Fatty Acid-free Bovine Serum Albumin Research Articles

Effect of fatty acids and pH on the binding of BSA to silica and polystyrene.

Bovine serum albumin (BSA) is a frequently used blocking agent for in-vitro diagnostic (IVD) applications. Fatty acid stabilized (FAS) and fatty acid free (FAF) BSA are the common grades of BSA commercially offered for blocking. It is known that both fatty acid presence and pH influence the conformation of BSA. Previous adsorption studies have examined the effect of fatty acids or pH on BSA blocking, but little data is available for how these factors together impact function. In this study, we used Quartz Crystal Microbalance with Dissipation (QCM-D) to further understand of how pH and fatty acids impact BSA adsorption to IVD relevant surfaces.

Effects of exogenous C18 unsaturated fatty acids on milk lipid synthesis in bovine mammary epithelial cells.

We determined the effects of a combination of C18 unsaturated fatty acids (C18-UFAs) consisting of oleic, linoleic, and linolenic acids on milk lipogenesis in bovine mammary epithelial cells (BMECs). By orthogonal experiments to determine cellular triacylglycerol (TAG) accumulation, a combination of 200 μmol/l C18 : 1, 50 μmol/l C18 : 2, and 2 μmol/l C18 : 3 was selected as C18-UFAs combination treatment, and culture in medium containing fatty acid-free bovine serum albumin was used as the control. The expression of genes related to milk lipid synthesis and intracellular FA composition was measured. The results showed that cytosolic TAG formation was higher under C18-UFAs treatment than under control treatment. The mRNA expression of acetyl-CoA carboxylase-α (ACACA), fatty acid synthase (FASN), and peroxisome proliferator-activated receptor gamma (PPARG) did not differ between treatments. The abundance of stearoyl-CoA desaturase (SCD) and acyl-CoA synthetase long-chain family member 1 (ACSL1) was higher, whereas that of sterol regulatory element binding transcription factor 1 (SREBF-1) was lower after C18-UFAs treatment compared to control treatment. The C16 : 0 and SFA content was decreased following C18-UFAs treatment compared to control treatment, while the cis-9 C18 : 1 and UFA content was increased. In conclusion, C18-UFAs could stimulate triglyceride accumulation, increase the cellular UFA concentration, and regulate lipogenic genes in BMECs.

Conformational flexibility of fatty acid-free bovine serum albumin proteins enables superior antifouling coatings

Bovine serum albumin (BSA) protein is widely used to fabricate antifouling coatings for nanobiotechnology applications. Numerous BSA protein options are commercially available and obtained through different purification methods, however, there is no guidance on which ones are preferable for antifouling coatings. Herein, we investigate the real-time fabrication of antifouling coatings composed of BSA proteins obtained through different purification methods, and report significant differences in ultrathin film coating properties and ability to mitigate serum biofouling and to prevent nanoparticle-induced immune reactions. Mechanistic studies unravel the source of these performance variations, which are related to some BSA proteins containing fatty acid stabilizers while other BSA proteins are fatty acid-free depending on the purification method. Fatty acid-free BSA proteins exhibit greater conformational flexibility and less charge repulsion, which allow them to form more rigidly attached and tightly packed coatings on flat surfaces and nanoparticles that result in superior application performance.

Mortality From Coronavirus Disease 2019 Increases With Unsaturated Fat and May Be Reduced by Early Calcium and Albumin Supplementation

Mortality From Coronavirus Disease 2019 Increases With Unsaturated Fat and May Be Reduced by Early Calcium and Albumin Supplementation

Albumin and palmitate overload increases osteopontin production in renal tubule epithelial cells

Osteopontin (OPN) is one of the proinflammatory cytokines upregulated in the kidneys of diabetic animals and patients with diabetic nephropathy (DN). An increase in albuminuria and albumin‐bound fatty acids presents a proinflammatory environment to the proximal tubules in proteinuric kidney diseases including DN. This study was designed to examine if fatty acid overload or albumin itself could stimulate OPN production by renal tubular epithelial cells. Renal tubular OPN expression and secretion were examined in Zucker diabetic (ZD) rats and NRK‐52E cell line exposure to bovine serum albumin (BSA) or BSA overloaded with saturated fatty acid palmitate. Gene microarray and real‐time PCR confirmed an albuminuria‐associated upregulation of OPN transcripts in kidney cortex of ZD rats at the age of 7–20 weeks. Immunofluorescence staining of kidney sections revealed an increase in OPN protein in damaged proximal tubules of diabetic rats. Western blot analysis detected a progressive increase in urinary OPN levels in the ZD rats, which was prevented by antiproteinuric treatment with losartan, an angiotensin II receptor blocker. When exposed to fatty acid‐free bovine serum albumin (BSA, 10 mg/ml), NRK‐52E cells exhibited an increase in OPN protein expression. Palmitate (250 μM) further enhanced albumin‐induced OPN in NRK‐52E cells. These results demonstrate a stimulatory effect of albumin and its conjugated fatty acids on OPN expression in renal tubule epithelial cells. Thus, besides lowering albuminuria/proteinuria, mitigating circulating fatty acid levels may be an important factor in preventing renal inflammation in proteinuric diseases.Support or Funding InformationNIH SC1DK112151, NIH/NCRR/RCMI 8G12MD007602 & 8U54MD007588This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of the ratios of acetate and β-hydroxybutyrate on the expression of milk fat- and protein-related genes in bovine mammary epithelial cells

The objective of this study was to evaluate the effects of the different ratios of acetate and β-hydroxybutyrate (BHBA) on cell viability, triacylglycerol (TAG) content, and mRNA expression of the genes related to lipid and protein synthesis in bovine mammary epithelial cells (BMECs). Primary cells were isolated from the mammary glands of Holstein dairy cows and were passaged twice. Then, the cells were cultured with different ratios of acetate and BHBA (1 : 3, 1 : 2, 1 : 1, 2 : 1, 3 : 1, 4 : 1, and 1 : 1, Group 1 to Group 7, respectively) for 48 h, and the fetal bovine serum in the culture media was replaced with fatty acid-free bovine serum albumin (BSA) (1 g/l). The control culture media contained only fatty acid-free BSA without unsaturated fatty acids (0mM). Cell viability was not affected by adding different ratios of acetate and BHBA, but TAG accumulation was significantly influenced by supplementing the culture media with different ratios of acetate and BHBA. The expression levels of genes related to milk fat (FASN, ACACA, CD36, SCD, FABP3, LPL, PPARG, and SPEBF1) and milk protein-related genes (CSN1S1, CSN3, mTOR, 4E-BP1, S6KB1, STAT5, JAK2, and LEPTIN) were significantly affected by the addition of different ratios of acetate and BHBA to the BMECs. Our results suggested that Groups 3 and 4 (1 : 1 and 2 : 1) had a stronger acceleration of milk fat synthesis, and Group 4 (2 : 1) had the strongest effect. The expression of the CSN1S1 and LEPTIN mRNAs was more effectively promoted in Groups 3 and 4 (1 : 1 and 2 : 1), and Group 3 (1 : 1) had the strongest acceleration. Expressions of genes related to milk protein synthesis (mTOR, 4E-BP1, S6KB1, JAK2, and STAT5) were up-regulated using a ratio of acetate and BHBA of 2 : 1. Taken together, the 2 : 1 ratio of acetate and BHBA had the best effect for both the milk fat synthesis and milk protein synthesis genes. However, further studies are necessary to elucidate the mechanism for regulating milk fat and protein synthesis by different ratios of acetate and BHBA.

Influence of Small Amphiphiles on Aqueous Dispersions of <i>β</i>-Lactoglobulin A and Bovine Serum Albumin: (2) Surface Energy and Emulsifying Properties

Influence of amphiphiles on surface energy (γ), emulsifying activity index (EAI) and emulsion stability (ES) of fatty-acid free bovine serum albumin (FAF-BSA), bovine serum albumin (BSA), and β-lactoglobulin A (β-LGA) was studied. Contact angle measurements (θ) of sessile drops of protein-surfactant mixtures were determined using water and α-bromonaphthalene as solvents of known parameters to calculate γ. All surfactants except zwitterionic Z8 increased γ. The greatest increase was by anionic SDS. All surfactants significantly reduced EAI in spite of increased γ. ES was reduced by all surfactants except cationic TDTM which, conversely, enhanced ES by up to 186% for BSA and β-LGA.

Influence of Small Amphiphiles on Aqueous Dispersions of <i>β</i>-Lactoglobulin A and Bovine Serum Albumin: (1) Intermolecular Interactions

Hydrophilic and hydrophobic (φ) interactions among amphiphiles play critical roles in interfacial properties of proteins and other smaller amphiphiles and affect the creation and stability of foams and emulsions in food systems. Contribution of small amphiphiles on H-bonding and hydrophobic (φ) interactions at a model interface comprising of a water-hydrophobized surface interface as reflected by contact angle (θ) of fatty acid free bovine serum albumin (FAF-BSA), bovine serum albumin (BSA), and β-lactoglobulin variant A (β-LGA) was investigated. Amphiphiles were used with either protein in neutral water or α-bromonaphtalene (α-BrN) (22°C) to obtain θ-H2O and θ-α-BrN measurements, respectively. θ-α-BrN reflected influence of φ-interactions on θ since α-BrN molecules do not partake significantly in H-bonding. Ionic nature of the amphiphiles had no significant effect. Dramatic difference was between zwitterionic Z8 and Z12. At 1%, Z8 significantly increased H-bonding in BSA and β-LGA by 26% and 55%, respectively, whereas Z12, which is more hydrophobic, decreased it by 50% and 21%. At the same concentration, φ-interactions were enhanced by Z8 for BSA by 37% and by all amphiphiles except Z12 for FAF-BSA.

Different in vitro culture systems affect the birth weight of lambs from vitrified ovine embryos

It has been reported that different in vitro culture systems affect the birth weight of lambs. The aim of this study was to test body weight and lambing rate of lambs born from five different in vitro culture systems after vitrification. Oocytes of Sarda sheep were matured in TCM-199 plus 0.4% bovine serum albumin (BSA) using systems: (i) 4 mg/ml fatty acid-free BSA (BSA4); (ii) 8 mg/ml fatty acid-free BSA (BSA8); (iii) BSA8-hyaluronan (BSA8-HA); (iv) BSA8-charcoal-stripped FBS (BSA8-CH); or (v) with 10% fetal bovine serum (FBS; serum) and fertilized with fresh semen. The presumptive zygotes were cultured up to the blastocyst stage with BSA8, BSA8-HA, BSA8-CH or serum or BSA4. In the third and fifth days of culture 5% charcoal-stripped FBS was added into BSA8-CH and serum, while 8 mg/ml or 4 mg/ml fatty acid-free BSA was added as BSA8, BSA8-HA and BSA4 respectively; 6 mg/ml HA was added to BSA8-HA. In total, 240 vitrified blastocysts were transferred into synchronized ewes. The lambing rate was not significant different between BSA groups or between serum groups (BSA8-CH and serum), while serum groups showed significant lower values when compared with BSA groups. Only BSA8 groups produced heavy lambs (≥4.5 kg) with a significant difference between BSA4 and BSA8 groups (P < 0.05).

A3 Domain Region 1803–1818 Contributes to the Stability of Activated Factor VIII and Includes a Binding Site for Activated Factor IX

A recent chemical footprinting study in our laboratory suggested that region 1803-1818 might contribute to A2 domain retention in activated factor VIII (FVIIIa). This site has also been implicated to interact with activated factor IX (FIXa). Asn-1810 further comprises an N-linked glycan, which seems incompatible with a role of the amino acids 1803-1818 for FIXa or A2 domain binding. In the present study, FVIIIa stability and FIXa binding were evaluated in a FVIII-N1810C variant, and two FVIII variants in which residues 1803-1810 and 1811-1818 are replaced by the corresponding residues of factor V (FV). Enzyme kinetic studies showed that only FVIII/FV 1811-1818 has a decreased apparent binding affinity for FIXa. Flow cytometry analysis indicated that fluorescent FIXa exhibits impaired complex formation with only FVIII/FV 1811-1818 on lipospheres. Site-directed mutagenesis revealed that Phe-1816 contributes to the interaction with FIXa. To evaluate FVIIIa stability, the FVIII/FV chimeras were activated by thrombin, and the decline in cofactor function was followed over time. FVIII/FV 1803-1810 and FVIII/FV 1811-1818 but not FVIII-N1810C showed a decreased FVIIIa half-life. However, when the FVIII variants were activated in presence of FIXa, only FVIII/FV 1811-1818 demonstrated an enhanced decline in cofactor function. Surface plasmon resonance analysis revealed that the FVIII variants K1813A/K1818A, E1811A, and F1816A exhibit enhanced dissociation after activation. The results together demonstrate that the glycan at 1810 is not involved in FVIII cofactor function, and that Phe-1816 of region 1811-1818 contributes to FIXa binding. Both regions 1803-1810 and 1811-1818 contribute to FVIIIa stability.

Type VIII Collagen Mediates Vessel Wall Remodeling after Arterial Injury and Fibrous Cap Formation in Atherosclerosis

Collagens in the atherosclerotic plaque signal regulation of cell behavior and provide tensile strength to the fibrous cap. Type VIII collagen, a short-chain collagen, is up-regulated in atherosclerosis; however, little is known about its functions invivo. We studied the response to arterial injury and the development of atherosclerosis in type VIII collagen knockout mice (Col8(-/-) mice). After wire injury of the femoral artery, Col8(-/-) mice had decreased vessel wall thickening and outward remodeling when compared with Col8(+/+) mice. We discovered that apolipoprotein E (ApoE) is an endogenous repressor of the Col8a1 chain, and, therefore, in ApoE knockout mice, type VIII collagen was up-regulated. Deficiency of type VIII collagen in ApoE(-/-) mice (Col8(-/-);ApoE(-/-)) resulted in development of plaques with thin fibrous caps because of decreased smooth muscle cell migration and proliferation and reduced accumulation of fibrillar type I collagen. In contrast, macrophage accumulation was not affected, and the plaques had large lipid-rich necrotic cores. We conclude that in atherosclerosis, type VIII collagen is up-regulated in the absence of ApoE and functions to increase smooth muscle cell proliferation and migration. This is an important mechanism for formation of a thick fibrous cap to protect the atherosclerotic plaque from rupture.

Blastocyst rate of in vitro embryo production in sheep is affected by season

It has been reported that the number and quality of in vitro produced embryos is season related. This study was conducted to assess the effect of season on cleavage, blastocyst and lambing rates of in vitro produced ovine embryos during 3 years of collection data. Ovaries of Sarda sheep were collected from a slaughterhouse. In total, 5035 oocytes were recovered and matured in TCM-199 with 4 mg/ml bovine serum albumin (BSA), 100 μM cysteamine, 0.3 mM Na pyruvate, 0.1 UI/ml recombinant follicle-stimulating hormone (r-FSH), 0.1 UI/ml recombinant luteinising hormone (r-LH), and 1 μg/ml estradiol-17β. Matured oocytes were fertilized with fresh semen in synthetic oviductal fluid (SOF) with 20% heat inactivated estrous sheep serum. The presumptive zygotes were cultured for 6-7 days (blastocyst stage) in SOF medium supplemented with 1% Basel Medium Eagle (BME), 1% Minimum Essential Medium, 1 mM glutamine and 8 mg/ml fatty acid-free BSA. The embryos produced were vitrified and a total of 165 blastocysts (80 from the breeding season and 85 from the anoestrous season) were transferred in pairs into recipient ewes during the reproductive period. There were no significant differences in cleavage rates between seasons in any of the 3 years examined (84% versus 83%, 81% versus 80% and 80% versus 79%, respectively). The blastocyst rate varied significantly between seasons in 2005 and 2007 (P < 0.05), and in 2006 (P < 0.001). There were no differences in pregnancy and lambing rates between embryos during anoestrous versus during the breeding season. In conclusion, only the blastocyst rate appeared to have been affected by season, possibly due to variation in the number of developmentally competent oocytes.

Proteomic Identification of Betaig-h3 as a Lysophosphatidic Acid-Induced Secreted Protein of Human Mesenchymal Stem Cells: Paracrine Activation of A549 Lung Adenocarcinoma Cells by Betaig-h3

Lysophosphatidic acid (LPA) is enriched in the serum and malignant effusion of cancer patients and plays a key role in tumorigenesis and metastasis. LPA-activated mesenchymal stem cells promote tumorigenic potentials of cancer cells through a paracrine mechanism. LPA-conditioned medium (LPA CM) from human adipose tissue-derived mesenchymal stem cells (hASCs) elicited adhesion and proliferation of A549 human lung adenocarcinoma cells. To identify proteins involved in the LPA-stimulated paracrine functions of hASCs, we analyzed the LPA CM using liquid-chromatography tandem mass spectrometry-based shotgun proteomics. We identified βig-h3, an extracellular matrix protein that is implicated in tumorigenesis and metastasis, as an LPA-induced secreted protein in hASCs. LPA-induced βig-h3 expression was abrogated by pretreating hASCs with the LPA receptor(1/3) inhibitor Ki16425 or small interfering RNA-mediated silencing of endogenous LPA(1). LPA-induced βig-h3 expression was blocked by treating the cells with the Rho kinase inhibitor Y27632, implying that LPA-induced βig-h3 expression is mediated by the LPA(1)- Rho kinase pathway. Immunodepletion or siRNA-mediated silencing of βig-h3 abrogated LPA CM-stimulated adhesion and proliferation of A549 cells, whereas retroviral overexpression of βig-h3 in hASCs potentiated it. Furthermore, recombinant βig-h3 protein stimulated the proliferation and adhesion of A549 human lung adenocarcinoma cells. These results suggest that hASC-derived βig-h3 plays a key role in tumorigenesis by stimulating the adhesion and proliferation of cancer cells and it can be applicable as a biomarker and therapeutic target for lung cancer.

95 SHEEP ZYGOTES CULTURED WITH HYALURONAN, BOVINE SERUM ALBUMIN, OR SERUM AND THEIR EFFECTS ON PRENATAL AND POSTNATAL EMBRYO PRODUCTION AND DEVELOPMENT

Mammalian pre-implantation embryos are sensitive to their environment and numerous publications suggest that post-fertilization culture is the most critical period affecting embryo quality and subsequent embryo development. This study was conducted to examine the viability, twining, sex ratio and birthweight (BW) after transfer of fresh and vitrified in vitro-produced sheep embryos derived from oocytes cultured in a semi- or defined medium supplemented with fatty-acid–free bovine serum albumin (BSA) with or without hyaluronan (HA) versus fetal bovine serum (FBS) as a control. A total of 649 oocytes were matured in TCM-199 containing 2 mM glutamine, 4 mg mL–1 BSA, 100 μM cysteamine, 0.3 mM sodium pyruvate, 1 μg mL–1 oestradiol-17β and fertilized with fresh semen prepared with SOF, enriched with 20% oestrus sheep serum. Presumptive zygotes were cultured for 48 h in SOF supplemented with 1% BME-essential and 1% MEM-nonessential amino acids, 1 mM glutamine and 8 mg mL–1 BSA. On the 3rd day of culture, presumptive zygotes were divided randomly into 3 groups: (1) BSA+HA, medium supplemented with 8 mg mL–1 BSA and 6 mg mL–1 HA, (2) BSA, medium supplemented with 8 mg mL–1 BSA only, or (3) control, medium supplemented with 5% charcoal-stripped fetal bovine serum (FBS). On the 5th day of culture, media were replaced accordingly in all groups. Embryos that reached the expanded blastocyst stage by 6 or 7 days/group were recorded and transferred as fresh or vitrified. The cleavage rate, blastocyst formation, pregnancies (PR) at 40, 60 and 80 days, lambing, twinning, sex ratio and BW were all recorded. Among all groups, no significant differences were observed in cleavage and blastocyst rates. The proportion of zygotes that developed to blastocysts at 6 days was significantly higher after culture with BSA+HA vs BSA and control (67/92, 72.8%), (46/118, 39.0%) and (29/72, 40.3%), respectively. Compared with the control group, vitrified BSA blastocysts were associated with increased (P &lt; 0.05) PRs after 60 and 80 days and lambing rates. In comparison, there were no significant differences in pregnancy and lambing rate after transfer of fresh blastocysts. There was a positive correlation between age of embryo at transfer and higher BW. The mean BWs were higher after transfer of 7-day vs 6-day embryos (3.95 and 3.37 kg), respectively. However, there were no significant differences in twining and sex ratio after transfer fresh/or vitrified treated embryos versus control group. In conclusion, the results indicate that sheep zygotes cultured with BSA+HA had an increased development to blastocysts at 6 days, but the rate of blastocyst formation was not increased, PR and lambing rate were comparable with the BSA and control groups. In addition, transfer of vitrified embryos produced in the presence of BSA significantly increased the PR at 60, 80 days and lambing rate versus control group.

Identification of UDP-Glucuronosyltransferases Responsible for the Glucuronidation of Darexaban, an Oral Factor Xa Inhibitor, in Human Liver and Intestine

Darexaban maleate is a novel oral direct factor Xa inhibitor, which is under development for the prevention of venous thromboembolism. Darexaban glucuronide was the major component in plasma after oral administration of darexaban to humans and is the pharmacologically active metabolite. In this study, we identified UDP-glucuronosyltransferases (UGTs) responsible for darexaban glucuronidation in human liver microsomes (HLM) and human intestinal microsomes (HIM). In HLM, the K(m) value for darexaban glucuronidation was >250 μM. In HIM, the reaction followed substrate inhibition kinetics, with a K(m) value of 27.3 μM. Among recombinant human UGTs, UGT1A9 showed the highest intrinsic clearance for darexaban glucuronidation, followed by UGT1A8, -1A10, and -1A7. All other UGT isoforms were inactive toward darexaban. The K(m) value of recombinant UGT1A10 for darexaban glucuronidation (34.2 μM) was comparable to that of HIM. Inhibition studies using typical UGT substrates suggested that darexaban glucuronidation in both HLM and HIM was mainly catalyzed by UGT1A8, -1A9, and -1A10. Fatty acid-free bovine serum albumin (2%) decreased the unbound K(m) for darexaban glucuronidation from 216 to 17.6 μM in HLM and from 35.5 to 18.3 μM in recombinant UGT1A9. Recent studies indicated that the mRNA expression level of UGT1A9 is extremely high among UGT1A7, -1A8, -1A9, and -1A10 in human liver, whereas that of UGT1A10 is highest in the intestine. Thus, the present results strongly suggest that darexaban glucuronidation is mainly catalyzed by UGT1A9 and UGT1A10 in human liver and intestine, respectively. In addition, UGT1A7, -1A8, and -1A9 play a minor role in human intestine.

In vitro maturation supplements affect developmental competence of bovine cumulus–oocyte complexes and embryo quality after vitrification

Oocyte quality affects subsequent embryo development and quality. We examined the impact of bovine oocyte in vitro maturation (IVM) conditions on subsequent embryo yield, quality and cryosurvival. Cumulus–oocyte complexes (COCs) were sampled for cytological and gene expression analysis after IVM in TCM199 supplemented with 10% fetal bovine serum (FBS), 4mg/ml of fatty-acid-free bovine serum albumin (FAFBSA), 4mg/ml of polyvinylpyrrolidone (PVP), FAFBSA with epidermal growth factor (EGF, 100ng/ml) and insulin-like growth factor 1 (IGF-I, 100ng/ml) (FAFBSAGF), PVP with EGF and IGF-I (PVPGF) or PVP with single strength BME and MEM amino acids (PVPAA). The remaining COCs were fertilized. On day 7 (IVF=day 0) quality 1 blastocysts were vitrified or analyzed for glucose transporter 1 (Glut-1) expression levels. The remaining blastocysts (days 7–9) were evaluated for morphology and total cell counts. After warming, survival and hatching rates were evaluated followed by total cell counts and Glut-1 expression levels. Only PVPGF IVM resulted in embryo production rates comparable to those recorded with FBS IVM. Growth factors with FAFBSA and amino acids with PVP reduced embryo production rates whereas the effect of the growth factors with PVP was negligible. Insulin-like growth factor 2 binding protein 3 (IGF2BP3) and beta cell translocation gene 4 (BTG4) were revealed as potential candidates for oocyte developmental competence, and secreted protein, acidic and rich in cysteine (SPARC) for cumulus cell expansion. There were no differences among treatments in hatching rates of vitrified embryos after warming. However, total cell numbers and Glut-1 expression levels at 72h were affected.

High-sucrose diet increases ROS generation, FFA accumulation, UCP2 level, and proton leak in liver mitochondria

Obesity, a risk factor for insulin resistance, contributes to the development of type 2 diabetes and cardiovascular diseases. The relationship between increased levels of free fatty acids in the liver mitochondria, mitochondrial function, and ROS generation in rat model of obesity induced by a high-sucrose diet was not sufficiently established. We determined how the bioenergetic functions and ROS generation of the mitochondria respond to a hyperlipidemic environment. Mitochondria from sucrose-fed rats generated H(2)O(2) at a higher rate than the control mitochondria. Adding fatty acid-free bovine serum albumin to mitochondria from sucrose-fed rats significantly reduced the rate of H(2)O(2) generation. In contrast, adding exogenous oleic or linoleic acid exacerbated the rate of H(2)O(2) generation in both sucrose-fed and control mitochondria, and the mitochondria from sucrose-fed rats were more sensitive than the control mitochondria. The increased rate of H(2)O(2) generation in sucrose-fed mitochondria corresponded to decreased levels of reduced GSH and vitamin E and increased levels of Cu/Zn-SOD in the intermembrane space. There was no difference between the levels of lipid peroxidation and protein carbonylation in the two types of mitochondria. In addition to the normal activity of Mn-SOD, GPX and catalase detected an increased activity of complex II, and upregulation of UCP2 was observed in mitochondria from sucrose-fed rats, all of which may accelerate respiration rates and reduce generation of ROS. In turn, these effects may protect the mitochondria of sucrose-fed rats from oxidative stress and preserve their function and integrity. However, in whole liver these adaptive mechanisms of the mitochondria were inefficient at counteracting redox imbalances and inhibiting oxidative stress outside of the mitochondria.

A Hierarchical Whole-body Modeling Approach Elucidates the Link between in Vitro Insulin Signaling and in Vivo Glucose Homeostasis

Type 2 diabetes is a metabolic disease that profoundly affects energy homeostasis. The disease involves failure at several levels and subsystems and is characterized by insulin resistance in target cells and tissues (i.e. by impaired intracellular insulin signaling). We have previously used an iterative experimental-theoretical approach to unravel the early insulin signaling events in primary human adipocytes. That study, like most insulin signaling studies, is based on in vitro experimental examination of cells, and the in vivo relevance of such studies for human beings has not been systematically examined. Herein, we develop a hierarchical model of the adipose tissue, which links intracellular insulin control of glucose transport in human primary adipocytes with whole-body glucose homeostasis. An iterative approach between experiments and minimal modeling allowed us to conclude that it is not possible to scale up the experimentally determined glucose uptake by the isolated adipocytes to match the glucose uptake profile of the adipose tissue in vivo. However, a model that additionally includes insulin effects on blood flow in the adipose tissue and GLUT4 translocation due to cell handling can explain all data, but neither of these additions is sufficient independently. We also extend the minimal model to include hierarchical dynamic links to more detailed models (both to our own models and to those by others), which act as submodules that can be turned on or off. The resulting multilevel hierarchical model can merge detailed results on different subsystems into a coherent understanding of whole-body glucose homeostasis. This hierarchical modeling can potentially create bridges between other experimental model systems and the in vivo human situation and offers a framework for systematic evaluation of the physiological relevance of in vitro obtained molecular/cellular experimental data.

Human Lysophosphatidylcholine Acyltransferases 1 and 2 Are Located in Lipid Droplets Where They Catalyze the Formation of Phosphatidylcholine

Phosphatidylcholine (PC) is synthesized by two different pathways, the Lands cycle and the Kennedy pathway. The recently identified key enzymes of the Lands cycle, lysophosphatidylcholine acyltransferase 1 and 2 (LPCAT1 and -2), were reported to localize to the endoplasmic reticulum and to function in lung surfactant production and in inflammation response. Here, we show in various mammalian cell lines that both enzymes additionally localize to lipid droplets (LDs), which consist of a core of neutral lipids surrounded by a monolayer of phospholipid, mainly PC. This dual localization is enabled by the monotopic topology of these enzymes demonstrated in this study. Furthermore, we show that LDs have the ability to locally synthesize PC and that this activity correlates with the LPCAT1 and -2 expression level. This suggests that LPCAT1 and -2 have, in addition to their known function in specialized cells, a ubiquitous role in LD-associated lipid metabolism.

Structural and Functional Analysis of Tomosyn Identifies Domains Important in Exocytotic Regulation

Tomosyn is a 130-kDa cytosolic R-SNARE protein that associates with Q-SNAREs and reduces exocytotic activity. Two paralogous genes, tomosyn-1 and -2, occur in mammals and produce seven different isoforms via alternative splicing. Here, we map the structural differences between the yeast homologue of m-tomosyn-1, Sro7, and tomosyn genes/isoforms to identify domains critical to the regulation of exocytotic activity to tomosyn that are outside the soluble N-ethylmaleimide-sensitive attachment receptor motif. Homology modeling of m-tomosyn-1 based on the known structure of yeast Sro7 revealed a highly conserved functional conformation but with tomosyn containing three additional loop domains that emanate from a β-propeller core. Notably, deletion of loops 1 and 3 eliminates tomosyn inhibitory activity on secretion without altering its soluble N-ethylmaleimide-sensitive attachment receptor pairing with syntaxin1A. By comparison, deletion of loop 2, which contains the hypervariable splice region, did not reduce the ability of tomosyn to inhibit regulated secretion. However, exon variation within the hypervariable splice region resulted in significant differences in protein accumulation of tomosyn-2 isoforms. Functional analysis of s-tomosyn-1, m-tomosyn-1, m-tomosyn-2, and xb-tomosyn-2 demonstrated that they exert similar inhibitory effects on elevated K(+)-induced secretion in PC12 cells, although m-tomosyn-2 was novel in strongly augmenting basal secretion. Finally, we report that m-tomosyn-1 is a target substrate for SUMO 2/3 conjugation and that mutation of this small ubiquitin-related modifier target site (Lys-730) enhances m-tomosyn-1 inhibition of secretion without altering interaction with syntaxin1A. Together these results suggest that multiple domains outside the R-SNARE of tomosyn are critical to the efficacy of inhibition by tomosyn on exocytotic secretion.