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The limited availability of human donor organs suitable for transplantation has resulted in ever-increasing patient waiting lists globally. Xenotransplantation is considered a potential option, but is yet to reach clinical practice. Although remarkable progress has been made in overcoming immunological rejection, issues with functionality are still to be resolved. Bioengineering approaches have been used to create cardiac tissues with optimized functions. The use of decellularized xenogeneic cardiac tissues seeded with donor-derived cardiac cells may prove to be a viable strategy as supporting structures of the native tissue such as vasculature can be utilized. Here we used sequential perfusion to decellularize adult rat hearts. The acellular scaffolds were reseeded with human endothelial cells, human fibroblasts, human mesenchymal stem cells, and cardiac cells derived from human-induced pluripotent stem cells. The ability of the resultant recellularized rat scaffolds to activate human naïve neutrophils in vitro was investigated to measure xenogeneic recognition. Our results demonstrate that in contrast to cadaveric xenogeneic hearts, acellular and recellularized xenogeneic scaffolds did not activate human naïve neutrophils and suggest that decellularization removes the xenogeneic antigens that lead to human naïve neutrophil activation thus allowing human cells to populate the now "allogenized" xenogeneic scaffolds.

Background The ever-increasing prevalence of diabetes and associated comorbidities serves to highlight the necessity of biologically relevant small-animal models to investigate its etiology, pathology and treatment. Although the C57BL/6 J model is amongst the most widely used mouse model due to its susceptibility to diet-induced obesity (DIO), there are a number of limitations namely [1] that unambiguous fasting hyperglycemia can only be achieved via dietary manipulation and/or chemical ablation of the pancreatic beta cells. [2] Heterogeneity in the obesogenic effects of hypercaloric feeding has been noted, together with sex-dependent differences, with males being more responsive. The KK mouse strain has been used to study aspects of the metabolic syndrome and prediabetes. We recently conducted a study which characterized the differences in male and female glucocentric parameters between the KK/HlJ and C57BL/6 J strains as well as diabetes-related behavioral differences (Inglis et al. 2019). In the present study, we further characterize these models by examining strain- and sex-dependent differences in pancreatic and adrenal gene expression using Affymetrix microarray together with endocrine-associated serum analysis. Results In addition to strain-associated differences in insulin tolerance, we found significant elevations in KK/HlJ mouse serum leptin, insulin and aldosterone. Additionally, glucagon and corticosterone were elevated in female mice of both strains. Using 2-factor ANOVA and a significance level set at 0.05, we identified 10,269 pancreatic and 10,338 adrenal genes with an intensity cut-off of ≥2.0 for all 4 experimental groups. In the pancreas, gene expression upregulated in the KK/HlJ strain related to increased insulin secretory granule biofunction and pancreatic hyperplasia, whereas ontology of upregulated adrenal differentially expressed genes (DEGs) related to cell signaling and neurotransmission. We established a network of functionally related DEGs commonly upregulated in both endocrine tissues of KK/HlJ mice which included the genes coding for endocrine secretory vesicle biogenesis and regulation: PCSK2, PCSK1N, SCG5, PTPRN, CHGB and APLP1. We also identified genes with sex-biased expression common to both strains and tissues including the paternally expressed imprint gene neuronatin. Conclusion Our novel results have further characterized the commonalities and diversities of pancreatic and adrenal gene expression between the KK/HlJ and C57BL/6 J strains as well as differences in serum markers of endocrine physiology.

Copyright © 2020 Murugaiah, Varghese, Saleh, Tsolaki, Alrokayan, Khan, Collison, Sim, Nal, Al-Mohanna and Kishore. The complement system is an ancient innate immune defence mechanism that can recognise molecular patterns on the invading pathogens. Factor H, as an inhibitor of the alternative pathway, down-regulates complement activation on the host cell surface. Locally synthesised factor H at the site of infection/injury, including lungs, can act as a pattern recognition molecule without involving complement activation. Here, we report that factor H, a sialic acid binder, interacts with influenza A virus (IAV) and modulates IAV replication, as observed by an upregulation of matrix protein 1 (M1) expression in H3N2-infected A549 cells, while downregulating M1 in H1N1 subtype-infected cells. Far-western blot revealed that factor H binds hemagglutinin (HA, ~70kDa), neuraminidase (NA, ~60kDa), and M1 (~25kDa). IAV-induced transcriptional levels of IFN-α, TNF-α, IL-12, IL-6, IFN-α, while RANTES were reduced following factor H treatment for the H1N1 subtype at 6 h post-infection. However, for the H3N2 subtype, mRNA levels of these pro-inflammatory cytokines were enhanced. Recombinant form of vaccinia virus complement control protein (VCP), which like factor H, contains CCP modules and has complement-regulatory activity, mirrored the results obtained with factor H. Both factor H (25%) and VCP (45%) were found to reduce luciferase reporter activity in MDCK cells transduced with H1N1 pseudotyped lentiviral particles. Factor H (50%) and VCP (30%) enhanced the luciferase reporter activity for H3N2, suggesting an entry inhibitory role of factor H and VCP against H1N1, but not H3N2. Thus, factor H can modulate IAV infection and inflammatory response independent of its complement-related functions. King Saud University, Riyadh, International Scientific Partnership Programme (ISPP) ISPP-145.

Aims Hypoxia-induced adipokine release has been attributed mainly to HIF-1α. Here we investigate the role of intracellular calcium and NF-kB in the hypoxia-dependent release of leptin, VEGF, IL-6 and the hypoxia-induced inhibition of adiponectin release in human adipocytes. Main methods We used intracellular calcium imaging to compare calcium status in preadipocytes and in adipocytes. We subjected both cell types to hypoxic conditions and measured the release of adipokines induced by hypoxia in the presence and absence of HIF-1α inhibitor YC-1, NF-κB inhibitor SN50 and intracellular calcium chelator BAPTA-AM. Key findings We demonstrate reduced intracellular calcium oscillations and increased oxidative stress as the cells transitioned from preadipocytes to adipocytes. We show that differentiation of preadipocytes to adipocytes is associated with distinct morphological changes in the mitochondria. We also show that hypoxia-induced secretion of leptin, VEGF, IL-6 and hypoxia-induced inhibition of adiponectin secretion are independent of HIF-1α expression. The hypoxia-induced leptin, VEGF and IL-6 release are [Ca++]i dependent whereas adiponectin is NF-kB dependent. Significance Our work suggests a major role for [Ca++]i in preadipocyte differentiation to adipocytes and that changes in mitochondrial morphology in the adipocytes might underlie the reduced calcium oscillations observed in the adipocytes. It also demonstrates that multiple signaling pathways are associated with the hypoxia-induced adipokine secretion.

The data set presented here is associated with the article “Intracellular calcium and NF-kB regulate hypoxia-induced leptin, VEGF, IL-6 and adiponectin secretion in human adipocytes” (Al-Anazi et al., 2018). Data illustrate hypoxia-induced VEGF and leptin expression in human adipocytes treated with the calcium chelator BAPTA-AM (1 µM). It also shows NF-κB p65 induced expression by hypoxia. Preadipocytes were differentiated for 14 days and then subjected to 0.5–1.5% oxygen in the presence and absence of BAPTA-AM or the NF-κB inhibitor SN50 for 48 h prior to RNA isolation and PCR analysis.

Background Pulmonary fibrosis is one of the leading indications for lung transplantation. The disease, which is of unknown aetiology, can be progressive, resulting in distortion of the extracellular matrix (ECM), inflammation, fibrosis and eventual death. Methods 13 patients born to consanguineous parents from two unrelated families presenting with interstitial lung disease were clinically investigated. Nine patients developed respiratory failure and subsequently died. Molecular genetic investigations were performed on patients' whole blood or archived tissues, and cell biological investigations were performed on patient-derived fibroblasts. Results The combination of a unique pattern of early-onset lung fibrosis (at 12–15 years old) with distinctive radiological findings, including 1) traction bronchiectasis, 2) intralobular septal thickening, 3) shrinkage of the secondary pulmonary lobules mainly around the bronchovascular bundles and 4) early type 2 respiratory failure (elevated blood carbon dioxide levels), represents a novel clinical subtype of familial pulmonary fibrosis. Molecular genetic investigation of families revealed a hypomorphic variant in S100A3 and a novel truncating mutation in S100A13, both segregating with the disease in an autosomal recessive manner. Family members that were either heterozygous carriers or wild-type normal for both variants were unaffected. Analysis of patient-derived fibroblasts demonstrated significantly reduced S100A3 and S100A13 expression. Further analysis demonstrated aberrant intracellular calcium homeostasis, mitochondrial dysregulation and differential expression of ECM components. Conclusion Our data demonstrate that digenic inheritance of mutations in S100A3 and S100A13 underlie the pathophysiology of pulmonary fibrosis associated with a significant reduction of both proteins, which suggests a calcium-dependent therapeutic approach for management of the disease., New evidence links an atypical form of pulmonary fibrosis with digenic mutations in the genes for calcium binding proteins S100A3 and S100A13. This implicates calcium homeostasis in the aetiology and pathogenesis of pulmonary fibrosis. http://bit.ly/2LyUQwb

Rationale Aspartame (L-aspartyl phenylalanine methyl ester) is a non-nutritive sweetener (NNS) approved for use in more than 6000 dietary products and pharmaceuticals consumed by the general public including adults and children, pregnant and nursing mothers. However a recent prospective study reported a doubling of the risk of being overweight amongst 1-year old children whose mothers consumed NNS-sweetened beverages daily during pregnancy. We have previously shown that chronic aspartame (ASP) exposure commencing in utero may detrimentally affect adulthood adiposity status, glucose metabolism and aspects of behavior and spatial cognition, and that this can be modulated by developmental N-methyl-D-aspartate receptor (NMDAR) blockade with the competitive antagonist CGP 39551 (CGP). Since glucose homeostasis and certain aspects of behavior and locomotion are regulated in part by the NMDAR-rich hypothalamus, which is part of the hypothalamic-pituitary-adrenal- (HPA) axis, we have elected to examine changes in hypothalamic and adrenal gene expression in response to ASP exposure in the presence or absence of developmental NMDAR antagonism with CGP, using Affymetrix microarray analysis. Results Using 2-factor ANOVA we identified 189 ASP-responsive differentially expressed genes (DEGs) in the adult male hypothalamus and 2188 in the adrenals, and a further 23 hypothalamic and 232 adrenal genes significantly regulated by developmental treatment with CGP alone. ASP exposure robustly elevated the expression of a network of genes involved in hypothalamic neurosteroidogenesis, together with cell stress and inflammatory genes, consistent with previous reports of aspartame-induced CNS stress and oxidative damage. These genes were not differentially expressed in ASP mice with CGP antagonism. In the adrenal glands of ASP-exposed mice, GABA and Glutamate receptor subunit genes were amongst those most highly upregulated. Developmental NMDAR antagonism alone had less effect on adulthood gene expression and affected mainly hypothalamic neurogenesis and adrenal steroid metabolism. Combined ASP + CGP treatment mainly upregulated genes involved in adrenal drug and cholesterol metabolism. Conclusion ASP exposure increased the expression of functional networks of genes involved in hypothalamic neurosteroidogenesis and adrenal catecholamine synthesis, patterns of expression which were not present in ASP-exposed mice with developmental NMDAR antagonism.

Background Lipid metabolism dysfunction leading to excess fat deposits (obesity) may cause tumor (cancer) development. Both obesity and cancer are the epicenter of important medical issues. Lipid metabolism and cell death/proliferation are controlled by biochemical and molecular pathways involving many proteins, and organelles; alteration in these pathways leads to fat accumulation or tumor growth. Mammalian Kruppel-like factors, KLFs play key roles in both lipid metabolism and tumor development. Scope of review Substantial epidemiological and clinical studies have established strong association of obesity with a number of human cancers. However, we need more experimental verification to determine the exact role of this metabolic alteration in the context of tumor development. A clear understanding of molecules, pathways and the mechanisms involved in lipid metabolism and cell death/proliferation will have important implications in pathogenesis, and prevention of these diseases. Major conclusion The regulatory role of KLFs, in both cell death/proliferation and lipid metabolism suggests a common regulation of both processes. This provides an excellent model for delivering a precise understanding of the mechanisms linking altered expression of KLFs to obesity and tumor development. General significance Currently, mouse and rats are the models of choice for investigating disease mechanisms and pharmacological therapies but a genetic model is needed for a thorough examination of KLF function in vivo during the development of an organism. The worm Caenorhabditis elegans is an ideal model to study the connectivity between lipid metabolism and cell death/proliferation.

Introduction Small-animal models are the most widely used preclinical model for studying the etiology, pathology and treatment of diabetes, prediabetes and diabetic comorbidities. Diabetic patients are burdened with higher rates of depression, anxiety and cognitive decline due to inadequate control of blood glucose levels, vascular damage and aberrant CNS insulin signaling. The C57BL/6J model is amongst the most widely used mouse model due to its susceptibility to diet-induced obesity (DIO). This strain has also been well-characterized in behavioral research studies. However the C57BL/6J model has a number of limitations: [1] overt fasting hyperglycemia can only be induced by dietary manipulation and/or chemical ablation of the pancreatic beta cells. [2] There is heterogeneity in the obesogenic response to hypercaloric feeding as well as sex-dependent differences, with males being more responsive. The KK inbred strain has been used to study aspects of the metabolic syndrome and prediabetes due to inherent glucose intolerance, hyperinsulinemia and insulin resistance. However KK/HlJ mice are less well-characterized and there have been fewer behavioral studies reported. The aim of this study was to examine differences in male and female glucocentric parameters between KK/HlJ and C57BL/6J mice, and to compare their performance in a variety of standard behavioral tests relating to general, anxiogenic and cognitive paradigms. Methods Strain differences in male and female KK/HlJ and C57BL/6J mouse adiposity, glucose and insulin parameters were studied together with group differences in standard Open Field, Object Recognition, Elevated Plus Maze, Light-Dark Transition, Porsolt test, Marble Burying, Social Recognition and Morris Water Maze tests. Correlations between behavioral variables were analyzed. Results and conclusion In addition to being uniformly larger, hyperinsulinemic and more insulin intolerant than C57BL/6J mice, we observed marked strain and sex-differences in KK/HlJ behavior. KK/HlJ mice exhibited less locomotor and vertical exploratory behavior in comparison to C57BL/6J, whereas object exploration and novel object discrimination were superior in KK/HlJ mice. Female KK/HlJ mice were faster swimmers, whereas the males exhibited greater spatial cognition and place-learning during the MWM test.

BackgroundPulmonary fibrosis is one of the leading indications for lung transplantation. The disease, which is of unknown aetiology, can be progressive, resulting in distortion of the extracellular matrix (ECM), inflammation, fibrosis and eventual death.Methods13 patients born to consanguineous parents from two unrelated families presenting with interstitial lung disease were clinically investigated. Nine patients developed respiratory failure and subsequently died. Molecular genetic investigations were performed on patients' whole blood or archived tissues, and cell biological investigations were performed on patient-derived fibroblasts.ResultsThe combination of a unique pattern of early-onset lung fibrosis (at 12–15 years old) with distinctive radiological findings, including 1) traction bronchiectasis, 2) intralobular septal thickening, 3) shrinkage of the secondary pulmonary lobules mainly around the bronchovascular bundles and 4) early type 2 respiratory failure (elevated blood carbon dioxide levels), represents a novel clinical subtype of familial pulmonary fibrosis. Molecular genetic investigation of families revealed a hypomorphic variant in S100A3 and a novel truncating mutation in S100A13, both segregating with the disease in an autosomal recessive manner. Family members that were either heterozygous carriers or wild-type normal for both variants were unaffected. Analysis of patient-derived fibroblasts demonstrated significantly reduced S100A3 and S100A13 expression. Further analysis demonstrated aberrant intracellular calcium homeostasis, mitochondrial dysregulation and differential expression of ECM components.ConclusionOur data demonstrate that digenic inheritance of mutations in S100A3 and S100A13 underlie the pathophysiology of pulmonary fibrosis associated with a significant reduction of both proteins, which suggests a calcium-dependent therapeutic approach for management of the disease.

We have previously showed that lifetime exposure to aspartame, commencing in utero via the mother's diet, may impair insulin tolerance and cause behavioral deficits in adulthood via mechanisms which are incompletely understood. The role of the CNS in regulating glucose homeostasis has been highlighted by recent delineation of the gut-brain axis, in which N -methyl- d -aspartic acid receptors (NMDARs) are important in maintaining glucose homeostasis, in addition to regulating certain aspects of behavior. Since the gut-brain axis can be modulated by fetal programming, we hypothesized that early-life NMDAR antagonism may affect aspartame-induced glucose deregulation in adulthood, and may alter the aspartame behavioral phenotype. Accordingly, C57Bl/6J mice were chronically exposed to aspartame commencing in utero , in the presence and absence of maternal administration of the competitive NMDAR antagonist CGP 39551, from conception until weaning. Drug/diet interactions in adulthood glucocentric and behavioral parameters were assessed. Aspartame exposure elevated blood glucose and impaired insulin-induced glucose disposal during an insulin tolerance test, which could be normalized by NMDAR antagonism. The same effects were not observed in control diet mice, suggesting an early-life drug/diet interaction. Behavioral analysis of adult offspring indicated that NMDAR antagonism of control diet mice caused hyperlocomotion and impaired spatial navigation. Conversely hypolocomotion, reduced exploratory activity and increased anxiety-related behavior were apparent in aspartame diet mice with early-life NMDAR antagonism. Conclusion: significant drug/diet interactions in glucocentric and behavioral parameters were identified in aspartame-exposed mice with early-life NMDAR antagonism. This suggests a possible involvement of early NMDAR interactions in aspartame-impaired glucose homeostasis and behavioral deficits.

Nonalcoholic fatty liver disease begins with a relatively benign hepatic steatosis, often associated with increased adiposity, but may progress to a more severe nonalcoholic steatohepatitis with inflammation. A subset of these patients develops progressive fibrosis and ultimately cirrhosis. Various dietary components have been shown to contribute to the development of liver disease, including fat, sugars, and neonatal treatment with high doses of monosodium glutamate (MSG). However, rodent models of progressive disease have been disappointing, and alternative animal models of diet-induced liver disease would be desirable, particularly if they contribute to our knowledge of changes in gene expression as a result of dietary manipulation. The domestic cat has previously been shown to be an appropriate model for examining metabolic changes–associated human diseases such as diabetes. Our aim was therefore to compare changes in hepatic gene expression induced by dietary MSG, with that of a diet containing Trans-fat and high fructose corn syrup (HFCS), using a feline model. MSG treatment increased adiposity and promoted hepatic steatosis compared to control (P

Nonalcoholic fatty liver disease (NAFLD) is associated with obesity and insulin resistance. It is also a predisposing factor for type 2 diabetes. Dietary factors are believed to contribute to all three diseases. NAFLD is characterized by increased intrahepatic fat and mitochondrial dysfunction, and its etiology may be attributed to excessive fructose intake. Consumption of high fructose corn syrup-55 (HFCS-55) stands at up to 15% of the average total daily energy intake in the United States, and is linked to weight gain and obesity. The aim of this study was to establish whether HFCS-55 could contribute to the pathogenesis of NAFLD, by examining the effects of HFCS-55 on hepatocyte lipogenesis, insulin signaling, and cellular function, in vitro and in vivo. Exposure of hepatocytes to HFCS-55 caused a significant increase in hepatocellular triglyceride (TG) and lipogenic proteins. Basal production of reactive oxygen metabolite (ROM) was increased, together with a decreased capacity to respond to an oxidative challenge. HFCS-55 induced a downregulation of the insulin signaling pathway, as indicated by attenuated (ser473)phosphorylation of AKT1. The c-Jun amino-terminal kinase (JNK), which is intimately linked to insulin resistance, was also activated; and this was accompanied by an increase in endoplasmic reticulum (ER) stress and intracellular free calcium perturbation. Hepatocytes exposed to HFCS-55 exhibited mitochondrial dysfunction and released cytochrome C (CytC) into the cytosol. Hepatic steatosis and mitochondrial disruption was induced in vivo by a diet enriched with 20% HFCS 55; accompanied by hypoadiponectinemia and elevated fasting serum insulin and retinol-binding protein-4 (RBP4) levels. Taken together our findings indicate a potential mechanism by which HFCS-55 may contribute to the pathogenesis of NAFLD.

The effects of dietary monosodium glutamate (MSG) on trans-fatty acid (TFA)-induced nonalcoholic fatty liver disease (NAFLD) are addressed in an animal model. We used Affymetrix microarray analysis to investigate hepatic gene expression and the contribution of visceral white adipose tissue (WAT) to diet-induced NAFLD. Trans-fat feeding increased serum leptin, FFA, HDL-cholesterol (HDL-C), and total cholesterol (T-CHOL) levels, while robustly elevating the expression of genes involved in hepatic lipogenesis, including the transcription factor sterol-regulatory element binding protein 1c. Histological examination revealed hepatic macrosteatosis in TFA-fed animals. Conversely, dietary MSG at doses similar to human average daily intake caused hepatic microsteatosis and the expression of beta-oxidative genes. Serum triglyceride, FFA, and insulin levels were elevated in MSG-treated animals. The abdominal cavities of TFA- or MSG-treated animals had increased WAT deposition compared with controls. Microarray analysis of WAT gene expression revealed increased lipid biosynthetic gene expression, together with a 50% decrease in the key transcription factor Ppargc1a. A combination of TFA+MSG resulted in the highest levels of serum HDL-C, T-CHOL, and leptin. Microarray analysis of TFA+MSG-treated livers showed elevated expression of markers of hepatic inflammation, lipid storage, cell damage, and cell cycle impairment. TFA+MSG mice also had a high degree of WAT deposition and lipogenic gene expression. Levels of Ppargc1a were further reduced to 25% by TFA+MSG treatment. MSG exacerbates TFA-induced NAFLD.

Recruitment of leukocytes from bloodstream to extrahematic sites is tightly regulated by a variety of adhesion molecules that are expressed on the leukocytes and the vessel walls. In this manuscript, wedescribe the interactions between natural killer (NK) cells and activated, autologous platelets under physiologic flow. We found that surface-adherent human platelets are capable of recruiting human NK cells from flow and that this recruitment is characterized by an initial tethering followed by a rolling phase. Both phases were dependent on the adhesion molecule P-selectin and its counter-ligand on the NK cells (P-selectin glycoprotein ligand 1). Activation of rolling NK cells with inflammatory mediators commonly found in atherosclerotic plaques (interleukin-12 and leukotriene B4) causes immediate cessation of the rolling process and conversion to stationary adhesion. Blocking antibodies to the adhesion molecules membrane-activated complex-1 and leukocyte function antigen-1 inhibited this conversion. Our data suggest that platelets deposited at sites of vascular injury may provide an alternative substrate to endothelial cells for initial recruitment of NK cells to the vessel wall. This may result in extravasation of the NK cells if the appropriate chemotactic signal is applied. These data implicate the P-selectin and integrin family of adhesion molecules in the recruitment of NK cells to atherosclerotic sites.

The cytokine interleukin (IL)-12 plays a bridging role between innate and adaptive immunity. Here, we demonstrate that treatment of neutrophils with IL-12 leads to a transient increase in intracellular-free calcium [Ca++]i levels, which is necessary for the production of reactive oxygen metabolites (ROM). This production is associated with the activation and nuclear translocation of the transcription factor nuclear factor (NF)-κB and is inhibited in the presence of the intracellular calcium chelator 1,2-bis(O-amminophenoxy) ethane-N,N-N′,N′-tetraacetic acid-acetoxymethyl ester and the ROM production inhibitor diphenyl iodonium. We show that IL-12 causes a significant increase in total mRNA levels, which appear dependent on the generated ROM. In addition IL-12 induces the de novo synthesis and production of IL-8 and tumor necrosis factor α (TNF-α) in a calcium- and ROM-dependent manner. Our data demonstrate a direct role for IL-12 in the activation of human neutrophils and suggest a ROM-dependent interplay between IL-12-induced [Ca++]i transient and the release of IL-8 and TNF-α through NF-κB activation.

The accumulation of advanced glycation end products (AGEs) in the tissue and serum of subjects with diabetes has been linked to the pathogenesis of vascular complications. Because diabetes may be also complicated by increased susceptibility to recurrent infection, we investigated the effects of AGEs on human neutrophils, because their burst of activity immediately upon engagement of pathogens or other inflammatory triggers is critical to host response. We demonstrate the presence of receptor for advanced glycation end products (RAGE) at the message and protein levels. We also demonstrate that AGE albumin (but not control albumin) binds with high affinity to human neutrophils (Kd of 3.7±0.4 nM). The binding was blocked almost completely by excess soluble RAGE, anti-RAGE antibodies, or antibodies to CML-modified albumin. AGE albumin induced a dose-dependent increase in intracellular-free calcium as well as actin polymerization. Further, AGE albumin inhibited transendothelial migration and Staphylococcus aureus-induced but not fMLP-induced production of reactive oxygen metabolite. Moreover, although AGE albumin enhanced neutrophil phagocytosis of S. aureus, it inhibited bacterial killing. We conclude that functional RAGE is present on the plasma membrane of human neutrophils and is linked to Ca2+ and actin polymerization, and engagement of RAGE impairs neutrophil functions.

The molecular mechanisms underlying the geometrical changes of the left ventricle during the progression to heart failure and recovery are not well defined.Here we investigate the involvement of matrixins and cardiokines in an ovine model of pressure-induced left ventricular failure (LVF).Fifteen sheep underwent supracoronary aortic banding with an inflatable cuff. A controlled and progressive increase of LV pressure was monitored echocardiographically. Endomyocardial biopsies were collected throughout the development of LVF and subsequent recovery after pressure unloading.Thirteen sheep developed LVF with a subsequent recovery. Peak left ventricular hypertrophy (LVH) and dilatation (LVD) occurred at 31.5 ± 1.6 weeks and 102.7 ± 2.2 weeks post-banding respectively, with an increase in LV internal diameter in diastole (LVIDd 5.11 ± 0.12 compared to the control 3.37 ± 0.07 cm, p0.001), with preserved LV ejection fraction (LVEF). Reduced LVEF became evident 116.5 ± 2.7 weeks post-banding. Clinical and echocardiographic improvements were observed following deflation of the aortic banding cuff. By 138.1 ± 3.1 weeks cardiac performance recovered with restoration of LVEF. Significant changes in the expression of matrix metalloproteinases (MMP)-1, -2, -3, vascular endothelial cell growth factor (VEGF), fibroblast growth factor (FGF)-2, interferon (INF)-α-2 and soluble CD40 ligand (sCD40L) were observed throughout the progression to failure and recovery.We used an ovine model to study reversible LV remodelling without interruption and found significant changes in matrixin and cardiokine expression during LV progression to failure and recovery.

Fluorinated analogues of propranolol, namely trifluoroethyl propranolol (F3), pentafluoropropyl propranolol (F5), and heptafluorobutyl propranolol (F7), were found to induce reactive oxygen metabolite (ROM) production in human neutrophils in a dose-dependent manner. Preincubation of neutrophils with the calcium chelator BAPTA-AM or the tyrosine kinase inhibitor genistein inhibited this ROM production. Direct measurements of intracellular calcium revealed that these analogues caused a transient increase in intracellular calcium. In addition, these fluorinated analogues of propranolol caused a transient increase in actin polymerization. The effects of these compounds were found to be dependent upon the degree of fluorination of the parent compound. Propranolol, on the other hand, had no direct effect on ROM, calcium, or actin polymerization when added alone to neutrophils, although it did modify responses of cells to various stimuli. Whereas ROM production induced by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine was enhanced in a dose-dependent manner, the response to the particulate stimulus, latex beads, was abolished.

Background. Interaction between vascularized xeno- graft and host immune system is thought to occur via Galactose a (1,3) Galactose (Gala 1,3 gal) structures decorating the xenograft. Methods. We raised anti-Gala 1,3 gal-BSA polyclonal antibodies in baboons and investigated effect(s) of these antibodies as well as soluble Gala 1,3 gal-BSA on human naive natural killer (NK) cell interactions with porcine aortic endothelial cells. Results. We demonstrate that human naive (un- stimulated) NK cells recognize xenogeneic endothelial cells under conditions where binding to the Gala 1,3 gal structures is minimized by the presence of block- ing anti-Gala 1,3 gal IgG or soluble Gala 1-3 gal and in the absence of xenoreactive natural antibodies and complement. After xenogeneic encounter both endo- thelial cells and human NK cells are activated. Endo- thelial cell activation is rapid and is manifested ini- tially by an intraendothelial calcium transient and subsequently by expression of P-selectin and vascular endothelial cell adhesion molecule-1 on the xenoendo- thelium surface. NK cell activation is manifested by increased expression of perforin and increased cyto- toxicity towards the xenoendothelium. Neither recog- nition nor activation of the xenoendothelium was af- fected by the introduction of either anti-Gala 1,3 gal IgG or soluble Gala 1-3 gal. Conclusion. Our data provide evidence that innate immune cells, such as NK cells, recognize and activate xenoendothelial cells independently of Gala 1-3 gal structures and raise the possibility of novel interac- tive sites on both human naive NK cells and discor- dant xenogeneic endothelium.

Endoplasmic reticulum-associated amyloid beta-peptide (Abeta)-binding protein (ERAB)/L-3-hydroxyacyl-CoA dehydrogenase type II (HADH II) is expressed at high levels in Alzheimer's disease (AD)-affected brain, binds Abeta, and contributes to Abeta-induced cytotoxicity. Purified recombinant ERAB/HADH II catalyzed the NADH-dependent reduction of S-acetoacetyl-CoA with a Km of approximately 68 microM and a Vmax of approximately 430 micromol/min/mg. The contribution of ERAB/HADH II enzymatic activity to Abeta-mediated cellular dysfunction was studied by site-directed mutagenesis in the catalytic domain (Y168G/K172G). Although COS cells cotransfected to overexpress wild-type ERAB/HADH II and variant beta-amyloid precursor protein (betaAPP(V717G)) showed DNA fragmentation, cotransfection with Y168G/K172G-altered ERAB and betaAPP(V717G) was without effect. We thus asked whether the enzyme might recognize alcohol substrates of which the aldehyde products could be cytotoxic; ERAB/HADH II catalyzed oxidation of a variety of simple alcohols (C2-C10) to their respective aldehydes in the presence of NAD+ and NAD-dependent oxidation of 17beta-estradiol. Addition of micromolar levels of synthetic Abeta(1-40) to purified ERAB/HADH II inhibited, in parallel, reduction of S-acetoacetyl-CoA (Ki approximately 1.6 microM), as well as oxidation of 17beta-estradiol (Ki approximately 3.2 microM) and (-)-2-octanol (Ki approximately 2.6 microM). Because micromolar levels of Abeta were required to inhibit ERAB/HADH II activity, whereas Abeta binding to ERAB/HADH II occurred at much lower concentrations (Km approximately 40-70 nM), the latter more closely simulating Abeta levels within cells, Abeta perturbation of ERAB/HADH II was likely to result from mechanisms other than the direct modulation of enzymatic activity. Cells cotransfected to overexpress ERAB/HADH II and betaAPP(V717G) generated malondialdehyde-protein and 4-hydroxynonenal-protein epitopes, which were detectable only at the lowest levels in cells overexpressing either ERAB/HADH II or betaAPP(V717G) alone. Generation of such toxic aldehydes was not observed in cells contransfected to overexpress Y168G/K172G-altered ERAB and betaAPP(V717G). We conclude that the generalized alcohol dehydrogenase activity of ERAB/HADH II is central to the cytotoxicity observed in an Abeta-rich environment.

Significant immunological obstacles are to be negotiated before xenotransplantation becomes a clinical reality. An initial rejection of transplanted vascularized xenograft is attributed to Galα1,3Galβ1,4GlcNAc-R (Galα1,3-Gal)–dependent and –independent mechanisms. Hitherto, no receptor molecule has been identified that could account for Galα1,3-Gal–independent rejection. In this study, we identify the tetraspanin CD82 as a receptor molecule for the Galα1,3-Gal–independent mechanism. We demonstrate that, in contrast to human undifferentiated myeloid cell lines, differentiated cell lines are capable of recognizing xenogeneic porcine aortic endothelial cells in a calcium-dependent manner. Transcriptome-wide analysis to identify the differentially expressed transcripts in these cells revealed that the most likely candidate of the Galα1,3-Gal–independent recognition moiety is the tetraspanin CD82. Abs to CD82 inhibited the calcium response and the subsequent activation invoked by xenogeneic encounter. Our data identify CD82 on innate immune cells as a major “xenogenicity sensor” and open new avenues of intervention to making xenotransplantation a clinical reality.

Background Recent evidence suggests that the effects of certain food additives may be synergistic or additive. Aspartame (ASP) and Monosodium Glutamate (MSG) are ubiquitous food additives with a common moiety: both contain acidic amino acids which can act as neurotransmitters, interacting with NMDA receptors concentrated in areas of the Central Nervous System regulating energy expenditure and conservation. MSG has been shown to promote a neuroendocrine dysfunction when large quantities are administered to mammals during the neonatal period. ASP is a low-calorie dipeptide sweetener found in a wide variety of diet beverages and foods. However, recent reports suggest that ASP may promote weight gain and hyperglycemia in a zebrafish nutritional model. Methods We investigated the effects of ASP, MSG or a combination of both on glucose and insulin homeostasis, weight change and adiposity, in C57BL/6 J mice chronically exposed to these food additives commencing in-utero, compared to an additive-free diet. Pearson correlation analysis was used to investigate the associations between body characteristics and variables in glucose and insulin homeostasis. Results ASP alone (50 mg/Kgbw/day) caused an increase in fasting blood glucose of 1.6-fold, together with reduced insulin sensitivity during an Insulin Tolerance Test (ITT) P Conclusions Aspartame exposure may promote hyperglycemia and insulin intolerance. MSG may interact with aspartame to further impair glucose homeostasis. This is the first study to ascertain the hyperglycemic effects of chronic exposure to a combination of these commonly consumed food additives; however these observations are limited to a C57BL/6 J mouse model. Caution should be applied in extrapolating these findings to other species.

The incidence of obesity and type 2 diabetes mellitus (T2DM) is increasing, and new experimental models are required to investigate the diverse aspects of these polygenic diseases, which are intimately linked in terms of aetiology. Feline T2DM has been shown to closely resemble human T2DM in terms of its clinical, pathological and physiological features. Our aim was to develop a feline model of diet-induced weight gain, adiposity and metabolic deregulation, and to examine correlates of weight and body fat change, insulin homeostasis, lipid profile, adipokines and clinical chemistry, in order to study associations which may shed light on the mechanism of diet-induced metabolic dysregulation. We used a combination of partially hydrogenated vegetable shortening and high-fructose corn syrup to generate a high-fat–high-fructose diet. The effects of this diet were compared with an isoenergetic standard chow, either in the presence or absence of 1·125 % dietary monosodium glutamate (MSG). Dual-energy X-ray absorptiometry body imaging and a glucose tolerance test were performed. The present results indicate that dietary MSG increased weight gain and adiposity, and reduced insulin sensitivity (P P P P

Background The prevalence of obesity and overweight is increasing globally. Frequently coexisting with under-nutrition in developing countries, obesity is a major contributor to chronic disease, and will become a serious healthcare burden especially in countries with a larger percentage of youthful population. 35% of the population of Saudi Arabia are under the age of 16, and adult dietary preferences are often established during early childhood years. Our objective was to examine the dietary habits in relation to body-mass-index (BMI) and waist circumference (W_C), together with exercise and sleep patterns in a cohort of male and female Saudi school children, in order to ascertain whether dietary patterns are associated with obesity phenotypes in this population. Methods 5033 boys and 4400 girls aged 10 to 19 years old participated in a designed Food Frequency Questionnaire. BMI and W_C measurements were obtained and correlated with dietary intake. Results The overall prevalence of overweight and obesity was 12.2% and 27.0% respectively, with boys having higher obesity rates than girls (P ≤ 0.001). W_C and BMI was positively correlated with sugar-sweetened carbonated beverage (SSCB) intake in boys only. The association between male BMI and SSCB consumption was significant in a multivariate regression model (P < 0.0001). SSCB intake was positively associated with poor dietary choices in both males and females. Fast food meal intake, savory snacks, iced desserts and total sugar consumption correlated with SSCB intake in both boys (r = 0.39, 0.13, 0.10 and 0.52 respectively, P < 0.001) and girls (r = 0.45, 0.23, 0.16 and 0.55 respectively, P < 0.001). Older children reported eating significantly less fruit and vegetables than younger children; and less eggs, fish and cereals. Conversely, consumption of SSCB and sugar-sweetened hot beverages were higher in older versus younger children (P < 0.001). BMI and W_C were negatively correlated with hours of night-time sleep and exercise in boys, but only with night time sleep in girls, who also showed the lowest frequency of exercise. Conclusions A higher intake of SSCB is associated with poor dietary choices. Male SSCB intake correlates with a higher W_C and BMI. Limiting exposure to SSCB could therefore have a large public health impact.

It has previously been shown that patients with nonalcoholic fatty liver disease (NAFLD) exhibit alterations in both hepatic and adipose tissue metabolism, and the dietary factors that contribute to the pathogenesis of NAFLD are likely to be multifactorial. Using C57BL/6J mice, we examined whether chronic exposure to low-dose dietary monosodium glutamate (MSG), high-fructose corn syrup (HFCS), or a combination of the two, vs. control would affect metabolism and hepatic and visceral fat gene expression in adult male progeny. A maternal diet containing 20% HFCS and/or dietary MSG (97.2 +/- 6.3 mg/kg body weight (bw), provided in the drinking water) was offered ad libitum from 3 weeks before mating, and continued throughout gestation and weaning until the progeny reached 32 weeks of age. Liver and abdominal fat gene expression was compared with control animals fed isocaloric standard chow under identical conditions. HFCS induced hepatic steatosis and increased the expression of genes involved in carbohydrate and lipid metabolism. Conversely, dietary MSG elevated serum free fatty acids (FFAs), triglycerides (TGs), high-density lipoprotein-cholesterol (HDL-C), and insulin, together with the expression of hepatic genes involved in lipid metabolism and bile synthesis. The HFCS+MSG combination elevated hepatic TGs, serum FFAs, and TG levels. In visceral white adipose tissue, both MSG and HFCS diets increased the expression of transcription factor Srebf2 and decreased expression of Ppargc1a, while downregulating the expression of mitochondrial respiratory chain components. MSG increased the expression of several genes implicated in adipocytes differentiation. We hypothesize that HFCS may promote hepatic steatosis, whereas dietary MSG induces dyslipidemia and markers of insulin resistance.

Aims Recent evidence suggests that intake of excessive dietary fat, particularly saturated fat and trans -hydrogenated oils ( trans -fatty acids: TFA) can impair learning and memory. Central obesity, which can be induced by neonatal injections of monosodium Glutamate (MSG), also impairs learning and memory. To further clarify the effects of dietary fat and MSG, we treated C57BL/6 J mice with either a TFA-enriched diet, dietary MSG, or a combination of both and examined serum lipid profile and spatial memory compared to mice fed standard chow. Spatial learning was assessed at 6, 16 and 32 weeks of age in a Morris Water Maze (MWM). The subjects were given four days of training to find a hidden platform and a fifth day of reversal learning, in which the platform was moved to a new location. Results The TFA + MSG combination caused a central adiposity that was accompanied by impairment in locating the hidden platform in the MWM. Females in the TFA + MSG group showed a greater impairment compared to the other diet groups, and also showed elevated levels of fasting serum LDL-C and T-CHOL:HDL-C ratio, together with the lowest levels of HDL-C. Similarly, males in the TFA + MSG diet group were less successful than control mice at locating the hidden platform and had the highest level of abdominal adiposity and elevated levels of fasting serum LDL-C. Conclusion Dietary trans -fat combined with MSG increased central adiposity, promoted dyslipidemia and impaired spatial learning.

The role of innate immune cells in the recognition and activation of xenogeneic endothe- lium has always been considered secondary to the initial insult of xenoreactive natural antibodies (XNA) and complement. It was argued, however, that innate immune cells are capable of recognizing and activating xenogeneic endothelium in the ab- sence XNA and complement. Here, we show that porcine aortic endothelial cells (PAECs) activate human neutrophils directly. This contact-depen- dent activation causes a transient calcium rise lead- ing to increased reactive oxygen metabolite (ROM) production. Neutrophil gene-expression profiling using an adenylate uridylate-rich element-based microarray revealed a dramatic change in the neutrophil gene profiles upon exposure to PAECs. The PAEC-dependent neutrophil tran- scriptional activity was further confirmed by real- time polymerase chain reaction, which revealed a rapid increase in the mRNA message of a num- ber of inflammatory cytokines. The activation of human neutrophils by PAECs was independent of galactose 1,3-galactose (Gal1,3-gal) struc- tures, as inclusion of saturating concentrations of anti-Gal1,3-gal l antibodies had no signifi- cant effect. Furthermore, this activation was in- hibited in the presence of the calcium chelator 1,2-bis(O-aminophenyl-ethane-ethane)-N,N,N,N- tetraacetic acid-acetoxymethyl ester and the ROM inhibitor diphelylene iodonium. Our data illustrate the direct activation of innate immune cells by PAECs in the absence of XNA and complement and suggest alternative recognition sites between PAECs and hu- man innate immune cells. J. Leukoc. Biol. 78: 51-61; 2005.

The accumulation of advanced glycation end products (AGEs) in the tissue and serum of subjects with diabetes has been linked to the pathogenesis of vascular complications. Because diabetes may be also complicated by increased susceptibility to recurrent infection, we investigated the effects of AGEs on human neutrophils, because their burst of activity immediately upon engagement of pathogens or other inflammatory triggers is critical to host response. We demonstrate the presence of receptor for advanced glycation end products (RAGE) at the message and protein levels. We also demonstrate that AGE albumin (but not control albumin) binds with high affinity to human neutrophils (K(d) of 3.7 +/- 0.4 nM). The binding was blocked almost completely by excess soluble RAGE, anti-RAGE antibodies, or antibodies to CML-modified albumin. AGE albumin induced a dose-dependent increase in intracellular-free calcium as well as actin polymerization. Further, AGE albumin inhibited transendothelial migration and Staphylococcus aureus-induced but not fMLP-induced production of reactive oxygen metabolite. Moreover, although AGE albumin enhanced neutrophil phagocytosis of S. aureus, it inhibited bacterial killing. We conclude that functional RAGE is present on the plasma membrane of human neutrophils and is linked to Ca(2)(+) and actin polymerization, and engagement of RAGE impairs neutrophil functions.

Amyloid-beta is a neurotoxic peptide which is implicated in the pathogenesis of Alzheimer's disease. It binds an intracellular polypeptide known as ERAB, thought to be a hydroxysteroid dehydrogenase enzyme, which is expressed in normal tissues, but is overexpressed in neurons affected in Alzheimer's disease. ERAB immunoprecipitates with amyloid-beta, and when cell cultures are exposed to amyloid-beta, ERAB inside the cell is rapidly redistributed to the plasma membrane. The toxic effect of amyloid-beta on these cells is prevented by blocking ERAB and is enhanced by overexpression of ERAB. By interacting with intracellular amyloid-beta, ERAB may therefore contribute to the neuronal dysfunction associated with Alzheimer's disease.

Background The human diet has altered markedly during the past four decades, with the introduction of Trans hydrogenated fat, which extended the shelf-life of dietary oils and promoted a dramatic increase in elaidic acid (Trans-18.1) consumption. Food additives such as monosodium glutamate (MSG) and aspartame (ASP) were introduced to increase food palatability and reduce caloric intake. Nutrigenomics studies in small-animal models are an established platform for analyzing the interactions between various macro- and micronutrients. We therefore investigated the effects of changes in hepatic and adipose tissue gene expression induced by the food additives ASP, MSG or a combination of both additives in C57Bl/6 J mice fed a Trans fat-enriched diet. Methods Hepatic and adipose tissue gene expression profiles, together with body characteristics, glucose parameters, serum hormone and lipid profiles were examined in C57Bl/6 J mice consuming one of the following four dietary regimens, commencing in utero via the mother’s diet: [A] Trans fat (TFA) diet; [B] MSG + TFA diet; [C] ASP + TFA diet; [D] ASP + MSG + TFA diet. Results Whilst dietary MSG significantly increased hepatic triglyceride and serum leptin levels in TFA-fed mice, the combination of ASP + MSG promoted the highest increase in visceral adipose tissue deposition, serum free fatty acids, fasting blood glucose, HOMA-IR, total cholesterol and TNFα levels. Microarray analysis of significant differentially expressed genes (DEGs) showed a reduction in hepatic and adipose tissue PPARGC1a expression concomitant with changes in PPARGC1a-related functional networks including PPARα, δ and γ. We identified 73 DEGs common to both adipose and liver which were upregulated by ASP + MSG in Trans fat-fed mice; and an additional 51 common DEGs which were downregulated. Conclusion The combination of ASP and MSG may significantly alter adiposity, glucose homeostasis, hepatic and adipose tissue gene expression in TFA-fed C57Bl/6 J mice.

Sir, I read with considerable interest the epidemiological study by Insawang et al., which demonstrates an association between monosodium glutamate (MSG) intake and the prevalence of the Metabolic Syndrome in a rural Thai population [1]. It is important to point out that Insawang et al. did not claim that MSG causes the Metabolic Syndrome, they did however concluded that “elevated dietary MSG consumption is significantly associated with having the Metabolic Syndrome and being overweight in a Thai rural population”. The present commentary by Dr Rogers [2] concerning the research by Insawang et al. stresses throughout that there is no supporting evidence for a direct causal relationship between MSG intake and the prevalence of Metabolic Syndrome and overweight. The relevance of this oft-repeated statement is questionable since Insawang et al. never proposed a direct causal relationship between MSG intake and the Metabolic Syndrome. Although the authors of this epidemiological study are under no obligation to provide evidence for a causal relationship, a number of issues were raised which make interesting points for discussion. One concern broached by Dr Rogers was that the authors failed to mention in their Discussion a previous publication regarding the Jiangsu Nutritional Study of 1227 Chinese adults [3], which did not show an association between MSG and obesity or weight gain over 5 years. In point of fact, the

Digital fluorescence calcium imaging was used to investigate and identify the primary biological responses of human neutrophils to monomeric immunoglobulin E (IgE). Treatment of neutrophils with IgE caused a transient rise in the level of intracellular calcium that was inhibited by pertussis toxin. The calcium rise was due mainly to release from an intracellular membrane-enclosed store that is also sensitive to the chemotactic peptide formyl-Met-Leu-Phe. The IgE-induced calcium transient was independent of Fcγ receptors and of Fc receptor ligation. Our data suggest that the mere binding of IgE to neutrophils is sufficient to evoke a biological response without the need for IgE/receptor cross-linking.

Background A paucity of information on biological sex-specific differences in cardiac gene expression in response to diet has prompted this present nutrigenomics investigation. Sexual dimorphism exists in the physiological and transcriptional response to diet, particularly in response to high-fat feeding. Consumption of Trans-fatty acids (TFA) has been linked to substantially increased risk of heart disease, in which sexual dimorphism is apparent, with males suffering a higher disease rate. Impairment of the cardiovascular system has been noted in animals exposed to Monosodium Glutamate (MSG) during the neonatal period, and sexual dimorphism in the growth axis of MSG-treated animals has previously been noted. Processed foods may contain both TFA and MSG. Methods We examined physiological differences and changes in gene expression in response to TFA and/or MSG consumption compared to a control diet, in male and female C57BL/6J mice. Results Heart and % body weight increases were greater in TFA-fed mice, who also exhibited dyslipidemia (P < 0.05). Hearts from MSG-fed females weighed less than males (P < 0.05). 2-factor ANOVA indicated that the TFA diet induced over twice as many cardiac differentially expressed genes (DEGs) in males compared to females (P < 0.001); and 4 times as many male DEGs were downregulated including Gata4, Mef2d and Srebf2. Enrichment of functional Gene Ontology (GO) categories were related to transcription, phosphorylation and anatomic structure (P < 0.01). A number of genes were upregulated in males and downregulated in females, including pro-apoptotic histone deacetylase-2 (HDAC2). Sexual dimorphism was also observed in cardiac transcription from MSG-fed animals, with both sexes upregulating approximately 100 DEGs exhibiting sex-specific differences in GO categories. A comparison of cardiac gene expression between all diet combinations together identified a subset of 111 DEGs significant only in males, 64 DEGs significant in females only, and 74 transcripts identified as differentially expressed in response to dietary manipulation in both sexes. Conclusion Our model identified major changes in the cardiac transcriptional profile of TFA and/or MSG-fed mice compared to controls, which was reflected by significant differences in the physiological profile within the 4 diet groups. Identification of sexual dimorphism in cardiac transcription may provide the basis for sex-specific medicine in the future.

ADP-ribosylation factor (ARF), a small GTPase required for vesicle formation, has been identified as an activator of phospholipase D (PLD), thus implying that PLD is localized at intracellular organelles. HL60 cells were prelabelled with [14C]acetate for 72 h and, after disruption, fractionated on a linear sucrose gradient. ARF1-regulated PLD activity in each fraction was assessed by measurement of phosphatidylethanol production. Two peaks of activity were identified, coincident with markers for Golgi/endoplasmic reticulum/granules (endomembranes) and plasma membrane respectively. Analysis of the fractions using exogenous phosphatidylcholine as substrate confirmed the presence of ARF1-dependent PLD activity in endomembranes and plasma membrane, and also identified an additional activity in the cytosol. In formyl-Met-Leu-Phe-stimulated cells, PLD activity as assessed by phosphatidylethanol formation was also associated with both the plasma membrane and endomembranes. Since ARF1-regulated PLD activity requires phosphatidylinositol 4,5-bisphosphate (PIP2), the distributions of inositol lipids and the kinases responsible for lipid phosphorylation were examined. PIP2 was highly enriched at the plasma membrane, whereas phosphatidylinositol (PI) and phosphatidylinositol 4-phosphate (PI4P), the precursors for PIP2 synthesis, were found predominantly at endomembranes. The distribution of PI 4-kinase and PI4P 5-kinase activities confirmed the plasma membrane as the major site of PIP2 production. However, endomembranes possessed substantial PI 4-kinase activity and some PI4P 5-kinase activity, illustrating the potential for PIP2 synthesis. It is concluded that: (1) ARF1-regulated PLD activity is localized at endomembranes and the plasma membrane, (2) PIP2 is available at both membrane compartments to function as a cofactor for ARF-regulated PLD, and (3) in intact cells, formyl-Met-Leu-Phe stimulates PLD activity at endomembranes as well as plasma membrane.

Lipid metabolic disorder is a critical risk factor for metabolic syndrome, triggering debilitating diseases like obesity and diabetes. Both obesity and diabetes are the epicenter of important medical issues, representing a major international public health threat. Accumulation of fat in adipose tissue, muscles and liver and/or the defects in their ability to metabolize fatty acids, results in insulin resistance. This triggers an early pathogenesis of type 2 diabetes (T2D). In mammals, lipid metabolism involves several organs, including the brain, adipose tissue, muscles, liver, and gut. These organs are part of complex homeostatic system and communicate through hormones, neurons and metabolites. Our study dissects the importance of mammalian Krüppel-like factors in over all energy homeostasis. Factors controlling energy metabolism are conserved between mammals and Caenorhabditis elegans providing a new and powerful strategy to delineate the molecular pathways that lead to metabolic disorder. The C. elegans intestine is our model system where genetics, molecular biology, and cell biology are used to identify and understand genes required in fat metabolism. Thus far, we have found an important role of C. elegans KLF in FA biosynthesis, mitochondrial proliferation, lipid secretion, and β-oxidation. The mechanism by which KLF controls these events in lipid metabolism is unknown. We have recently observed that C. elegans KLF-3 selectively acts on insulin components to regulate insulin pathway activity. There are many factors that control energy homeostasis and defects in this control system are implicated in the pathogenesis of human obesity and diabetes. In this review we are discussing a role of KLF in human metabolic regulation.