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Metabolic diseases are closely linked to aberrant synthesis of endogenous fatty acids in the liver, called de novo lipogenesis (DNL), which is mediated by the enzyme fatty acid synthase (FASN). The composition of complex lipids consists of saturated or monosaturated fatty acids, which can be endogenously produced, and polyunsaturated fatty acids (PUFA), which are strictly dietary. Compositional differences between individuals are insufficiently understood and may influence the onset and progression of metabolic and cardiovascular diseases. Here we show that DNL critically determines the use of dietary PUFA. A patient with a hypofunctional heterozygous de novo Arg2177Cys variant in FASN exhibited an elevated composition of PUFA, which was phenocopied by pharmacological inhibition of FASN with TVB-2640 in patients with nonalcoholic steatohepatitis (NASH). In mice, the incorporation rate of supplemented omega-3 PUFA during an obesogenic diet was increased by genetic or pharmacologic reduction of DNL. Mechanistically, we show that the FASN variant exhibited a cysteine-dependent, non-enzymatic acetylation of FASN, which resulted in hyperubiquitinylation and decreased protein stability. The study reveals that PUFA storage is an active, enzymatic process controlled by FASN, DGAT2 and MFSD2A and that combining FASN inhibition and PUFA supplementation exerts additive beneficial metabolic effects. These findings provide evidence that the success of PUFA supplementation may depend on the rate of endogenous DNL and that combined PUFA supplementation and FASN inhibition may be a promising approach in metabolic disease.

Metabolic diseases such as obesity, type 2 diabetes mellitus and non-alcoholic steatohepatitis (NASH) are closely linked to aberrant synthesis of endogenous fatty acids in the liver, called de novo lipogenesis, which is mediated by the enzyme fatty acid synthase (FASN). Endogenously synthesized fatty acids are either saturated or monounsaturated and can adversely affect metabolic health through mechanisms that are not sufficiently understood. Here we show that lipogenesis critically determines the use of food-derived, metabolically beneficial polyunsaturated fatty acids (PUFA). We found a patient with a hypofunctional heterozygous de novo Arg2177Cys mutation in FASN, which resulted in a cysteine-dependent, non-enzymatic acetylation of FASN and decreased protein stability. Remarkably, the patient showed markedly elevated levels of polyunsaturated fatty acids, whereas patients with high FASN expression in the liver showed diminished levels. Moreover, pharmacological intervention with the FASN-inhibitor TVB-2640 in patients with NASH was sufficient to increase the rate of PUFA use in circulating lipids. In line, the incorporation rate of supplemented omega-3 PUFA in obesogenic diet could be dramatically increased by reducing the lipogenesis rate in mice. Mechanistically, we show that fatty acid storage preferences are determined by an active, enzymatic process dependent on FASN and DGAT2. Our results demonstrate that low lipogenesis rates increase the efficiency of PUFA incorporation into complex lipids, whereas high lipogenesis rates lead to low PUFA use. This has critical clinical implications for the prediction of low therapeutic success of omega-3 supplementation in patients with high lipogenesis and provides evidence for the urgent development of combined therapy options targeting lipogenesis and PUFA supplementation.

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

Palmitoylation cycling involves successive addition and removal of lipid from proteins to regulate cell structure and membrane interactions. Cycling requires depalmitoylation, mediated mostly by the enzyme acyl-protein thioesterase 1 (APT1). To test the hypothesis that depalmitoylation is critical for normal glucose metabolism, we generated global and islet-specific (using tamoxifen-inducible Pdx1 Cre) APT1 knockout mice. Both models on a chow diet had enhanced glucose tolerance due to increased insulin secretion. Pancreatic insulin content was unaffected by APT1 deficiency. Isolated islets from APT1-deficient models demonstrated increased glucose-stimulated insulin secretion under static and dynamic conditions. Quantitative histomorphometry demonstrated β cell hypertrophy in islets from global APT1 KO mice. Calcium dynamics in isolated islets were unaffected by APT1 deficiency, suggesting that increased insulin secretion is mediated downstream of depolarization-dependent calcium influx. APT1 knockdown in replicates of human islets from two donors showed increased insulin secretion. At 4 weeks of age, global APT1 KO db/db mice had improved glucose tolerance due to increased insulin secretion compared to littermate db/db mice. However, by 16 weeks of age APT1-deficient db/db mice had severe glucose intolerance, hypoinsulinemia, and β cell atrophy compared to littermate db/db mice. Islet palmitoylation proteomics identified putative APT1 substrates including Scamp1, -2 and -3. Scamp1 was validated as a palmitoylated protein, APT1 deficiency would be predicted to impair Scamp1 function, and Scamp1 knockdown in insulinoma cells increased insulin secretion. These results suggest that deficiency of APT1, an enzyme susceptible to post-translational regulation in the setting of insulin resistance, causes hypersecretion of insulin that leads to β cell failure, modeling the clinical evolution of human type 2 diabetes. Disclosure G. Dong: None. X. Wei: None. L. Yin: None. C. Feng: None. S. Adak: None. Z.A. Shyr: None. Q. Zhang: None. S. Morikawa: None. R. Asada: None. F. Urano: Stock/Shareholder; Self; CytRx Corporation. Other Relationship; Self; AETAS Pharma, Amylyx Pharmaceuticals, Mitochon, National Center for Advancing Translational Sciences, Novus Biologicals?. M.S. Remedi: None. C.F. Semenkovich: None.

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.

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., Endogenous fatty acid generation is dispensable for retinal development, but required for maintaining microdomain lipid composition that sustains vision through trophic signaling pathways.

SummaryFatty 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.

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 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.

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.

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-1β 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.

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.

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.