Legionella pneumophila usurps host cell lipids for vacuole expansion and bacterial growth.

Vacuolar pathogens reside in membrane-bound compartments within host cells. Maintaining the integrity of this compartment is paramount to bacterial survival and replication as it protects against certain host surveillance mechanisms that function to eradicate invading pathogens. Preserving this compartment during bacterial replication requires expansion of the vacuole membrane to accommodate the increasing number of bacteria, and yet, how this is accomplished remains largely unknown. Here, we show that the vacuolar pathogen Legionella pneumophila exploits multiple sources of host cell fatty acids, including inducing host cell fatty acid scavenging pathways, in order to promote expansion of the replication vacuole and bacteria growth. Conversely, when exogenous lipids are limited, the decrease in host lipid availability restricts expansion of the replication vacuole membrane, resulting in a higher density of bacteria within the vacuole. Modifying the architecture of the vacuole prioritizes bacterial growth by allowing the greatest number of bacteria to remain protected by the vacuole membrane despite limited resources for its expansion. However, this trade-off is not without risk, as it can lead to vacuole destabilization, which is detrimental to the pathogen. However, when host lipid resources become extremely scarce, for example by inhibiting host lipid scavenging, de novo biosynthetic pathways, and/or diverting host fatty acids to storage compartments, bacterial replication becomes severely impaired, indicating that host cell fatty acid availability also directly regulates L. pneumophila growth. Collectively, these data demonstrate dual roles for host cell fatty acids in replication vacuole expansion and bacterial proliferation, revealing the central functions for these molecules and their metabolic pathways in L. pneumophila pathogenesis.

Decreased sarcoplasmic reticulum phospholipids in human skeletal muscle are associated with metabolic syndrome.

Metabolic syndrome affects more than one in three adults and is associated with increased risk of diabetes, cardiovascular disease, and all-cause mortality. Muscle insulin resistance is a major contributor to the development of the metabolic syndrome. Studies in mice have linked skeletal muscle sarcoplasmic reticulum (SR) phospholipid composition to sarcoplasmic/endoplasmic reticulum Ca2+-ATPase activity and insulin sensitivity. To determine if the presence of metabolic syndrome alters specific phosphatidylcholine (PC) and phosphatidylethanolamine (PE) species in human SR, we compared SR phospholipid composition in skeletal muscle from sedentary subjects with metabolic syndrome and sedentary control subjects without metabolic syndrome. Both total PC and total PE were significantly decreased in skeletal muscle SR of sedentary metabolic syndrome patients compared with sedentary controls, particularly in female participants, but there was no difference in the PC:PE ratio between groups. Total SR PC levels, but not total SR PE levels or PC:PE ratio, were significantly negatively correlated with BMI, waist circumference, total fat, visceral adipose tissue, triglycerides, fasting insulin, and homeostatic model assessment for insulin resistance. These findings are consistent with the existence of a relationship between skeletal muscle SR PC content and insulin resistance in humans.

Glucose-mediated de novo lipogenesis in photoreceptors drives early diabetic retinopathy.

Diabetic retinopathy (DR) is an increasingly frequent cause of blindness across populations; however, the events that initiate pathophysiology of DR remain elusive. Strong preclinical and clinical evidence suggests that abnormalities in retinal lipid metabolism caused by diabetes may account for the origin of this disease. A major arm of lipid metabolism, de novo biosynthesis, is driven by elevation in available glucose, a common thread binding all forms of vision loss in diabetes. Therefore, we hypothesized that aberrant retinal lipid biogenesis is an important promoter of early DR. In murine models, we observed elevations of diabetes-associated retinal de novo lipogenesis ∼70% over control levels. This shift was primarily because of activation of fatty acid synthase (FAS), a rate-limiting enzyme in the biogenic pathway. Activation of FAS was driven by canonical glucose-mediated disinhibition of acetyl-CoA carboxylase, a major upstream regulatory enzyme. Mutant mice expressing gain-of-function FAS demonstrated increased vulnerability to DR, whereas those with FAS deletion in rod photoreceptors maintained preserved visual responses upon induction of diabetes. Excess retinal de novo lipogenesis-either because of diabetes or because of FAS gain of function-was associated with modestly increased levels of palmitate-containing phosphatidylcholine species in synaptic membranes, a finding with as yet uncertain significance. These findings implicate glucose-dependent increases in photoreceptor de novo lipogenesis in the early pathogenesis of DR, although the mechanism of deleterious action of this pathway remains unclear.

Endothelial Palmitoylation Cycling Coordinates Vessel Remodeling in Peripheral Artery Disease.

RATIONALE: Peripheral artery disease, common in metabolic syndrome and diabetes mellitus, responds poorly to medical interventions and is characterized by chronic vessel immaturity leading to lower extremity amputations. OBJECTIVE: To define the role of reversible palmitoylation at the endothelium in the maintenance of vascular maturity. METHODS AND RESULTS: Endothelial knockout of the depalmitoylation enzyme APT-1 (acyl-protein thioesterase 1) in mice impaired recovery from chronic hindlimb ischemia, a model of peripheral artery disease. Endothelial APT-1 deficiency decreased fibronectin processing, disrupted adherens junctions, and inhibited in vitro lumen formation. In an unbiased palmitoylation proteomic screen of endothelial cells from genetically modified mice, R-Ras, known to promote vessel maturation, was preferentially affected by APT-1 deficiency. R-Ras was validated as an APT-1 substrate, and click chemistry analyses demonstrated increased R-Ras palmitoylation in cells with APT-1 deficiency. APT-1 enzyme activity was decreased in endothelial cells from db/db mice. Hyperglycemia decreased APT-1 activity in human umbilical vein endothelial cells, due, in part, to altered acetylation of the APT-1 protein. Click chemistry analyses demonstrated increased R-Ras palmitoylation in the setting of hyperglycemia. Altered R-Ras trafficking, increased R-Ras palmitoylation, and fibronectin retention were found in diabetes mellitus models. Loss of R-Ras depalmitoylation caused by APT-1 deficiency constrained R-Ras membrane trafficking, as shown by total internal reflection fluorescence imaging. To rescue cellular phenotypes, we generated an R-Ras molecule with an inserted hydrophilic domain to circumvent membrane rigidity caused by defective palmitoylation turnover. This modification corrected R-Ras membrane trafficking, restored fibronectin processing, increased adherens junctions, and rescued defective lumen formation induced by APT-1 deficiency. CONCLUSIONS: These results suggest that endothelial depalmitoylation is regulated by the metabolic milieu and controls plasma membrane partitioning to maintain vascular homeostasis.

Endothelial ether lipids link the vasculature to blood pressure, behavior, and neurodegeneration.

Vascular disease contributes to neurodegeneration, which is associated with decreased blood pressure in older humans. Plasmalogens, ether phospholipids produced by peroxisomes, are decreased in Alzheimer's disease, Parkinson's disease, and other neurodegenerative disorders. However, the mechanistic links between ether phospholipids, blood pressure, and neurodegeneration are not fully understood. Here, we show that endothelium-derived ether phospholipids affect blood pressure, behavior, and neurodegeneration in mice. In young adult mice, inducible endothelial-specific disruption of PexRAP, a peroxisomal enzyme required for ether lipid synthesis, unexpectedly decreased circulating plasmalogens. PexRAP endothelial knockout (PEKO) mice responded normally to hindlimb ischemia but had lower blood pressure and increased plasma renin activity. In PEKO as compared with control mice, tyrosine hydroxylase was decreased in the locus coeruleus, which maintains blood pressure and arousal. PEKO mice moved less, slept more, and had impaired attention to and recall of environmental events as well as mild spatial memory deficits. In PEKO hippocampus, gliosis was increased, and a plasmalogen associated with memory was decreased. Despite lower blood pressure, PEKO mice had generally normal homotopic functional connectivity by optical neuroimaging of the cerebral cortex. Decreased glycogen synthase kinase-3 phosphorylation, a marker of neurodegeneration, was detected in PEKO cerebral cortex. In a co-culture system, PexRAP knockdown in brain endothelial cells decreased glycogen synthase kinase-3 phosphorylation in co-cultured astrocytes that was rescued by incubation with the ether lipid alkylglycerol. Taken together, our findings suggest that endothelium-derived ether lipids mediate several biological processes and may also confer neuroprotection in mice.

Dynamic analysis of the epidermal growth factor (EGF) receptor-ErbB2-ErbB3 protein network by luciferase fragment complementation imaging.

ErbB3 is a member of the ErbB family of receptor tyrosine kinases. It is unique because it is the only member of the family whose kinase domain is defective. As a result, it is obliged to form heterodimers with other ErbB receptors to signal. In this study, we characterized the interaction of ErbB3 with the EGF receptor and ErbB2 and assessed the effects of Food and Drug Administration-approved therapeutic agents on these interactions. Our findings support the concept that ErbB3 exists in preformed clusters that can be dissociated by NRG-1ß and that it interacts with other ErbB receptors in a distinctly hierarchical fashion. Our study also shows that all pairings of the EGF receptor, ErbB2, and ErbB3 form ligand-independent dimers/oligomers. The small-molecule tyrosine kinase inhibitors erlotinib and lapatinib differentially enhance the dimerization of the various ErbB receptor pairings, with the EGFR/ErbB3 heterodimer being particularly sensitive to the effects of erlotinib. The data suggest that the physiological effects of these drugs may involve not only inhibition of tyrosine kinase activity but also a dynamic restructuring of the entire network of receptors.

Fatty acid synthesis suppresses dietary polyunsaturated fatty acid use.

Dietary polyunsaturated fatty acids (PUFA) are increasingly recognized for their health benefits, whereas a high production of endogenous fatty acids - a process called de novo lipogenesis (DNL) - is closely linked to metabolic diseases. Determinants of PUFA incorporation into complex lipids are insufficiently understood and may influence the onset and progression of metabolic diseases. Here we show that fatty acid synthase (FASN), the key enzyme of DNL, critically determines the use of dietary PUFA in mice and humans. Moreover, the combination of FASN inhibition and PUFA-supplementation decreases liver triacylglycerols (TAG) in mice fed with high-fat diet. Mechanistically, FASN inhibition causes higher PUFA uptake via the lysophosphatidylcholine transporter MFSD2A, and a diacylglycerol O-acyltransferase 2 (DGAT2)-dependent incorporation of PUFA into TAG. Overall, the outcome of PUFA supplementation may depend on the degree of endogenous DNL and combining PUFA supplementation and FASN inhibition might be a promising approach to target metabolic disease.

Hepatic palmitoyl-proteomes and acyl-protein thioesterase protein proximity networks link lipid modification and mitochondria.

Acyl-protein thioesterases 1 and 2 (APT1 and APT2) reverse S-acylation, a potential regulator of systemic glucose metabolism in mammals. Palmitoylation proteomics in liver-specific knockout mice shows that APT1 predominates over APT2, primarily depalmitoylating mitochondrial proteins, including proteins linked to glutamine metabolism. miniTurbo-facilitated determination of the protein-protein proximity network of APT1 and APT2 in HepG2 cells reveals APT proximity networks encompassing mitochondrial proteins including the major translocases Tomm20 and Timm44. APT1 also interacts with Slc1a5 (ASCT2), the only glutamine transporter known to localize to mitochondria. High-fat-diet-fed male mice with dual (but not single) hepatic deletion of APT1 and APT2 have insulin resistance, fasting hyperglycemia, increased glutamine-driven gluconeogenesis, and decreased liver mass. These data suggest that APT1 and APT2 regulation of hepatic glucose metabolism and insulin signaling is functionally redundant. Identification of substrates and protein-protein proximity networks for APT1 and APT2 establishes a framework for defining mechanisms underlying metabolic disease.

Palmitoylation couples insulin hypersecretion with ß cell failure in diabetes.

Hyperinsulinemia often precedes type 2 diabetes. Palmitoylation, implicated in exocytosis, is reversed by acyl-protein thioesterase 1 (APT1). APT1 biology was altered in pancreatic islets from humans with type 2 diabetes, and APT1 knockdown in nondiabetic islets caused insulin hypersecretion. APT1 knockout mice had islet autonomous increased glucose-stimulated insulin secretion that was associated with prolonged insulin granule fusion. Using palmitoylation proteomics, we identified Scamp1 as an APT1 substrate that localized to insulin secretory granules. Scamp1 knockdown caused insulin hypersecretion. Expression of a mutated Scamp1 incapable of being palmitoylated in APT1-deficient cells rescued insulin hypersecretion and nutrient-induced apoptosis. High-fat-fed islet-specific APT1-knockout mice and global APT1-deficient db/db mice showed increased ß cell failure. These findings suggest that APT1 is regulated in human islets and that APT1 deficiency causes insulin hypersecretion leading to ß cell failure, modeling the evolution of some forms of human type 2 diabetes.

The tethering arm of the EGF receptor is required for negative cooperativity and signal transduction.

The EGF receptor is a classical receptor-tyrosine kinase. In the absence of ligand, the receptor adopts a closed conformation in which the dimerization arm of subdomain II interacts with the tethering arm in subdomain IV. Following the binding of EGF, the receptor opens to form a symmetric, back-to-back dimer. Although it is clear that the dimerization arm of subdomain II is central to the formation of receptor dimers, the role of the tethering arm of subdomain IV (residues 561-585) in this configuration is not known. Here we use (125)I-EGF binding studies to assess the functional role of the tethering arm in the EGF receptor dimer. Mutation of the three major residues that contribute to tethering (D563A,H566A,K585A-EGF receptor) did not significantly alter either the ligand binding properties or the signaling properties of the EGF receptor. By contrast, breaking the Cys(558)-Cys(567) disulfide bond through double alanine replacements or deleting the loop entirely led to a decrease in the negative cooperativity in EGF binding and was associated with small changes in downstream signaling. Deletion of the Cys(571)-Cys(593) disulfide bond abrogated cooperativity, resulting in a high affinity receptor and increased sensitivity of downstream signaling pathways to EGF. Releasing the Cys(571)-Cys(593) disulfide bond resulted in extreme negative cooperativity, ligand-independent kinase activity, and impaired downstream signaling. These data demonstrate that the tethering arm plays an important role in supporting cooperativity in ligand binding. Because cooperativity implies subunit-subunit interactions, these results also suggest that the tethering arm contributes to intersubunit interactions within the EGF receptor dimer.

The membrane-proximal intracellular domain of the epidermal growth factor receptor underlies negative cooperativity in ligand binding.

The binding of EGF induces dimerization of its receptor, leading to the stimulation of its intracellular tyrosine kinase activity. Kinase activation occurs within the context of an asymmetric dimer in which one kinase domain serves as the activator for the other kinase domain but is not itself activated. How ligand binding is related to the formation and dynamics of this asymmetric dimer is not known. The binding of EGF to its receptor is negatively cooperative--that is, EGF binds with lower affinity to the second site on the dimer than to the first site on the dimer. In this study, we analyzed the binding of (125)I-EGF to a series of EGF receptor mutants in the intracellular juxtamembrane domain and demonstrate that the most membrane-proximal portion of this region plays a significant role in the genesis of negative cooperativity in the EGF receptor. The data are consistent with a model in which the binding of EGF to the first site on the dimer induces the formation of one asymmetric kinase dimer. The binding of EGF to the second site is required to disrupt the initial asymmetric dimer and allow the formation of the reciprocal asymmetric dimer. Thus, some of the energy of binding to the second site is used to reorient the first asymmetric dimer, leading to a lower binding affinity and the observed negative cooperativity.

Functional and epigenetic phenotypes of humans and mice with DNMT3A Overgrowth Syndrome.

Germline pathogenic variants in DNMT3A were recently described in patients with overgrowth, obesity, behavioral, and learning difficulties (DNMT3A Overgrowth Syndrome/DOS). Somatic mutations in the DNMT3A gene are also the most common cause of clonal hematopoiesis, and can initiate acute myeloid leukemia (AML). Using whole genome bisulfite sequencing, we studied DNA methylation in peripheral blood cells of 11 DOS patients and found a focal, canonical hypomethylation phenotype, which is most severe with the dominant negative DNMT3AR882H mutation. A germline mouse model expressing the homologous Dnmt3aR878H mutation phenocopies most aspects of the human DOS syndrome, including the methylation phenotype and an increased incidence of spontaneous hematopoietic malignancies, suggesting that all aspects of this syndrome are caused by this mutation.

Dynamic and electrokinetic behavior of erythrocyte membrane in diabetes mellitus and diabetic cardiovascular disease.

The dynamic and electrokinetic properties of erythrocyte membrane are explored as significant indices involved in the association of diabetes and diabetic cardiovascular disease. Lipid peroxidation studies reveal malondialdehyde concentration to reach a maximum in diabetic cardiovascular patients. Lower fluidity of erythrocyte membrane implies declined ability of erythrocyte to deform in pathogenic state, which is supported by decreased osmotic resistance. Membrane protein profile modification detected by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) indicates a significant reduction in the quantity of ankyrin protein band 2.1 in diabetic subjects. In addition the reduction in an immunoreactive band against polyclonal anti-ankyrin antibody during Western blot analysis confirms the modification of ankyrin protein in diseased erythrocyte (reported for the first time). The electrokinetic behavior of erythrocyte membrane is monitored by laser Doppler velocimetry mode of the Nano-ZS. Changes in zeta potential values of the red blood cell membrane are consistent with decreased membrane fluidity in diseased erythrocytes (reported for the first time). Membrane potential values of control, diabetic and diabetic cardiovascular erythrocytes are -37.24+/-1.5 mV, -28.44+/-1.34 mV, and -22.21+/-1.21 mV respectively indicating a gradual lowering of zeta potential when erythrocyte membrane undergoes progressive changes - from simple agglomeration to fluid gel formation - and finally to a rigid gel.

Circulating serum fatty acid synthase is elevated in patients with diabetes and carotid artery stenosis and is LDL-associated.

BACKGROUND AND AIMS: Diabetes is an independent risk factor for carotid artery stenosis (CAS). Fatty acid synthase (FAS), an essential de novo lipogenesis enzyme, has increased activity in the setting of diabetes that leads to altered lipid metabolism. Circulating FAS (cFAS) was recently observed in the blood of patients with hyperinsulinemia and cancer. We thought to evaluate the origin of cFAS and its role in diabetes-associated CAS. METHODS: Patients with diabetes and no diabetes, undergoing carotid endarterectomy (CEA) for CAS, were prospectively enrolled for collection of plaque and fasting serum. FPLC was used to purify lipoprotein fractions, and ELISA was used to quantify cFAS content and activity. Immunoprecipitation (IP) was used to evaluate the affinity of cFAS to LDL-ApoB. RESULTS: Patients with CAS had higher cFAS activity (p < 0.01), and patients with diabetes had higher cFAS activity than patients with no diabetes (p < 0.05). cFAS activity correlated with serum glucose (p = 0.03, r2 = 0.35), and cFAS content trended with plaque FAS content (p = 0.06, r2 = 0.69). cFAS was predominantly in LDL cholesterol fractions of patients with CAS (p < 0.001), and IP of cFAS demonstrated pulldown of ApoB. Similar to patients with diabetes, db/db mice had highest levels of serum cFAS (p < 0.01), and fasL-/- (tissue-specific liver knockdown of FAS) mice had the lowest levels of cFAS (p < 0.001). CONCLUSIONS: Serum cFAS is higher in patients with diabetes and CAS, appears to originate from the liver, and is LDL cholesterol associated. We postulate that LDL may be serving as a carrier for cFAS that contributes to atheroprogression in carotid arteries of patients with diabetes.

Retinal de novo lipogenesis coordinates neurotrophic signaling to maintain vision.

Membrane lipid composition is central to the highly specialized functions of neurological tissues. In the retina, abnormal lipid metabolism causes severe forms of blindness, often through poorly understood neuronal cell death. Here, we demonstrate that deleting the de novo lipogenic enzyme fatty acid synthase (FAS) from the neural retina, but not the vascular retina, results in progressive neurodegeneration and blindness with a temporal pattern resembling rodent models of retinitis pigmentosa. Blindness was not rescued by protection from light-evoked activity; by eating a diet enriched in palmitate, the product of the FAS reaction; or by treatment with the PPARα agonist fenofibrate. Vision loss was due to aberrant synaptic structure, blunted responsiveness to glial-derived neurotrophic factor and ciliary neurotrophic factor, and eventual apoptotic cell loss. This progressive neurodegeneration was associated with decreased membrane cholesterol content, as well as loss of discrete n-3 polyunsaturated fatty acid- and saturated fatty acid-containing phospholipid species within specialized membrane microdomains. Neurotrophic signaling was restored by exogenous cholesterol delivery. These findings implicate de novo lipogenesis in neurotrophin-dependent cell survival by maintaining retinal membrane configuration and lipid composition, and they suggest that ongoing lipogenesis may be required to prevent cell death in many forms of retinopathy.

Enhanced oxygen releasing capacity and oxidative stress in diabetes mellitus and diabetes mellitus-associated cardiovascular disease: a comparative study.

BACKGROUND: Macrovascular disease, especially cardiovascular accounts for most of the mortality in patients with type 2 diabetes mellitus. We compared oxidative stress, thermal stability and oxygen releasing capacity of hemoglobin in patients with type 2 diabetes mellitus and diabetes-associated cardiovascular disease. METHODS: The study was performed on 38 control subjects, 31 diabetics, 36 diabetics with cardiovascular complications, and 33 non-diabetic cardiovascular subjects. RESULTS: Enhanced oxidative stress was shown by an increased protein carbonyl content observed both in plasma and in hemolysate of the diseased samples in type 2 diabetes and diabetes-associated cardiovascular diseased patients. Altered levels of cytoprotective enzymes were shown by decreased catalase activity, increased glutathione reductase activity and unaltered superoxide dismutase activity. Peroxidative activity of diseased hemoglobin was much higher compared to healthy controls indicating possible structural changes in pathologic hemoglobin molecule as a result of disease induced oxidative stress. This result is in good agreement with the observation that thermal stability of pathologic hemoglobin was also found to be less compared to control subjects. Enhanced oxygen releasing capacity of tetrameric oxyhemoglobin was monitored in presence of the drug Trifluoperazine in pathologic red blood cells, maximum increment being noticed in diabetic cardiovascular diseased subjects. CONCLUSION: Hyperglycemia-induced oxidative stress was responsible to affect the thermal stability and oxygen releasing capacity of hemoglobin and the effect is more pronounced in diabetes-associated cardiovascular disease.

Peroxisomal lipid synthesis regulates inflammation by sustaining neutrophil membrane phospholipid composition and viability.

Fatty acid synthase (FAS) is altered in metabolic disorders and cancer. Conventional FAS null mice die in utero, so effects of whole-body inhibition of lipogenesis following development are unknown. Inducible global knockout of FAS (iFASKO) in mice was lethal due to a disrupted intestinal barrier and leukopenia. Conditional loss of FAS was associated with the selective suppression of granulopoiesis without disrupting granulocytic differentiation. Transplantation of iFASKO bone marrow into wild-type mice followed by Cre induction resulted in selective neutrophil depletion, but not death. Impaired lipogenesis increased ER stress and apoptosis in neutrophils by preferentially decreasing peroxisome-derived membrane phospholipids containing ether bonds. Inducible global knockout of PexRAP, a peroxisomal enzyme required for ether lipid synthesis, also produced neutropenia. FAS knockdown in neutrophil-like HL-60 cells caused cell loss that was partially rescued by ether lipids. Inhibiting ether lipid synthesis selectively constrains neutrophil development, revealing an unrecognized pathway in immunometabolism.

Co-existence of risk and protective haplotypes of Calpain 10 gene to type 2 diabetes in the eastern Indian population.

Linkage and association studies have detected a role for Calpain-10 (CAPN10) polymorphisms in susceptibility to T2DM in many populations. This study aimed to evaluate possible associations between three SNPs in the CAPN10 (UCSNPs -43, -19 and -63) gene and T2DM in the east Indian population. The distribution of genotype frequency of UCSNP-63 varied significantly between T2DM patients and controls under a dominant model. The uncommon (T) allele (OR = 3.74, 95% CI: 1.44-9.7) of the UCSNP-63 and haplotype 112 (OR = 3.4, 95% CI: 1.17-9.9) were associated with increased risk of T2DM. On the contrary, the most common haplotype 121 (OR = 0.70 95% CI: 0.50-0.99) was associated with a reduced risk for T2DM. In our population a novel 111/112-haplotype combination created by the CAPN10 UCSNP-43, -19 and -63 was associated with risk of T2DM. Haplotypes 112 and 121 with opposite genetic influences also co-exist in our population.