Ligation of α-Dystroglycan on Podocytes Induces Intracellular Signaling: A New Mechanism for Podocyte Effacement?

Background

α-Dystroglycan is a negatively charged glycoprotein that covers the apical and basolateral membrane of the podocyte. Its transmembrane binding to the cytoskeleton is regulated via tyrosine phosphorylation (pY892) of β-dystroglycan. At the basolateral side α-dystroglycan binds the glomerular basement membrane. At the apical membrane, it plays a role in the maintenance of the filtration slit. In this study, we evaluated whether ligation of α-dystroglycan with specific antibodies or natural ligands induces intracellular signaling, and whether there is an effect on podocyte architecture.

Methodology/Principal Findings

Conditionally immortalized podocytes were exposed in vitro to antibodies to α-dystroglycan, and to fibronectin, biglycan, laminin and agrin. Intracellular calcium fluxes, phosphorylation of β-dystroglycan and podocyte architecture were studied. Antibodies to α-dystroglycan could specifically induce calcium signaling. Fibronectin also induced calcium signaling, and led to dephosphorylation of pY892 in β-dystroglycan. Ligation of α-dystroglycan resulted in an altered actin architecture, a decreased number of podocyte pedicles and a more flattened appearance of the podocyte.

Conclusions/Significance

We conclude that ligation of α-dystroglycan on podocytes induces intracellular calcium signaling, which leads to an altered cytoskeleton architecture akin to the situation of foot process effacement. In particular the ability of fibronectin to induce intracellular signaling events is of interest, since the expression and excretion of this protein is upregulated in several proteinuric diseases. Therefore, fibronectin-induced signaling via dystroglycan may be a novel mechanism for foot process effacement in proteinuric diseases.     

MDPSCL2: A New Protecting Group for Chemoselective Synthesis of 2′-O-Alkylated Guanosines

Abstract

An improved strategy for the synthesis of 2′-O-methyl-guanosine (6) and 2′-MOE-guanosine (8) is reported. The regioselectivity of the alkylation was attained using a novel silicon-based protecting group, methylene-bis (diisopropyl-silylchloride) (MDPSCl₂, 2). The alkylation proceeded in a chemoselective manner using NaHMDS as the base and MeCl or MOE-Br as the appropriate electrophiles.     

Inhibition of O2−- and HO - Mediated Processes by a New Class of Free Radical Scavengers: The N-ACYL Dehydroalanines

N-phenylacetyl dehydroalanines are captodative olefins. They inhibit two processes mediated by superoxide anion (O₂-) in a concentration dependent manner: reduction of NBT to blue formazan and oxidation of epinephrine to adrenochrome. They also inhibit in a dose related way the degradation of deoxyribose produced during either the Fenton reaction or the radiolysis of water, which are the two experimental sources of hydroxyl radical (HO^?) production. Based on the results obtained with superoxide dismutase, mannitol, thiourea, and uric acid, we postulate that these competitive inhibitory effects suggest a reaction between the dehydroalanine derivatives and the two oxygen derived radicals. Hydroxyl free radical is scavenged more efficiently than superoxide anion. Substitution of the phenyl ring by methoxy groups does not modify significantly the activity. These molecules possess three target active sites which can react with free radicals.     

Inhibition of Klebsiella β-Lactamases (SHV-1 and KPC-2) by Avibactam: A Structural Study

β-Lactamase inhibition is an important clinical strategy in overcoming β-lactamase-mediated resistance to β-lactam antibiotics in Gram negative bacteria. A new β-lactamase inhibitor, avibactam, is entering the clinical arena and promising to be a major step forward in our antibiotic armamentarium. Avibactam has remarkable broad-spectrum activity in being able to inhibit classes A, C, and some class D β-lactamases. We present here structural investigations into class A β-lactamase inhibition by avibactam as we report the crystal structures of SHV-1, the chromosomal penicillinase of Klebsiella pneumoniae, and KPC-2, an acquired carbapenemase found in the same pathogen, complexed with avibactam. The 1.80 Å KPC-2 and 1.42 Å resolution SHV-1 β-lactamase avibactam complex structures reveal avibactam covalently bonded to the catalytic S70 residue. Analysis of the interactions and chair-shaped conformation of avibactam bound to the active sites of KPC-2 and SHV-1 provides structural insights into recently laboratory-generated amino acid substitutions that result in resistance to avibactam in KPC-2 and SHV-1. Furthermore, we observed several important differences in the interactions with amino acid residues, in particular that avibactam forms hydrogen bonds to S130 in KPC-2 but not in SHV-1, that can possibly explain some of the different kinetic constants of inhibition. Our observations provide a possible reason for the ability of KPC-2 β-lactamase to slowly desulfate avibactam with a potential role for the stereochemistry around the N1 atom of avibactam and/or the presence of an active site water molecule that could aid in avibactam desulfation, an unexpected consequence of novel inhibition chemistry.     

Acid Hydrolysis of Wheat Gluten Induces Formation of New Epitopes but Does Not Enhance Sensitizing Capacity by the Oral Route: A Study in “Gluten Free” Brown Norway Rats

Background

Acid hydrolyzed wheat proteins (HWPs) are used in the food and cosmetic industry as emulsifiers. Cases of severe food allergic reactions caused by HWPs have been reported. Recent data suggest that these reactions are caused by HWPs produced by acid hydrolysis.

Objectives

To examine the sensitizing capacity of gluten proteins per se when altered by acid or enzymatic hydrolysis relative to unmodified gluten in rats naïve to gluten.

Methods

High IgE-responder Brown Norway (BN) rats bred on a gluten-free diet were sensitized without the use of adjuvant to three different gluten products (unmodified, acid hydrolyzed and enzymatic hydrolyzed). Rats were sensitized by intraperitoneal (i.p.) immunization three times with 200 µg gluten protein/rat or by oral dosing for 35 days with 0.2, 2 or 20 mg gluten protein/rat/day. Sera were analyzed for specific IgG and IgE and IgG-binding capacity by ELISA. IgE functionality was measured by rat basophilic leukemia (RBL) assay.

Results

Regardless of the route of dosing, all products had sensitizing capacity. When sensitized i.p., all three gluten products induced a strong IgG1 response in all animals. Acid hydrolyzed gluten induced the highest level of specific IgE but with a low functionality. Orally all three gluten products induced specific IgG1 and IgE but with different dose-response relations. Sensitizing rats i.p. or orally with unmodified or enzymatic hydrolyzed gluten induced specific IgG1 responses with similar binding capacity which was different from that of acid hydrolyzed gluten indicating that acid hydrolysis of gluten proteins induces formation of ‘new’ epitopes.

Conclusions

In rats not tolerant to gluten acid hydrolysis of gluten enhances the sensitizing capacity by the i.p. but not by the oral route. In addition, acid hydrolysis induces formation of new epitopes. This is in contrast to the enzymatic hydrolyzed gluten having an epitope pattern similar to unmodified gluten.     

Non-equivalent Ligand Selectivity of Agonist Sites in (α4β2)2α4 Nicotinic Acetylcholine Receptors: A KEY DETERMINANT OF AGONIST EFFICACY*

The α4β2 nicotinic acetylcholine receptor (nAChR) is the most abundant nAChR type in the brain, and this receptor type exists in alternate (α4β2)₂α4 and (α4β2)₂β2 forms, which are activated by agonists with strikingly differing efficacies. Recent breakthroughs have identified an additional operational agonist binding site in the (α4β2)₂α4 nAChR that is responsible for the signature sensitivity of this receptor to activation by agonists, yet the structural mechanisms determining agonist efficacy at this receptor type are not yet fully understood. In this study, we characterized the ligand selectivity of the individual agonist sites of the (α4β2)₂α4 nAChR to determine whether differences in agonist selectivity influence agonist efficacy. Applying the substituted cysteine accessibility method to individual agonist sites in concatenated (α4β2)₂α4 receptors, we determined the agonist selectivity of the agonist sites of the (α4β2)₂α4 receptor. We show that (a) accessibility of substituted cysteines to covalent modification by methanesulfonate reagent depends on the agonist site at which the modification occurs and (b) that agonists such as sazetidine-A and TC-2559 are excluded from the site at the α4/α4 interface. Given that additional binding to the agonist site in the α4/α4 interface increases acetylcholine efficacy and that agonists excluded from the agonist site at the α4/α4 interface behave as partial agonists, we conclude that the ability to engage all agonist sites in (α4β2)₂α4 nAChRs is a key determinant of agonist efficacy. The findings add another level of complexity to the structural mechanisms that govern agonist efficacy in heteromeric nAChRs and related ligand-gated ion channels.     

Aquifex aeolicus 3-Deoxy-d-manno-2-Octulosonic Acid 8-Phosphate Synthase: A New Class of KDO 8-P Synthase?

The relationship between 3-deoxy-D-manno-2-octulosonic acid 8-phosphate (KDO 8-P) synthase and 3-deoxy-D-arabino-2-heptulosonic acid 7-phosphate (DAH 7-P) synthase has not been adequately addressed in the literature. Based on recent reports of a metal requiring KDO 8-P synthase and the newly solved X-ray crystal structures of both Escherichia coli KDO 8-P synthase and DAH 7-P synthase, we begin to address the evolutionary kinship between these catalytically similar enzymes. Using a maximum likelihood-based grouping of 29 KDO 8-P synthase sequences, we demonstrate the existence of a new class of KDO 8-P synthase, the members of which we propose to require a metal cofactor for catalysis. Similarly, we hypothesize a class of DAH 7-P synthase that does not have the metal requirement of the heretofore model E. coli enzyme. Based on this information and a careful investigation of the reported X-ray crystal structures, we also propose that KDO 8-P synthase and DAH 7-P synthase are the product of a divergent evolutionary process from a common ancestor.     

A New Crystal Form of the Fab Fragment of a Monoclonal Antibody to Human Interleukin-2: The Three-Dimensional Structure at 2.7 Å Resolution

The three-dimensional structure of the antigen-binding fragment of a monoclonal antibody to human interleukin-2 in a new crystal form (space group P2₁2₁2₁; unit cell parameters: a = 42.82 Å, b = 90.68 Å, and c = 139.82 Å) was determined by the X-ray molecular replacement method at the resolution of 2.7 Å. The protein folding and the stereochemistry of its antigen-binding site were comparatively analyzed.     

Proteomic Analysis of C2C12 Myoblast and Myotube Exosome-Like Vesicles: A New Paradigm for Myoblast-Myotube Cross Talk?

Exosomes are nanometer-sized microvesicles formed in multivesicular bodies (MVBs) during endosome maturation. Exosomes are released from cells into the microenvironment following fusion of MVBs with the plasma membrane. During the last decade, skeletal muscle-secreted proteins have been identified with important roles in intercellular communications. To investigate whether muscle-derived exosomes participate in this molecular dialog, we determined and compared the protein contents of the exosome-like vesicles (ELVs) released from C2C12 murine myoblasts during proliferation (ELV-MB), and after differentiation into myotubes (ELV-MT). Using a proteomic approach combined with electron microscopy, western-blot and bioinformatic analyses, we compared the protein repertoires within ELV-MB and ELV-MT. We found that these vesicles displayed the classical properties of exosomes isolated from other cell types containing components of the ESCRT machinery of the MVBs, as well as numerous tetraspanins. Specific muscle proteins were also identified confirming that ELV composition also reflects their muscle origin. Furthermore quantitative analysis revealed stage-preferred expression of 31 and 78 proteins in ELV-MB and ELV-MT respectively. We found that myotube-secreted ELVs, but not ELV-MB, reduced myoblast proliferation and induced differentiation, through, respectively, the down-regulation of Cyclin D1 and the up-regulation of myogenin. We also present evidence that proteins from ELV-MT can be incorporated into myoblasts by using the GFP protein as cargo within ELV-MT. Taken together, our data provide a useful database of proteins from C2C12-released ELVs throughout myogenesis and reveals the importance of exosome-like vesicles in skeletal muscle biology.     

A potential role of the GRO-α/CXCR2 system in Sjögren’s syndrome: regulatory effects of pro-inflammatory cytokines

Chemokines, small pro-inflammatory cytokines, are involved in migration of inflammatory cells in inflamed tissues and recent studies established their role in angiogenesis, hematopoiesis, cancer and autoimmune conditions. Growth related oncogene-alpha (GRO-α), a member of the CXC chemokine family, and its receptor CXCR2 are involved in the inflammatory processes. Since there is no previous report that supports a possible role of GRO-α/CXCR2 receptor complex during inflammation and neovascularization existing in the autoimmune disease Sjögren’s syndrome (SS), in this study, we examined CXCR2 and its ligand GRO-α expression in SS tissues. Immunohistochemistry revealed that GRO-α and its receptor CXCR2 were expressed at high levels in diseased tissues compared to healthy controls. In addition, human salivary gland epithelial cells (SGEC) cultures were submitted to a pro-inflammatory microenvironment using cytokines IL-6 and TNF-α in order to demonstrate that CXCR2 may change its initial expression pattern to another under inflammatory condition. The data show an increased expression of CXCR2 depending on the inflammatory cytokine used in culture in a time-dependent manner. Furthermore, silencing of the pro-angiogenic chemokine GRO-α is proportionally correlated with decreased expression of CXCR2 in pro-inflammatory cytokine-stimulated SGEC indicating the GRO-α/CXCR2 complex as a novel therapeutic target for the chronic inflammatory disease Sjögren’s syndrome.     

When to Start Antiretroviral Therapy in Children Aged 2–5 Years: A Collaborative Causal Modelling Analysis of Cohort Studies from Southern Africa

Background

There is limited evidence on the optimal timing of antiretroviral therapy (ART) initiation in children 2–5 y of age. We conducted a causal modelling analysis using the International Epidemiologic Databases to Evaluate AIDS–Southern Africa (IeDEA-SA) collaborative dataset to determine the difference in mortality when starting ART in children aged 2–5 y immediately (irrespective of CD4 criteria), as recommended in the World Health Organization (WHO) 2013 guidelines, compared to deferring to lower CD4 thresholds, for example, the WHO 2010 recommended threshold of CD4 count <750 cells/mm³ or CD4 percentage (CD4%) <25%.

Methods and Findings

ART-naïve children enrolling in HIV care at IeDEA-SA sites who were between 24 and 59 mo of age at first visit and with ≥1 visit prior to ART initiation and ≥1 follow-up visit were included. We estimated mortality for ART initiation at different CD4 thresholds for up to 3 y using g-computation, adjusting for measured time-dependent confounding of CD4 percent, CD4 count, and weight-for-age z-score. Confidence intervals were constructed using bootstrapping.The median (first; third quartile) age at first visit of 2,934 children (51% male) included in the analysis was 3.3 y (2.6; 4.1), with a median (first; third quartile) CD4 count of 592 cells/mm³ (356; 895) and median (first; third quartile) CD4% of 16% (10%; 23%). The estimated cumulative mortality after 3 y for ART initiation at different CD4 thresholds ranged from 3.4% (95% CI: 2.1–6.5) (no ART) to 2.1% (95% CI: 1.3%–3.5%) (ART irrespective of CD4 value). Estimated mortality was overall higher when initiating ART at lower CD4 values or not at all. There was no mortality difference between starting ART immediately, irrespective of CD4 value, and ART initiation at the WHO 2010 recommended threshold of CD4 count <750 cells/mm³ or CD4% <25%, with mortality estimates of 2.1% (95% CI: 1.3%–3.5%) and 2.2% (95% CI: 1.4%–3.5%) after 3 y, respectively. The analysis was limited by loss to follow-up and the unavailability of WHO staging data.

Conclusions

The results indicate no mortality difference for up to 3 y between ART initiation irrespective of CD4 value and ART initiation at a threshold of CD4 count <750 cells/mm³ or CD4% <25%, but there are overall higher point estimates for mortality when ART is initiated at lower CD4 values. Please see later in the article for the Editors'' Summary     

Soluble ST2 Associates with Diabetes but Not Established Cardiovascular Risk Factors: A New Inflammatory Pathway of Relevance to Diabetes?

Preliminary data mostly from animal models suggest the sST2/IL-33 pathway may have causal relevance for vascular disease and diabetes and thus point to a potential novel inflammatory link to cardiometabolic disease. However, the characterisation of sST2 levels in terms of metabolic or vascular risk in man is completely lacking. We sought to address this gap via a comprehensive analysis of risk factor and vascular correlates of sST2 in a cross-sectional study (pSoBid). We measured sST2 in plasma in 639 subjects and comprehensively related it to cardiovascular and diabetes risk factors and imaged atherosclerosis measures. Circulating sST2 levels increased with age, were lower in women and in highest earners. After adjusting for age and gender, sST2 levels associated strongly with markers of diabetes, including triglycerides [effect estimate (EE) per 1 standard deviation increase in sST2∶1.05 [95%CI 1.01,1.10]), liver function (alanine aminotransaminase [ALT] and γ-glutamyl transferase [GGT]: EE 1.05 [1.01,1.09] and 1.13 [1.07,1.19] respectively), glucose (1.02 [1.00,1.03]) and sICAM-1 (1.05 [1.02,1.07]). However, sST2 levels were not related to smoking, cholesterol, blood pressure, or atheroma (carotid intima media thickness, plaque presence). These results suggest that sST2 levels, in individuals largely without vascular disease, are related principally to markers associated with diabetes and ectopic fat and add support for a role of sST2 in diabetes. Further mechanistic studies determining how sST2 is linked to diabetes pathways may offer new insights into the inflammatory paradigm for type 2 diabetes.     

The Indolinone MAZ51 Induces Cell Rounding and G2/M Cell Cycle Arrest in Glioma Cells without the Inhibition of VEGFR-3 Phosphorylation: Involvement of the RhoA and Akt/GSK3β Signaling Pathways

MAZ51 is an indolinone-based molecule originally synthesized as a selective inhibitor of vascular endothelial growth factor receptor (VEGFR)-3 tyrosine kinase. This study shows that exposure of two glioma cell lines, rat C6 and human U251MG, to MAZ51 caused dramatic shape changes, including the retraction of cellular protrusions and cell rounding. These changes were caused by the clustering and aggregation of actin filaments and microtubules. MAZ51 also induced G2/M phase cell cycle arrest. This led to an inhibition of cellular proliferation, without triggering significant cell death. These alterations induced by MAZ51 occurred with similar dose- and time-dependent patterns. Treatment of glioma cells with MAZ51 resulted in increased levels of phosphorylated GSK3β through the activation of Akt, as well as increased levels of active RhoA. Interestingly, MAZ51 did not affect the morphology and cell cycle patterns of rat primary cortical astrocytes, suggesting it selectively targeted transformed cells. Immunoprecipitation–western blot analyses indicated that MAZ51 did not decrease, but rather increased, tyrosine phosphorylation of VEGFR-3. To confirm this unanticipated result, several additional experiments were conducted. Enhancing VEGFR-3 phosphorylation by treatment of glioma cells with VEGF-C affected neither cytoskeleton arrangements nor cell cycle patterns. In addition, the knockdown of VEGFR-3 in glioma cells did not cause morphological or cytoskeletal alterations. Furthermore, treatment of VEGFR-3-silenced cells with MAZ51 caused the same alterations of cell shape and cytoskeletal arrangements as that observed in control cells. These data indicate that MAZ51 causes cytoskeletal alterations and G2/M cell cycle arrest in glioma cells. These effects are mediated through phosphorylation of Akt/GSK3β and activation of RhoA. The anti-proliferative activity of MAZ51 does not require the inhibition of VEGFR-3 phosphorylation, suggesting that it is a potential candidate for further clinical investigation for treatment of gliomas, although the precise mechanism(s) underlying its effects remain to be determined.     

Association between Class III Obesity (BMI of 40–59 kg/m2) and Mortality: A Pooled Analysis of 20 Prospective Studies

Background

The prevalence of class III obesity (body mass index [BMI]≥40 kg/m²) has increased dramatically in several countries and currently affects 6% of adults in the US, with uncertain impact on the risks of illness and death. Using data from a large pooled study, we evaluated the risk of death, overall and due to a wide range of causes, and years of life expectancy lost associated with class III obesity.

Methods and Findings

In a pooled analysis of 20 prospective studies from the United States, Sweden, and Australia, we estimated sex- and age-adjusted total and cause-specific mortality rates (deaths per 100,000 persons per year) and multivariable-adjusted hazard ratios for adults, aged 19–83 y at baseline, classified as obese class III (BMI 40.0–59.9 kg/m²) compared with those classified as normal weight (BMI 18.5–24.9 kg/m²). Participants reporting ever smoking cigarettes or a history of chronic disease (heart disease, cancer, stroke, or emphysema) on baseline questionnaires were excluded. Among 9,564 class III obesity participants, mortality rates were 856.0 in men and 663.0 in women during the study period (1976–2009). Among 304,011 normal-weight participants, rates were 346.7 and 280.5 in men and women, respectively. Deaths from heart disease contributed largely to the excess rates in the class III obesity group (rate differences = 238.9 and 132.8 in men and women, respectively), followed by deaths from cancer (rate differences = 36.7 and 62.3 in men and women, respectively) and diabetes (rate differences = 51.2 and 29.2 in men and women, respectively). Within the class III obesity range, multivariable-adjusted hazard ratios for total deaths and deaths due to heart disease, cancer, diabetes, nephritis/nephrotic syndrome/nephrosis, chronic lower respiratory disease, and influenza/pneumonia increased with increasing BMI. Compared with normal-weight BMI, a BMI of 40–44.9, 45–49.9, 50–54.9, and 55–59.9 kg/m² was associated with an estimated 6.5 (95% CI: 5.7–7.3), 8.9 (95% CI: 7.4–10.4), 9.8 (95% CI: 7.4–12.2), and 13.7 (95% CI: 10.5–16.9) y of life lost. A limitation was that BMI was mainly ascertained by self-report.

Conclusions

Class III obesity is associated with substantially elevated rates of total mortality, with most of the excess deaths due to heart disease, cancer, and diabetes, and major reductions in life expectancy compared with normal weight. Please see later in the article for the Editors'' Summary     

Phosphorylation Sites Within α4 Subunits of α4β2 Neuronal Nicotinic Receptors: A Comparison of Substrate Specificities for cAMP-Dependent Protein Kinase (PKA) and Protein Kinase C (PKC)

The present study determined whether putative phosphorylation sites within the M3/M4 cytoplasmic domain of the human 4 subunit of 42 neuronal nicotinic receptors are substrates for cAMP-dependent protein kinase (PKA) or protein kinase C (PKC). Five peptides corresponding to predicted phosphorylation sequences were synthesized, and phosphorylation was compared with standard peptide substrates for each kinase, that is, Kemptide for PKA and glycogen synthase (GS) 1-8 for PKC. VRCRSRSI had the highest affinity for PKA, with a Km of 44.5 M; Kemptide had a Km of 7.7 M. LMKRPSVVK and KARSLSVQH were also phosphorylated by PKA, but had lower affinities of 593 M and 2896 M, respectively. LMKRPSVVK had the highest affinity for PKC with a Km of 182 M; GS 1–8 had a Km of 2.1 M. VRCRSRSI had a comparative affinity for PKC with a Km of 327 M. PCKCTCKK was not phosphorylated by PKA, but was a substrate for PKC with a Km of 1392 M, whereas PGPSCKSP was not phosphorylated by either kinase. Based on these findings, results suggest that Ser-362 and Ser-486 on the human 4 subunit may be phosphorylated by either PKA or PKC, Ser-467 is a putative PKA site, and Thr-532 represents a likely PKC substrate; Ser-421 does not appear to be phosphorylated by either kinase.     

Fatty Kidney Disease: A New Renal And Endocrine Clinical Entity? Describing the Role of the Kidney in Obesity,Metabolic Syndrome,and Type 2 Diabetes

Objective: To determine whether fatty kidney disease deserves be designated as a distinct clinical entity similar to fatty liver disease.Methods: Analysis and interpretation of the literature in a novel conceptual framework.Results: The kidney contributes to hyperglycemia, hypertension, inflammatory cytokines, and thus to diabetes and metabolic syndrome. Fat accumulation in and around the kidney drives this process and contributes to progression of chronic kidney disease itself. Weight loss improves these complications of fatty kidney. Diagnosis currently must be inferred from comorbidities but ultimately should be made by imaging once the importance of fatty kidney disease is established, much like fatty liver disease.Conclusion: Fatty kidney disease merits designation as a specific clinical entity similar to fatty liver disease. Greater attention to this may help encourage research into ameliorating the negative consequences of fatty kidney disease and developing new therapies.Abbreviations: BP = blood pressure; CKD = chronic kidney disease; CT = computed tomography; ESRD = end-stage renal disease; FFA = free fatty acid; FKD = fatty kidney disease; GFR = glomerular filtration rate; MetS = metabolic syndrome; MRI = magnetic resonance imaging; NAFLD = nonalcoholic fatty liver disease; RAAS = renin-angiotensin system; SGLT2 = sodium-glucose cotransporter 2; SNS = sympathetic nervous system; T2D = type 2 diabetes; TG = triglyceride