsaturated fatty acid docosahexaenoic acid reactive oxygen species fluorescent bovine serum modified Eagle's bovine serum differentiation factor receptor domain-containing adaptor inducing 6-)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetylester. Toll-like receptor lipopolysaccharide polyunsaturated fatty acid docosahexaenoic acid reactive oxygen species fluorescent bovine serum modified Eagle's bovine serum differentiation factor receptor domain-containing adaptor inducing 6-)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetylester. (LPS) from the ligand for the TLR4 PubMed Scopus Google of and (1Uematsu S. Akira S. J. Mol. Med. 2006; 84: 712-725Crossref PubMed Scopus (346) Google Scholar, 2Kawai T. Akira S. Trends Mol. Med. 2007; 13: 460-469Abstract PubMed Scopus Google LPS a with in serum leading to the of of LPS to which are to LPS to S. 2006; PubMed Scopus Google Lipid which of the of acylated with saturated fatty The of these saturated fatty acids are by saturated fatty of these saturated fatty acids from Lipid A not results in of Lipid A an the Lipid A PubMed Scopus Google Scholar, J. Med. PubMed Scopus Google or Lipid fatty acids are to and an J. J. PubMed Scopus Google Scholar, PubMed Google In addition, lipoproteins are to and to in PubMed Scopus Google Together, these results that saturated fatty acids acylated on Lipid A or bacterial lipoproteins play critical roles in ligand recognition and receptor activation for TLR4 and TLR2. that the of bacterial with of of acylated saturated fatty acids by and S. J. PubMed Scopus Google Scholar, S. J. Google a with or which the molecular of or T. Akira S. 13: PubMed Scopus Google Scholar, S. T. Akira S. J. PubMed Scopus Google Scholar, J. Med. PubMed Scopus Google So that for TLRs are acylated by saturated fatty from our previous studies demonstrated that saturated fatty acids TLR4 and polyunsaturated fatty acids saturated fatty and activation of TLR4 J. J. PubMed Scopus Google Scholar, J. Lipid PubMed Scopus Google In addition, the saturated fatty acid lauric acid the n-3 docosahexaenoic acid activation J. PubMed Scopus Google Together, these results that and TLR4 signaling pathways and target gene are reciprocally by saturated and polyunsaturated fatty However, the mechanism for by fatty acids not understood. TLR4 to lipid raft are with LPS and in J. PubMed Google in an of NADPH TLR4 recruitment to lipid rafts T. J. Med. 2006; PubMed Scopus Google a lipid raft the of T. J. Med. 2006; PubMed Scopus Google that lipid rafts are for TLR4-mediated and target gene expression. Lipid rafts are a of lipid by in Lipid rafts a Mol. PubMed Scopus Google Lipid rafts have a lipid that in and PubMed Scopus Google The in saturated fatty with J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google that saturated fatty lipid raft the n-3 signaling from lipid rafts by lipid and the to the of signaling J. J. PubMed Scopus Google that TLRs or (1Uematsu S. Akira S. J. Mol. Med. 2006; 84: 712-725Crossref PubMed Scopus (346) Google Scholar, 2Kawai T. Akira S. Trends Mol. Med. 2007; 13: 460-469Abstract PubMed Scopus Google TLR4 the of the receptor from our previous studies that the molecular target by which saturated fatty acids and n-3 reciprocally modulate TLR4 activation the receptor or the leading to the receptor activation of the downstream signaling J. J. PubMed Scopus Google Scholar, J. Lipid PubMed Scopus Google we the of TLR4 activation mediated by regulation of the dimerization and recruitment of TLR4 into lipid rafts, and these in an that chronic one of the key for the development and progression of many chronic and that the of the of development of diseases. induced by Toll-like receptors by recognizing invading and downstream signaling pathways that to the of of gene (1Uematsu S. Akira S. J. Mol. Med. 2006; 84: 712-725Crossref PubMed Scopus (346) Google Scholar, 2Kawai T. Akira S. Trends Mol. Med. 2007; 13: 460-469Abstract PubMed Scopus Google However, not and from a that TLRs by from and saturated fatty acids J. PubMed Scopus Google Scholar, J. PubMed Scopus Google that TLRs not induced by that TLRs and downstream signaling pathways provide for and the chronic diseases. from and studies TLRs to the development and progression of many chronic and J. PubMed Scopus Google Scholar, J. J. 2007; PubMed Scopus Google Scholar, J. Akira S. PubMed Scopus Google Scholar, J. 2006; PubMed Scopus Google studies demonstrated for the first time that saturated fatty acids and n-3 polyunsaturated fatty acids reciprocally modulate TLR4 activation J. Med. PubMed Scopus Google Scholar, J. J. PubMed Scopus Google Scholar, J. Lipid PubMed Scopus Google Scholar, J. PubMed Scopus Google The of that saturated fatty acid-induced and or are mediated by the activation of TLR4 J. J. 2007; PubMed Scopus Google Scholar, J. 2006; PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, S. J. 2007; PubMed Scopus Google Scholar, T. J. S. S. S. 2007; PubMed Scopus Google However, the molecular mechanism by which these fatty acids modulate the activation of TLRs not from our previous studies that the molecular target of the n-3 polyunsaturated fatty acid DHA in TLR4 activation the receptor or the events of downstream leading to the activation of TLR4 J. Med. PubMed Scopus Google Scholar, J. J. PubMed Scopus Google Scholar, J. Lipid PubMed Scopus Google Scholar, J. PubMed Scopus Google was demonstrated that TLR4 to lipid raft are with the LPS J. PubMed Google The activation of to recruitment of downstream adaptor PubMed Scopus Google Scholar, J. PubMed Scopus Google TLRs are to lipid rafts, activation by enhanced recruitment of into lipid which by of and TLRs or PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, T. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, PubMed Scopus Google TLR4 with or The and T. Akira S. 13: PubMed Scopus Google Scholar, S. T. Akira S. J. PubMed Scopus Google Scholar, J. Med. PubMed Scopus Google receptor dimerization and recruitment of TLR4 into lipid rafts to of the events leading to the activation of TLR4 signaling results demonstrate that saturated fatty acid lauric acid the n-3 polyunsaturated fatty acid DHA the dimerization and recruitment of TLR4 into lipid rafts. In addition, lauric acid DHA the association of TLR4 with MD-2 and downstream adaptor molecules, and the activation of and target gene expression. The of with the lipid raft of the of TLR4 in and lipid rafts. inhibited LPS- or lauric acid-induced activation of and the of TLR4 target acid, NADPH and DHA inhibited NADPH NADPH with and TLR4 dimerization induced by LPS or lauric Together, these results that the recruitment and dimerization of TLR4 into lipid rafts are the for the activation of the downstream signaling pathways and that lauric acid and DHA reciprocally modulate these TLR4 was not in non-lipid raft with LPS or lauric acid, that dimerization of TLR4 in the lipid rafts the non-lipid raft These results that the recruitment of TLR4 into lipid rafts coupled with the dimerization of TLR4. The results from our previous studies showed that saturated fatty acids and n-3 reciprocally modulate the dimerization of pattern recognition receptor that bacterial S. J. 2007; PubMed Scopus Google studies showed that LPS recruitment of TLR4 to lipid rafts NADPH oxidase-dependent T. J. Med. 2006; PubMed Scopus Google was that by TLR4 to lipid rafts in J. Med. 2006; PubMed Scopus Google In our lauric acid, DHA inhibited NADPH The NADPH or the TLR4 dimerization induced by LPS or lauric These results that lauric acid, DHA dimerization and recruitment of TLR4 to lipid rafts in a was that NADPH 4 with TLR4 in with and J. J. PubMed Scopus Google However, was that MyD88, the downstream of NADPH in J. PubMed Scopus Google not NADPH in the of TLR4 signaling and dimerization and recruitment of TLR4 to lipid rafts induced by LPS or lauric acid, that these one or a part of the signaling with TLR4 in lipid the saturated fatty acid lauric acid DHA dimerization and recruitment of TLR4 to lipid rafts, and lauric acid and DHA reciprocally modulate NADPH are The that Lipid A acylated with saturated fatty that of saturated fatty acids from Lipid A results in of J. J. PubMed Scopus Google Scholar, J. Lipid PubMed Scopus Google and that Lipid fatty acids are or an J. PubMed Scopus Google Scholar, J. PubMed Google that saturated fatty not fatty play the key role in LPS in in lipid rafts are acylated with saturated fatty acids that the of lipid rafts. results that LPS and lauric acid dimerization and recruitment of TLR4 to lipid rafts the that the saturated fatty acids acylated in Lipid A the of lipid rafts, and TLR4 to lipid rafts TLR4 with signaling to the downstream signaling was demonstrated that of with n-3 acid of fatty acid into in lipid rafts, which are with saturated fatty acids J. J. PubMed Scopus Google an of the fatty acid of in lipid rafts by n-3 to of signaling from lipid rafts, and downstream signaling pathways J. J. PubMed Scopus Google Scholar, J. PubMed Scopus Google results demonstrate that DHA LPS- or saturated fatty acid-induced dimerization and recruitment of TLR4 to lipid rafts, which the of TLR4 signaling a that n-3 the of lipid rafts by the fatty acid of that are to acylated with saturated fatty acid for the of lipid rafts. the of the of lipid rafts by DHA to of signaling to lipid results provide a new in understanding the mechanism by which saturated fatty acids and n-3 polyunsaturated fatty acids differentially modulate inflammatory responses the regulation of dimerization and recruitment of TLR4 to lipid rafts. that chronic one of the key for the development and progression of many chronic and that the of the of development of diseases. induced by Toll-like receptors by recognizing invading and downstream signaling pathways that to the of of gene (1Uematsu S. Akira S. J. Mol. Med. 2006; 84: 712-725Crossref PubMed Scopus (346) Google Scholar, 2Kawai T. Akira S. Trends Mol. Med. 2007; 13: 460-469Abstract PubMed Scopus Google However, not and from a that TLRs by from and saturated fatty acids J. PubMed Scopus Google Scholar, J. PubMed Scopus Google that TLRs not induced by that TLRs and downstream signaling pathways provide for and the chronic diseases. from and studies TLRs to the development and progression of many chronic and J. PubMed Scopus Google Scholar, J. J. 2007; PubMed Scopus Google Scholar, J. Akira S. PubMed Scopus Google Scholar, J. 2006; PubMed Scopus Google studies demonstrated for the first time that saturated fatty acids and n-3 polyunsaturated fatty acids reciprocally modulate TLR4 activation J. Med. PubMed Scopus Google Scholar, J. J. PubMed Scopus Google Scholar, J. Lipid PubMed Scopus Google Scholar, J. PubMed Scopus Google The of that saturated fatty acid-induced and or are mediated by the activation of TLR4 J. J. 2007; PubMed Scopus Google Scholar, J. 2006; PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, S. J. 2007; PubMed Scopus Google Scholar, T. J. S. S. S. 2007; PubMed Scopus Google However, the molecular mechanism by which these fatty acids modulate the activation of TLRs not from our previous studies that the molecular target of the n-3 polyunsaturated fatty acid DHA in TLR4 activation the receptor or the events of downstream leading to the activation of TLR4 J. Med. PubMed Scopus Google Scholar, J. J. PubMed Scopus Google Scholar, J. Lipid PubMed Scopus Google Scholar, J. PubMed Scopus Google was demonstrated that TLR4 to lipid raft are with the LPS J. PubMed Google The activation of to recruitment of downstream adaptor PubMed Scopus Google Scholar, J. PubMed Scopus Google TLRs are to lipid rafts, activation by enhanced recruitment of into lipid which by of and TLRs or PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, T. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, PubMed Scopus Google TLR4 with or The and T. Akira S. 13: PubMed Scopus Google Scholar, S. T. Akira S. J. PubMed Scopus Google Scholar, J. Med. PubMed Scopus Google receptor dimerization and recruitment of TLR4 into lipid rafts to of the events leading to the activation of TLR4 signaling results demonstrate that saturated fatty acid lauric acid the n-3 polyunsaturated fatty acid DHA the dimerization and recruitment of TLR4 into lipid rafts. In addition, lauric acid DHA the association of TLR4 with MD-2 and downstream adaptor molecules, and the activation of and target gene expression. The of with the lipid raft of the of TLR4 in and lipid rafts. inhibited LPS- or lauric acid-induced activation of and the of TLR4 target acid, NADPH and DHA inhibited NADPH NADPH with and TLR4 dimerization induced by LPS or lauric Together, these results that the recruitment and dimerization of TLR4 into lipid rafts are the for the activation of the downstream signaling pathways and that lauric acid and DHA reciprocally modulate these TLR4 was not in non-lipid raft with LPS or lauric acid, that dimerization of TLR4 in the lipid rafts the non-lipid raft These results that the recruitment of TLR4 into lipid rafts coupled with the dimerization of TLR4. The results from our previous studies showed that saturated fatty acids and n-3 reciprocally modulate the dimerization of pattern recognition receptor that bacterial S. J. 2007; PubMed Scopus Google studies showed that LPS recruitment of TLR4 to lipid rafts NADPH oxidase-dependent T. J. Med. 2006; PubMed Scopus Google was that by TLR4 to lipid rafts in J. Med. 2006; PubMed Scopus Google In our lauric acid, DHA inhibited NADPH The NADPH or the TLR4 dimerization induced by LPS or lauric These results that lauric acid, DHA dimerization and recruitment of TLR4 to lipid rafts in a was that NADPH 4 with TLR4 in with and J. J. PubMed Scopus Google However, was that MyD88, the downstream of NADPH in J. PubMed Scopus Google not NADPH in the of TLR4 signaling and dimerization and recruitment of TLR4 to lipid rafts induced by LPS or lauric acid, that these one or a part of the signaling with TLR4 in lipid rafts. the saturated fatty acid lauric acid DHA dimerization and recruitment of TLR4 to lipid rafts, and lauric acid and DHA reciprocally modulate NADPH are The that Lipid A acylated with saturated fatty that of saturated fatty acids from Lipid A results in of J. J. PubMed Scopus Google Scholar, J. Lipid PubMed Scopus Google and that Lipid fatty acids are or an J. PubMed Scopus Google Scholar, J. PubMed Google that saturated fatty not fatty play the key role in LPS in in lipid rafts are acylated with saturated fatty acids that the of lipid rafts. results that LPS and lauric acid dimerization and recruitment of TLR4 to lipid rafts the that the saturated fatty acids acylated in Lipid A the of lipid rafts, and TLR4 to lipid rafts TLR4 with signaling to the downstream signaling was demonstrated that of with n-3 acid of fatty acid into in lipid rafts, which are with saturated fatty acids J. J. PubMed Scopus Google an of the fatty acid of in lipid rafts by n-3 to of signaling from lipid rafts, and downstream signaling pathways J. J. PubMed Scopus Google Scholar, J. 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Toll-like receptor-4 (TLR4) can be activated by nonbacterial agonists, including saturated fatty acids. However, downstream signaling pathways activated by nonbacterial agonists are not known. Thus, we determined the downstream signaling pathways derived from saturated fatty acid-induced TLR4 activation. Saturated fatty acid (lauric acid)-induced NFkappaB activation was inhibited by a dominant-negative mutant of TLR4, MyD88, IRAK-1, TRAF6, or IkappaBalpha in macrophages (RAW264.7) and 293T cells transfected with TLR4 and MD2. Lauric acid induced the transient phosphorylation of AKT. LY294002, dominant-negative (DN) phosphatidylinositol 3-kinase (PI3K), or AKT(DN) inhibited NFkappaB activation, p65 transactivation, and cyclooxygenase-2 (COX-2) expression induced by lauric acid or constitutively active (CA) TLR4. AKT(DN) blocked MyD88-induced NFkappaB activation, suggesting that AKT is a MyD88-dependent downstream signaling component of TLR4. AKT(CA) was sufficient to induce NFkappaB activation and COX-2 expression. These results demonstrate that NFkappaB activation and COX-2 expression induced by lauric acid are at least partly mediated through the TLR4/PI3K/AKT signaling pathway. In contrast, docosahexaenoic acid (DHA) inhibited the phosphorylation of AKT induced by lipopolysaccharide or lauric acid. DHA also suppressed NFkappaB activation induced by TLR4(CA), but not MyD88(CA) or AKT(CA), suggesting that the molecular targets of DHA are signaling components upstream of MyD88 and AKT. Together, these results suggest that saturated and polyunsaturated fatty acids reciprocally modulate the activation of TLR4 and its downstream signaling pathways involving MyD88/IRAK/TRAF6 and PI3K/AKT and further suggest the possibility that TLR4-mediated target gene expression and cellular responses are also differentially modulated by saturated and unsaturated fatty acids.

Toll-like receptor 4 (TLR4) and TLR2 agonists from bacterial origin require acylated saturated fatty acids in their molecules. Previously, we reported that TLR4 activation is reciprocally modulated by saturated and polyunsaturated fatty acids in macrophages. However, it is not known whether fatty acids can modulate the activation of TLR2 or other TLRs for which respective ligands do not require acylated fatty acids. A saturated fatty acid, lauric acid, induced NFkappaB activation when TLR2 was co-transfected with TLR1 or TLR6 in 293T cells, but not when TLR1, 2, 3, 5, 6, or 9 was transfected individually. An n-3 polyunsaturated fatty acid (docosahexaenoic acid (DHA)) suppressed NFkappaB activation and cyclooxygenase-2 expression induced by the agonist for TLR2, 3, 4, 5, or 9 in a macrophage cell line (RAW264.7). Because dimerization is considered one of the potential mechanisms for the activation of TLR2 and TLR4, we determined whether the fatty acids modulate the dimerization. However, neither lauric acid nor DHA affected the heterodimerization of TLR2 with TLR6 as well as the homodimerization of TLR4 as determined by co-immunoprecipitation assays in 293T cells in which these TLRs were transiently overexpressed. Together, these results demonstrate that lauric acid activates TLR2 dimers as well as TLR4 for which respective bacterial agonists require acylated fatty acids, whereas DHA inhibits the activation of all TLRs tested. Thus, responsiveness of different cell types and tissues to saturated fatty acids would depend on the expression of TLR4 or TLR2 with either TLR1 or TLR6. These results also suggest that inflammatory responses induced by the activation of TLRs can be differentially modulated by types of dietary fatty acids.

Severe vitamin B(12) deficiency produces a cluster of neurological symptoms in infants, including irritability, failure to thrive, apathy, anorexia, and developmental regression, which respond remarkably rapidly to supplementation. The underlying mechanisms may involve delayed myelination or demyelination of nerves; alteration in the S-adenosylmethionine:S-adenosylhomocysteine ratio; imbalance of neurotrophic and neurotoxic cytokines; and/or accumulation of lactate in brain cells. This review summarizes the current knowledge concerning infantile vitamin B(12) deficiency, including a pooled analysis of case studies of infants born to mothers with untreated pernicious anemia or a strict vegetarian lifestyle and a discussion of the mechanisms that may underlie the manifestations of deficiency.

OBJECTIVE: Hyperglycemia-induced inflammation is central in diabetes complications, and monocytes are important in orchestrating these effects. Toll-like receptors (TLRs) play a key role in innate immune responses and inflammation. However, there is a paucity of data examining the expression and activity of TLRs in hyperglycemic conditions. Thus, in the present study, we examined TLR2 and TLR4 mRNA and protein expression and mechanism of their induction in monocytic cells under high-glucose conditions. RESEARCH DESIGN AND METHODS: High glucose (15 mmol/l) significantly induced TLR2 and TLR4 expression in THP-1 cells in a time- and dose-dependent manner (P < 0.05). High glucose increased TLR expression, myeloid differentiation factor 88, interleukin-1 receptor-associated kinase-1, and nuclear factor-kappaB (NF-kappaB) p65-dependent activation in THP-1 cells. THP-1 cell data were further confirmed using freshly isolated monocytes from healthy human volunteers (n = 10). RESULTS: Pharmacological inhibition of protein kinase C (PKC) activity and NADPH oxidase significantly decreased TLR2 and TLR4 mRNA and protein (P < 0.05). Knocking down both TLR2 and TLR4 in the cells resulted in a 76% (P < 0.05) decrease in high-glucose-induced NF-kappaB activity, suggesting an additive effect. Furthermore, PKC-alpha knockdown decreased TLR2 by 61% (P < 0.05), whereas inhibition of PKC-delta decreased TLR4 under high glucose by 63% (P < 0.05). Small inhibitory RNA to p47Phox in THP-1 cells abrogated high-glucose-induced TLR2 and TLR4 expression. Additional studies revealed that PKC-alpha, PKC-delta, and p47Phox knockdown significantly abrogated high-glucose-induced NF-kappaB activation and inflammatory cytokine secretion. CONCLUSIONS: Collectively, these data suggest that high glucose induces TLR2 and -4 expression via PKC-alpha and PKC-delta, respectively, by stimulating NADPH oxidase in human monocytes.

Insulin resistance progressing to type 2 diabetes mellitus (T2DM) is marked by a broad perturbation of macronutrient intermediary metabolism. Understanding the biochemical networks that underlie metabolic homeostasis and how they associate with insulin action will help unravel diabetes etiology and should foster discovery of new biomarkers of disease risk and severity. We examined differences in plasma concentrations of >350 metabolites in fasted obese T2DM vs. obese non-diabetic African-American women, and utilized principal components analysis to identify 158 metabolite components that strongly correlated with fasting HbA1c over a broad range of the latter (r = -0.631; p<0.0001). In addition to many unidentified small molecules, specific metabolites that were increased significantly in T2DM subjects included certain amino acids and their derivatives (i.e., leucine, 2-ketoisocaproate, valine, cystine, histidine), 2-hydroxybutanoate, long-chain fatty acids, and carbohydrate derivatives. Leucine and valine concentrations rose with increasing HbA1c, and significantly correlated with plasma acetylcarnitine concentrations. It is hypothesized that this reflects a close link between abnormalities in glucose homeostasis, amino acid catabolism, and efficiency of fuel combustion in the tricarboxylic acid (TCA) cycle. It is speculated that a mechanism for potential TCA cycle inefficiency concurrent with insulin resistance is "anaplerotic stress" emanating from reduced amino acid-derived carbon flux to TCA cycle intermediates, which if coupled to perturbation in cataplerosis would lead to net reduction in TCA cycle capacity relative to fuel delivery.

Weightloss is a major concern for the US population. Surveys consistently show that most adults are trying to lose or maintain weight (1). Nevertheless, the prevalence of overweight and obesity has increased steadily over the past 30 years. Currently, 50% of all adult Americans are considered overweight or obese (2,3). These numbers have serious public health implications. Excess weight is associated with increased mortality (4) and morbidity (5). It is associated with cardiovascular disease, type 2 diabetes, hypertension, stroke, gallbladder disease, osteoarthritis, sleep apnea and respiratory problems, and some types of cancer (6,7). Most people who are trying to lose weight are not using the recommended combination of reducing caloric intake and increasing physical activity (1). Although over 70% of persons reported using each of the following strategies at least once in 4 years, increased exercise (82.2%), decreased fat intake (78.7%), reduced food amount (78.2%,) and reduced calories (73.2%), the duration of any one of these behaviors was brief. Even the most common behaviors were used only 20% of the time (8). Obesity-related conditions are significantly improved with modest weight loss of 5% to 10%, even when many patients remain considerably overweight (6). The Institute of Medicine (9) defined successful long-term weight loss as a 5% reduction in initial body weight (IBW) that is maintained for at least 1 year. Yet data suggest that such losses are not consistent with patients’ goals and expectations. Foster (10) reported that in obese women (mean body mass index [BMI] of 36.3 ± 4.3) goal weights targeted, on average, a 32% reduction in IBW, implying expectations that are unrealistic for even the best available treatments. Interestingly, the most important factors that influenced the selection of goal weights were appearance and physical comfort rather than change in medical condition or weight suggested by a doctor or health care professional. Is it any wonder that overweight individuals are willing to try any new diet that promises quick, dramatic results more in line with their desired goals and expectations than with what good science supports? The proliferation of diet books is nothing short of phenomenal. A search of books on Amazon.com using the key words “weight loss” revealed 1214 matches. Of the top 50 best-selling diet books, 58% were published in 1999 or 2000 and 88% were published since 1997. Many of the top 20 best sellers at Amazon.com promote some form of carbohydrate (CHO) restriction (e.g., Dr. Atkins’ New Diet Revolution, The Carbohydrate Addict's Diet, Protein Power, Lauri's Low-Carb Cookbook). This dietary advice is counter to that promulgated by governmental agencies (US Department of Agriculture [USDA]/Department of Health and Human Services, National Institutes of Health) and nongovernmental organizations (American Dietetic Association, American Heart Association, American Diabetes Association, American Cancer Society, and Shape Up America!). What is really known about popular diets? Is the information scientifically sound? Are popular diets effective for weight loss and/or weight maintenance? What is the effect, if any, on composition of weight loss (fat vs. lean body mass), micronutrient (vitamin and mineral) status, metabolic parameters (e.g., blood glucose, insulin sensitivity, blood pressure, lipid levels, uric acid, and ketone bodies)? Do they affect hunger and appetite, psychological well-being, and reduction of risk for chronic disease (e.g., coronary heart disease, diabetes, and osteoporosis)? What are the effects of these diets on insulin and leptin, long-term hormonal regulators of energy intake and expenditure? The objective of this article is to review the scientific literature on various types of popular diets based on their macronutrient composition in an attempt to answer these questions (see Appendix for diet summaries). This article is limited to the effects of popular diets in overweight and obese adults; there are no good data on children and adolescents. Dietary claims are scrutinized, diets are analyzed, and information is compared with scientific data published in peer-reviewed journals. No published studies are excluded, despite inherent methodological problems (e.g., small or inadequate sample size, limited duration, lack of adequate controls and randomization, poor or minimal dietary collection and/or description of diets, and potential biases). However, the strength of the evidence supporting various conclusions made throughout the paper is based on the following grading system used by National Heart, Lung, and Blood Institute (NHLBI) (6) (Table 1). Diets are characterized below and in Tables 2 and 3. High-fat (55% to 65%), low-CHO (<100 g of CHO per day), high-protein diets (e.g., Dr. Atkins’ New Diet Revolution, Protein Power, Life Without Bread). Moderate-fat (20% to 30%), balanced nutrient reduction diets, high in CHO and moderate in protein (e.g., USDA Food Guide Pyramid, DASH diet, Weight Watchers). Low-fat (11% to 19%), and very-low-fat (VLF) (<10%), very-high-CHO, moderate-protein diets (e.g., Dr. Dean Ornish's Program for Reversing Heart Disease, Eat More, Weigh Less, The New Pritikin Program). Diets that reduce caloric intake result in weight loss. In the absence of physical activity, a diet that contains ∼1400 to 1500 kcal/d, regardless of macronutrient composition, results in weight loss. Individuals consuming high-fat, low-CHO diets may lose weight because the intake of protein and fat is self-limiting and overall caloric intake is decreased (11,12). Low-fat and VLF diets contain a high proportion of complex CHOs, fruits, and vegetables. They are naturally high in fiber and low in caloric density. Individuals consuming these types of diets consume fewer calories and lose weight (13–17). Balanced nutrient reduction diets contain moderate amounts of fat, CHO, and protein. When overall caloric intake is reduced, these diets result in loss of body weight and body fat (6,18). Importantly, moderate-fat, balanced nutrient reduction diets produce weight loss even when they are consumed ad libitum. In sum, all popular diets, as well as diets recommended by governmental and nongovernmental organizations, result in weight loss. However, it is important to note that weight loss is not the same as weight maintenance. Evidence Statement: Caloric balance is the major determinant of weight loss. Diets that reduce caloric intake result in weight loss. In the absence of physical activity, the optimal diet for weight loss contains ∼1400 to 1500 kcal/d, regardless of macronutrient composition. Evidence Category A. Evidence Statement: Free-living overweight individuals who self-select high-fat, low-CHO diets consume fewer calories and lose weight. Evidence Category C. Evidence Statement: Overweight individuals consuming high-fat, low-CHO, low-calorie diets under experimental conditions lose weight. Evidence Category C. Evidence Statement: Overweight individuals consuming moderate-fat, balanced nutrient reduction diets lose weight because they consume fewer calories. These diets can produce weight loss when consumed ad libitum. Evidence Category A. Evidence Statement: Overweight individuals consuming low-fat and VLF diets lose weight because they consume fewer calories. Evidence Category B. Evidence Statement: Weight loss on VLF diets may be the result of lifestyle modification, which may include decreased fat and energy intake, increased energy expenditure, or both. Evidence Category B. As body weight decreases, so does body fat and lean body mass. The optimal diet for weight loss is one that maximizes loss of body fat and minimizes loss of lean body mass. All low-calorie diets result in loss of body weight and body fat (6). Macronutrient composition does not seem to play a major role (19–22). In the short-term, however, high-fat, low-CHO ketogenic diets cause a greater loss of body water than body fat (23). When these diets end, water weight is regained (24). Eventually, however, all reduced calorie diets result in loss of body fat if sustained long term (25). Physical activity, an important factor with respect to lean body mass, should be promoted to enhance the effects of diet on body composition. Evidence Statement: All low-calorie diets result in loss of body weight and body fat. Macronutrient composition does not seem to play a major role. Evidence Category A Evidence Statement: In the short term, low-CHO ketogenic diets cause a greater loss of body water than body fat. Water weight is regained when the diet ends. If the diet is maintained long term, it results in loss of body fat. Evidence Category C. Proper food choices are always important when considering the nutritional quality of a diet. When individuals consume foods from all food groups, it is more likely that their diet will be nutritionally adequate. The moderate-fat, balanced nutrient reduction diet is optimal for ensuring adequate nutritional intake. However, poor food choices may result in inadequate levels of nutrients (e.g., calcium, iron, zinc), regardless of overall macronutrient composition. High-fat, low-CHO diets are nutritionally inadequate. They are low in vitamins E, A, thiamin, B6, folate, calcium, magnesium, iron, potassium, and dietary fiber, and require supplementation. These diets are high in saturated fat and cholesterol. VLF diets are low in vitamins E, B12, and zinc because meat and fat intake is low. Evidence Statement: With proper food choices, the moderate-fat, balanced nutrient reduction diet is nutritionally adequate. Evidence Category B. Evidence Statement: High-fat, low-CHO diets are nutritionally inadequate, and require supplementation. Evidence Category C. Evidence Statement: VLF diets are low in vitamins E, B12, and zinc. Evidence Category B. Low-CHO diets result in ketosis, and may cause a significant increase in blood uric acid concentrations. Blood lipid levels (e.g., total cholesterol [TC], low-density lipoprotein [LDL], high-density lipoprotein [HDL] and triglycerides [TGs]) decrease as body weight decreases (6,26,27). However, the macronutrient and fatty acid composition of energy-restricted diets can exert substantial effects on blood lipids. There are significantly greater decreases in LDL cholesterol during active weight loss when diets are low in saturated fatty acids. Changes in HDL cholesterol depend on dietary fat content and duration of energy restriction (28). Moderate-fat, balanced nutrient reduction diets reduce LDL-cholesterol and normalize the ratio of HDL/TC. Plasma TG levels also decrease with weight loss (6). Although they increase in response to short-term consumption of a VLF, high-CHO diet (29), the type of CHO consumed must be considered. High-fiber foods, including vegetables and legumes, do not lead to hypertriglyceridemia (30), and may easily be incorporated into moderate-fat, balanced nutrient reduction diets to help normalize plasma TG levels. Energy restriction, independent of diet composition, improves glycemic control (21,22,31–33). As body weight decreases, so does blood insulin and plasma leptin levels (21,34). Blood pressure decreases with weight loss, independent of diet composition (6,22,26). However, the DASH diet, high in fruits, vegetables, and low-fat dairy products effectively lowers blood pressure (35). Evidence Statement: High-fat, low-CHO diets result in ketosis. Evidence Category B. Evidence Statement: Metabolic profiles are improved with energy restriction and weight loss. Evidence Category A. Evidence Statement: Low-CHO diets that result in weight loss may also result in decreased blood lipid levels, decreased blood glucose and insulin levels, and decreased blood pressure. Evidence Category C. Evidence Statement: Low-fat and very low-fat diets reduce LDL-cholesterol, and may also decrease plasma TG levels, depending on diet composition. Evidence Category B. Evidence Statement: Moderate-fat, balanced nutrient reduction diets reduce LDL-cholesterol, normalize the ratio of HDL/TC, and normalize plasma TGs. Evidence Category A. Many factors influence hunger, appetite, and subsequent food intake. Macronutrient content of the diet is one, and it may not be the most important. Neurochemical factors (e.g., serotonin, endorphins, dopamine, hypothalamic neuropeptide transmitters), gastric signals (e.g., peptides, stomach distention), hedonistic qualities of food (e.g., taste, texture, smell), genetic, environmental (e.g., food availability, cost, cultural norms) and emotional factors (e.g., eating when bored, depressed, stressed, happy) must be considered. These parameters influence appetite primarily on a meal-to-meal basis. However, long-term body weight regulation seems to be controlled by hormonal signals from the endocrine pancreas and adipose tissue, i.e., insulin and leptin. Because insulin secretion and leptin production are influenced by the macronutrient content of the diet (36,37), effects of different diets on these long-term regulators of energy balance also need to be considered when investigating hunger and appetite. All fat-restricted diets provide a high degree of satiety. Subjects who consume fat-restricted diets do not complain of hunger, but of having “too much food” (38,39). These diets, high in fiber and water content are low in caloric density. Subjects who consume these diets develop a distaste for fat (40), which may be useful in long-term adherence to reduced fat, low-calorie diets. However, it is not clear that restricting fat provides any advantage over restricting CHOs. Ogden (41) reports weight loss maintainers used healthy eating habits and adhered to calorie-controlled diets. Long-term compliance to any diet means that short-term weight-loss has a chance to become long-term weight maintenance (42–44). Dietary compliance is likely a function of psychological issues (e.g., frequency of dietary counseling, coping with emotional eating, group support) rather than macronutrient composition, per se (42,45). Being conscious of one's behaviors, using social support, confronting problems directly, and using personally developed strategies may enhance long-term success (46). Ogden (41) notes that successful weight loss and maintenance may be predicted by an individuals’ belief system (e.g., that obesity is perceived as a problem that can be modified and if modifications bring changes in the short-term that are valued by the individual concerned). Evidence Statement: Many factors influence hunger, appetite, and subsequent food intake. There does not seem to be an optimal diet for reducing hunger. Evidence Category B. Evidence Statement: Long-term compliance is likely a function of psychological issues rather than macronutrient composition. Evidence Category B. Caloric balance (calories in vs. calories out), rather than macronutrient composition is the major determinant of weight loss. However, what is not clear is the effect of macronutrient content on long-term weight maintenance and adherence. Furthermore, it is not known whether maintenance of weight loss and dietary adherence is related to psychological issues (and brain neurochemistry), physiological parameters (e.g., hormones involved in body weight regulation such as insulin and leptin), physical activity, energy density, or some other factor(s). Controlled clinical trials of high-fat, low-CHO, and low-fat and VLF diets are needed to answer questions regarding long-term effectiveness (e.g., weight maintenance rather than weight loss) and potential long-term health benefits and/or detriments. Prevention of weight gain and weight maintenance are important goals. Scientifically validated, yet understandable information is clearly needed by millions of overweight and obese Americans who often find weight loss attainable, but maintaining weight loss nearly impossible.

Vitamin C is an essential dietary nutrient required as a co-factor for many enzymes, and humans are among the few animals that lack the ability to synthesize the compound from glucose. The reduced form of the vitamin, ascorbic acid, is an especially effective antioxidant owing to its high electron-donating power and ready conversion back to the active reduced form. Concentrations of the vitamin in body tissues and fluids are regulated through interactions of intestinal absorption, cellular transport, and excretion. The amount of vitamin C needed to prevent scurvy is very small and easily obtained in nearly all Western diets. There is great interest in the clinical roles of vitamin C because of evidence that oxidative damage is a root cause of, or at least associated with, many diseases. Population studies show that individuals with high intakes of vitamin C have lower risk of a number of chronic diseases, including heart disease, cancer, eye diseases, and neurodegenerative conditions. However, these results may simply reflect a more healthful diet or lifestyle for individuals with a high vitamin C intake. At present, data from controlled clinical trials have not established that higher intakes of vitamin C alone will help prevent chronic degenerative diseases. However, the evidence that ascorbic acid acts as an important antioxidant in many body tissues is convincing. The new higher Recommended Dietary Allowance (RDA) for vitamin C of 75 mg for women and 90 mg for men is, for the first time, based on the vitamin's role as an antioxidant as well as protection from deficiency. In healthy people, amounts greater than the RDA do not appear to be helpful. Vitamin C nutriture may be more important for people with certain diseases or conditions. High intakes of the vitamin are generally well tolerated; a Tolerable Upper Level was recently set at 2 g based on gastrointestinal upset that sometimes accompanies excessive intakes.

OBJECTIVE: Oral vaccine efficacy is low in less-developed countries, perhaps due to intestinal dysbiosis. This study determined if stool microbiota composition predicted infant oral and parenteral vaccine responses. METHODS: The stool microbiota of 48 Bangladeshi infants was characterized at 6, 11, and 15 weeks of age by amplification and sequencing of the 16S ribosomal RNA gene V4 region and by Bifidobacterium-specific, quantitative polymerase chain reaction. Responses to oral polio virus (OPV), bacille Calmette-Guérin (BCG), tetanus toxoid (TT), and hepatitis B virus vaccines were measured at 15 weeks by using vaccine-specific T-cell proliferation for all vaccines, the delayed-type hypersensitivity skin-test response for BCG, and immunoglobulin G responses using the antibody in lymphocyte supernatant method for OPV, TT, and hepatitis B virus. Thymic index (TI) was measured by ultrasound. RESULTS: Actinobacteria (predominantly Bifidobacterium longum subspecies infantis) dominated the stool microbiota, with Proteobacteria and Bacteroidetes increasing by 15 weeks. Actinobacteria abundance was positively associated with T-cell responses to BCG, OPV, and TT; with the delayed-type hypersensitivity response; with immunoglobulin G responses; and with TI. B longum subspecies infantis correlated positively with TI and several vaccine responses. Bacterial diversity and abundance of Enterobacteriales, Pseudomonadales, and Clostridiales were associated with neutrophilia and lower vaccine responses. CONCLUSIONS: Bifidobacterium predominance may enhance thymic development and responses to both oral and parenteral vaccines early in infancy, whereas deviation from this pattern, resulting in greater bacterial diversity, may cause systemic inflammation (neutrophilia) and lower vaccine responses. Vaccine responsiveness may be improved by promoting intestinal bifidobacteria and minimizing dysbiosis early in infancy.

Insulin receptor substrate (IRS) has been suggested as a molecular target of free fatty acids (FFAs) for insulin resistance. However, the signaling pathways by which FFAs lead to the inhibition of IRS function remain to be established. In this study, we explored the FFA-signaling pathway that contributes to serine phosphorylation and degradation of IRS-1 in adipocytes and in dietary obese mice. Linoleic acid, an FFA used in this study, resulted in a reduction in insulin-induced glucose uptake in 3T3-L1 adipocytes. This mimics insulin resistance induced by high-fat diet in C57BL/6J mice. The reduction in glucose uptake is associated with a decrease in IRS-1, but not IRS-2 or GLUT4 protein abundance. Decrease in IRS-1 protein was proceeded by IRS-1 (serine 307) phosphorylation that was catalyzed by serine kinases inhibitor kappaB kinase (IKK) and c-JUN NH2-terminal kinase (JNK). IKK and JNK were activated by linoleic acid and inhibition of the two kinases led to prevention of IRS-1 reduction. We demonstrate that protein kinase C (PKC) theta is expressed in adipocytes. In 3T3-L1 adipocytes and fat tissue, PKCtheta was activated by fatty acids as indicated by its phosphorylation status, and by its protein level, respectively. Activation of PKCtheta contributes to IKK and JNK activation as inhibition of PKCtheta by calphostin C blocked activation of the latter kinases. Inhibition of either PKCtheta or IKK plus JNK by chemical inhibitors resulted in protection of IRS-1 function and insulin sensitivity in 3T3-L1 adipocytes. These data suggest that: 1) activation of PKCtheta contributes to IKK and JNK activation by FFAs; 2) IKK and JNK mediate PKCtheta signals for IRS-1 serine phosphorylation and degradation; and 3) this molecular mechanism may be responsible for insulin resistance associated with hyperlipidemia.

This review describes current knowledge of the main causes of vitamin B12 and folate deficiency. The most common explanations for poor vitamin B12 status are a low dietary intake of the vitamin (i.e., a low intake of animal-source foods) and malabsorption. Although it has long been known that strict vegetarians (vegans) are at risk for vitamin B12 deficiency, evidence now indicates that low intakes of animal-source foods, such as occur in some lacto-ovo vegetarians and many less-industrialized countries, cause vitamin B12 depletion. Malabsorption of the vitamin is most commonly observed as food-bound cobalamin malabsorption due to gastric atrophy in the elderly, and probably as a result of Helicobacter pylori infection. There is growing evidence that gene polymorphisms in transcobalamins affect plasma vitamin B12 concentrations. The primary cause of folate deficiency is low intake of sources rich in the vitamin, such as legumes and green leafy vegetables, and the consumption of these foods may explain why folate status can be adequate in relatively poor populations. Other situations in which the risk of folate deficiency increases include lactation and alcoholism.

Incomplete β-oxidation of fatty acids in mitochondria is a feature of insulin resistance and type 2 diabetes mellitus (T2DM). Previous studies revealed that plasma concentrations of medium- and long-chain acylcarnitines (by-products of incomplete β-oxidation) are elevated in T2DM and insulin resistance. In a previous study, we reported that mixed D,L isomers of C12- or C14-carnitine induced an NF-κB-luciferase reporter gene in RAW 264.7 cells, suggesting potential activation of proinflammatory pathways. Here, we determined whether the physiologically relevant L-acylcarnitines activate classical proinflammatory signaling pathways and if these outcomes involve pattern recognition receptor (PRR)-associated pathways. Acylcarnitines induced the expression of cyclooxygenase-2 in a chain length-dependent manner in RAW 264.7 cells. L-C14 carnitine (5-25 μM), used as a representative acylcarnitine, stimulated the expression and secretion of proinflammatory cytokines in a dose-dependent manner. Furthermore, L-C14 carnitine induced phosphorylation of JNK and ERK, common downstream components of many proinflammatory signaling pathways including PRRs. Knockdown of MyD88, a key cofactor in PRR signaling and inflammation, blunted the proinflammatory effects of acylcarnitine. While these results point to potential involvement of PRRs, L-C14 carnitine promoted IL-8 secretion from human epithelial cells (HCT-116) lacking Toll-like receptors (TLR)2 and -4, and did not activate reporter constructs in TLR overexpression cell models. Thus, acylcarnitines have the potential to activate inflammation, but the specific molecular and tissue target(s) involved remain to be identified.

Elevated blood branched-chain amino acids (BCAA) are often associated with insulin resistance and type 2 diabetes, which might result from a reduced cellular utilization and/or incomplete BCAA oxidation. White adipose tissue (WAT) has become appreciated as a potential player in whole body BCAA metabolism. We tested if expression of the mitochondrial BCAA oxidation checkpoint, branched-chain α-ketoacid dehydrogenase (BCKD) complex, is reduced in obese WAT and regulated by metabolic signals. WAT BCKD protein (E1α subunit) was significantly reduced by 35-50% in various obesity models (fa/fa rats, db/db mice, diet-induced obese mice), and BCKD component transcripts significantly lower in subcutaneous (SC) adipocytes from obese vs. lean Pima Indians. Treatment of 3T3-L1 adipocytes or mice with peroxisome proliferator-activated receptor-γ agonists increased WAT BCAA catabolism enzyme mRNAs, whereas the nonmetabolizable glucose analog 2-deoxy-d-glucose had the opposite effect. The results support the hypothesis that suboptimal insulin action and/or perturbed metabolic signals in WAT, as would be seen with insulin resistance/type 2 diabetes, could impair WAT BCAA utilization. However, cross-tissue flux studies comparing lean vs. insulin-sensitive or insulin-resistant obese subjects revealed an unexpected negligible uptake of BCAA from human abdominal SC WAT. This suggests that SC WAT may not be an important contributor to blood BCAA phenotypes associated with insulin resistance in the overnight-fasted state. mRNA abundances for BCAA catabolic enzymes were markedly reduced in omental (but not SC) WAT of obese persons with metabolic syndrome compared with weight-matched healthy obese subjects, raising the possibility that visceral WAT contributes to the BCAA metabolic phenotype of metabolically compromised individuals.