The Normal Limits,Subclinical Significance,Related Metabolic Derangements and Distinct Biological Effects of Body Site-Specific Adiposity in Relatively Healthy Population

Background

The accumulation of visceral adipose tissue that occurs with normal aging is associated with increased cardiovascular risks. However, the clinical significance, biological effects, and related cardiometabolic derangements of body-site specific adiposity in a relatively healthy population have not been well characterized.

Materials and Methods

In this cross-sectional study, we consecutively enrolled 608 asymptomatic subjects (mean age: 47.3 years, 27% female) from 2050 subjects undergoing an annual health survey in Taiwan. We measured pericardial (PCF) and thoracic peri-aortic (TAT) adipose tissue volumes by 16-slice multi-detector computed tomography (MDCT) (Aquarius 3D Workstation, TeraRecon, San Mateo, CA, USA) and related these to clinical characteristics, body fat composition (Tanita 305 Corporation, Tokyo, Japan), coronary calcium score (CCS), serum insulin, high-sensitivity C-reactive protein (Hs-CRP) level and circulating leukocytes count. Metabolic risk was scored by Adult Treatment Panel III guidelines.

Results

TAT, PCF, and total body fat composition all increased with aging and higher metabolic scores (all p<0.05). Only TAT, however, was associated with higher circulating leukocyte counts (ß-coef.:0.24, p<0.05), serum insulin (ß-coef.:0.17, p<0.05) and high sensitivity C-reactive protein (ß-coef.:0.24, p<0.05). These relationships persisted after adjustment in multivariable models (all p<0.05). A TAT volume of 8.29 ml yielded the largest area under the receiver operating characteristic curve (AUROC: 0.79, 95%CI: 0.74–0.83) to identify metabolic syndrome. TAT but not PCF correlated with higher coronary calcium score after adjustment for clinical variables (all p<0.05).

Conclusion

In our study, we observe that age-related body-site specific accumulation of adipose tissue may have distinct biological effects. Compared to other adiposity measures, peri-aortic adiposity is more tightly associated with cardiometabolic risk profiles and subclinical atherosclerosis in a relatively healthy population.     

Human microvascular endothelial cell prostaglandin E1 synthesis during in vitro ischemia-reperfusion

Ischemia-reperfusion injury is a microvascular event documented in numerous in vivo animal models. In animal models, prostaglandin and prostaglandin analogues have been found to ameliorate reperfusion injury. These studies were undertaken to evaluate human microvascular endothelial PGE(1) synthesis during in vitro ischemia followed by reperfusion. Human (neonatal) microvascular endothelial cell (MEC) cultures (n = 6) were subjected to sequential 2 h periods of normoxia (20% O(2)), ischemia (1.5% O(2)), and reperfusion (20% O(2)). Prostaglandin E(2) synthesis in conditioned media was determined by ELISA. Steady state levels of MEC prostaglandin H synthase (PGHS)-1 and -2 mRNA were assessed at the end of each 2-h period using RT-PCR and a quantitative mRNA ELISA. MEC PGHS protein levels were analyzed using an ELISA. PGE(1) release increased significantly during the initial 30 min of ischemia, but rapidly fell below normoxic levels by 90 and 120 min. During reperfusion, PGE(1) release returned to normoxic levels at 30, 60, and 90 min, and exceeded normoxic levels at 120 min. PGHS-1 mRNA levels were undetectable during all experimental conditions. PGHS-2 mRNA levels were unchanged by ischemia, but were decreased by reperfusion. In contrast, PGHS-2 protein levels increased 3-fold during ischemia, and remained elevated during reperfusion. Human MEC do not express PGHS-1 mRNA in vitro. Prolonged ischemia decreases MEC PGE(1) synthesis, and stimulates increased PGHS-2 protein levels without altering the steady state levels of COX-2 mRNA. During reperfusion, increased PGHS-2 protein levels persist and are associated with stimulated PGE(2) secretion, despite relative decreases in PGHS-2 mRNA.     

Coordination and speciation of cadmium in corn seedlings and its effects on macro- and micronutrients uptake

The effect of cadmium (Cd) on both the absorption of important nutrients and the synthesis of low molecular weight thiols (LMWTs) was investigated in corn plants. The inductively coupled plasma-optical emission spectroscopy results demonstrated that the concentration of Cd in tissues (mainly in roots) increased as the concentration in the medium increased. In addition, the concentration of phosphorus increased in roots of Cd treated plants but remained at normal concentration in shoots. On the other hand, the uptake of sulfur (S) followed a similar trend as the Cd uptake. The concentration of S and the production of LMWT were found to increase significantly upon exposure to Cd. The results of the X-ray absorption spectroscopy analyses indicated that Cd within tissues was bound to S ligands with interatomic distances of 2.51–2.52 Å. These results confirm a strong linkage between S uptake and the production of LMWT upon exposure to Cd.     

On mechanical aspects of vesicular transport

From the chain of events leading to secretion we have identified and isolated stages in which mechanical and physical mechanics may play important roles. These include the vesicle motion towards the cell wall, drainage of the cytoplasmic fluid from the gap between the membranes, reorganization of the membrane constituents, failure of the membrane structure and coalescence into a new configuration. We suggest a unified mechanism, relevant to the neural, secretory and vascular systems, based on physical factors as flow, pressure and stress distributions, and membranes properties. The simulation of several stages of secretion is coupled with experimental observations. By use of the proposed hypothesis it is possible to explain some observed phenomena, such as spontaneous and induced secretion, membrane failure, protein lateral dislocation and the omega-shapes in electron microscopic exposures of fusion sites.     

The contributions of protein disulfide isomerase and its homologues to oxidative protein folding in the yeast endoplasmic reticulum

In vitro, protein disulfide isomerase (Pdi1p) introduces disulfides into proteins (oxidase activity) and provides quality control by catalyzing the rearrangement of incorrect disulfides (isomerase activity). Protein disulfide isomerase (PDI) is an essential protein in Saccharomyces cerevisiae, but the contributions of the catalytic activities of PDI to oxidative protein folding in the endoplasmic reticulum (ER) are unclear. Using variants of Pdi1p with impaired oxidase or isomerase activity, we show that isomerase-deficient mutants of PDI support wild-type growth even in a strain in which all of the PDI homologues of the yeast ER have been deleted. Although the oxidase activity of PDI is sufficient for wild-type growth, pulse-chase experiments monitoring the maturation of carboxypeptidase Y reveal that oxidative folding is greatly compromised in mutants that are defective in isomerase activity. Pdi1p and one or more of its ER homologues (Mpd1p, Mpd2p, Eug1p, Eps1p) are required for efficient carboxypeptidase Y maturation. Consistent with its function as a disulfide isomerase in vivo, the active sites of Pdi1p are partially reduced (32 +/- 8%) in vivo. These results suggest that PDI and its ER homologues contribute both oxidase and isomerase activities to the yeast ER. The isomerase activity of PDI can be compromised without affecting growth and viability, implying that yeast proteins that are essential under laboratory conditions may not require efficient disulfide isomerization.     

The N-terminal extension of S12 influences small ribosomal subunit assembly in Escherichia coli

The small subunit (SSU) of the ribosome of E. coli consists of a core of ribosomal RNA (rRNA) surrounded peripherally by ribosomal proteins (r-proteins). Ten of the 15 universally conserved SSU r-proteins possess nonglobular regions called extensions. The N-terminal noncanonically structured extension of S12 traverses from the solvent to intersubunit surface of the SSU and is followed by a more C-terminal globular region that is adjacent to the decoding center of the SSU. The role of the globular region in maintaining translational fidelity is well characterized, but a role for the S12 extension in SSU structure and function is unknown. We examined the effect of stepwise truncation of the extension of S12 in SSU assembly and function in vitro and in vivo. Examination of in vitro assembly in the presence of sequential N-terminal truncated variants of S12 reveals that N-terminal deletions of greater than nine amino acids exhibit decreased tRNA-binding activity and altered 16S rRNA architecture particularly in the platform of the SSU. While wild-type S12 expressed from a plasmid can rescue a genomic deletion of the essential gene for S12, rpsl; N-terminal deletions of S12 exhibit deleterious phenotypic consequences. Partial N-terminal deletions of S12 are slow growing and cold sensitive. Strains bearing these truncations as the sole copy of S12 have increased levels of free SSUs and immature 16S rRNA as compared with the wild-type S12. These differences are hallmarks of SSU biogenesis defects, indicating that the extension of S12 plays an important role in SSU assembly.     

Role of Dimerization in the Catalytic Properties of the Escherichia coli Disulfide Isomerase DsbC

The bacterial protein-disulfide isomerase DsbC is a homodimeric V-shaped enzyme that consists of a dimerization domain, two α-helical linkers, and two opposing thioredoxin fold catalytic domains. The functional significance of the two catalytic domains of DsbC is not well understood yet. We have engineered heterodimer-like DsbC derivatives covalently linked via (Gly₃-Ser) flexible linkers. We either inactivated one of the catalytic sites (CGYC), or entirely removed one of the catalytic domains while maintaining the putative binding area intact. Variants having a single active catalytic site display significant levels of isomerase activity. Furthermore, mDsbC[H45D]-dim[D53H], a DsbC variant lacking an entire catalytic domain but with an intact dimerization domain, also showed isomerase activity, albeit at lower levels. In addition, the absence of the catalytic domain allowed this protein to catalyze in vivo oxidation. Our results reveal that two catalytic domains in DsbC are not essential for disulfide bond isomerization and that a determining feature in isomerization is the availability of a substrate binding domain.Disulfide bonds are critical for the proper folding and structural stability of many exocytoplasmic proteins. The Dsb family of thiol:disulfide oxidoreductase enzymes catalyzes oxidative protein folding in the periplasm of Escherichia coli by means of two independent pathways (1–3). In the DsbA-DsbB oxidation pathway, DsbA, a very strong oxidant, catalyzes the formation of disulfide bonds on newly translocated proteins (4). The DsbA disulfide is rapidly recycled by DsbB, a membrane protein that transfers electrons from DsbA onto quinones (5–7). In the DsbC-DsbD isomerization pathway, non-native disulfides are reduced or rearranged by DsbC. DsbC is maintained in a reduced, catalytically active state via the transfer of electrons from the inner membrane protein DsbD that in turn accepts electrons from thioredoxin 1 and ultimately from NADPH (via thioredoxin reductase) within the cytoplasm (8, 9). Large kinetic barriers keep the oxidation and isomerization pathways isolated, preventing the establishment of a futile cycle of electron transfer. Accordingly, reactions between enzymes of the two pathways, for example the oxidation of DsbC by DsbB or the reduction of DsbA by DsbD, are 10³–10⁷-fold slower than the physiologically relevant DsbA-DsbB and DsbC-DsbD reactions (10). Nonetheless, the kinetic barrier between DsbB and DsbC can be breached by introducing mutations that result in structural changes in DsbC (11, 12).DsbC is a homodimer with each monomer comprising an N-terminal dimerization domain and a C-terminal thioredoxin-like catalytic domain fused by an α-helical linker. The crystal structure of DsbC reveals that the two monomers come together to form a V-shaped protein. The inner surface of the resulting cleft is patched with uncharged and hydrophobic residues suggesting an important role in the binding of substrate proteins. The active sites comprising the sequence Cys⁹⁸-Gly⁹⁹-Tyr¹⁰⁰-Cys¹⁰¹ in each of the monomeric subunits are located in the arms of the “V” facing each other (13). Isomerization involves an attack onto a substrate disulfide by Cys⁹⁸ resulting in the formation of a mixed disulfide, which then is resolved by either another cysteine from the substrate or by Cys¹⁰¹ from DsbC (14, 15). Besides its isomerase activity, DsbC is also known to display chaperone activity preventing protein aggregation during refolding (16). In E. coli, disulfide bond isomerization is the limiting step in the oxidative folding of many heterologous proteins that contain multiple cysteines. Overexpression of DsbC has been shown to enhance the yield of proteins such as human nerve growth factor, human tissue plasminogen activator (tPA)² and immunoglobulins (17–19).DsbC is topologically analogous to the eukaryotic protein-disulfide isomerase (PDI). The structural similarities between the two enzymes may have resulted from convergent evolution by thioredoxin-like domain replication in the case of PDI and domain recruitment in DsbC (20, 21). PDI comprises two thioredoxin-like catalytic domains (a and a′) separated by two non-catalytic domains (b and b′), in addition to a c domain (22). In PDI, the catalytic domains are different and functionally nonequivalent (23). Substrate binding is mediated primarily by the b′ domain; the two catalytic domains, a and a′, can catalyze oxidation of small model peptides indicating that they must also have low substrate binding affinity (24).The DsbC monomer is essentially devoid of RNase A isomerase activity (25). Sun and Wang (44) reported that DsbC with one catalytic site impaired by carboxymethylation is also essentially inactive but, in separate studies, Zapun et al. (26) did not detect cooperativity between the two catalytic sites indicating that they function independently of each other (26). Moreover, unlike PDI, the significance of the putative peptide binding cleft of DsbC on disulfide isomerization has not been ascertained. However, while DsbA or TrxA with a PDI active site dipeptide (CGHC) display very little isomerase activity in vitro and in vivo (27–29), we recently showed that upon fusion to a dimerization region that provides a putative substrate binding surface (the E. coli peptidyl proline isomerase FkpA) they acquire the ability to assist the folding of periplasmically expressed multidisulfide heterologous proteins (30).In the present work, we engineered heterodimer-like covalently linked DsbC derivatives in which one of the catalytic sites has been inactivated (Fig. 1A) or one of the catalytic domains has been entirely removed while maintaining the intact peptide binding cleft (which is normally formed by association of the N-terminal domains of the two monomers) (Fig. 3A). We show that DsbC forced monomers with one functional active site, or with one thioredoxin domain only, display significant isomerization activity. Interestingly, the latter variant is partially reduced in vivo indicating that the presence of both thioredoxin domains is important for the avoidance of protein oxidation by DsbB.Open in a separate windowFIGURE 1.A, protein structure of DsbC, and molecular models of mDsbC-mDsbC and the single active site covalently linked mutants. Dimerization domains are shown in gray, thioredoxin domains in black, and the active sites in white. B, gel filtration FPLC of DsbC and linked variants. Purified proteins were run on a Superdex^TM 200 column in PBS, 10% glycerol buffer.Open in a separate windowFIGURE 3.A, molecular model of mDsbC-dim. Dimerization domains are shown in gray, thioredoxin domain in black, and catalytic site in white. B, gel filtration FPLC of mDsbC-dim as compared with DsbC. Purified proteins were run on a Superdex^TM 200 column in PBS, 10% glycerol buffer. C, MALS measurement of the molar masses of the components of mDsbC-dim together with their hydrodynamic radii. The data show monomeric, dimeric, and tetrameric states. The relative concentrations were determined by the refractive index differences.