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An association between cardiovascular and periodontal disease may be due to lipopolysaccharide (LPS)-promoted release of inflammatory mediators, adverse alterations of the lipoprotein profile, and an imbalance in cholesterol homeostasis. Since periodontopathogenic potential differs between serotypes of a major periodontal pathogen, Actinobacillus actinomycetemcomitans, we studied the pro-atherogenic properties of LPS preparations from serotypes b and d strains on macrophages (RAW 264.7). A. actinomycetemcomitans LPS preparations induced a time-dependent release of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta). LPS induced foam cell formation and cholesteryl ester accumulation from native low density lipoprotein in the following order: A. actinomycetemcomitans strains JP2 (serotype b) > Y4 (serotype b) > IDH781 (serotype d). mRNA expression levels of scavenger receptor class B, type-I, and ATP-binding cassette transporter-1, receptors mediating cholesterol efflux from macrophages, were decreased by LPS preparations. The results suggest that the pro-atherogenic potential of A. actinomycetemcomitans LPS may depend on the infecting strain and correlate with the periodontopathogenic potential of the pathogen.

An association between cardiovascular and periodontal disease may be due to lipopolysaccharide (LPS)-promoted release of inflammatory mediators, adverse alterations of the lipoprotein profile, and an imbalance in cholesterol homeostasis. Since periodontopathogenic potential differs between serotypes of a major periodontal pathogen, Actinobacillus actinomycetemcomitans, we studied the pro-atherogenic properties of LPS preparations from serotypes b and d strains on macrophages (RAW 264.7). A. actinomycetemcomitans LPS preparations induced a time-dependent release of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). LPS induced foam cell formation and cholesteryl ester accumulation from native low density lipoprotein in the following order: A. actinomycetemcomitans strains JP2 (serotype b) > Y4 (serotype b) > IDH781 (serotype d). mRNA expression levels of scavenger receptor class B, type-I, and ATP-binding cassette transporter-1, receptors mediating cholesterol efflux from macrophages, were decreased by LPS preparations. The results suggest that the pro-atherogenic potential of A. actinomycetemcomitans LPS may depend on the infecting strain and correlate with the periodontopathogenic potential of the pathogen.

ORP1L is a member of the human oxysterol-binding protein (OSBP) family. ORP1L localizes to late endosomes (LEs)/lysosomes, colocalizing with the GTPases Rab7 and Rab9 and lysosome-associated membrane protein-1. We demonstrate that ORP1L interacts physically with Rab7, preferentially with its GTP-bound form, and provide evidence that ORP1L stabilizes GTP-bound Rab7 on LEs/lysosomes. The Rab7-binding determinant is mapped to the ankyrin repeat (ANK) region of ORP1L. The pleckstrin homology domain (PHD) of ORP1L binds phosphoinositides with low affinity and specificity. ORP1L ANK- and ANK+PHD fragments induce perinuclear clustering of LE/lysosomes. This is dependent on an intact microtubule network and a functional dynein/dynactin motor complex. The dominant inhibitory Rab7 mutant T22N reverses the LE clustering, suggesting that the effect is dependent on active Rab7. Transport of fluorescent dextran to LEs is inhibited by overexpression of ORP1L. Overexpression of ORP1L, and in particular the N-terminal fragments of ORP1L, inhibits vacuolation of LE caused by Helicobacter pylori toxin VacA, a process also involving Rab7. The present study demonstrates that ORP1L binds to Rab7, modifies its functional cycle, and can interfere with LE/lysosome organization and endocytic membrane trafficking. This is the first report of a direct connection between the OSBP-related protein family and the Rab GTPases.

The intracellular targeting determinants of oxysterol binding protein (OSBP)-related protein 3 (ORP3) were studied using a series of truncated and point mutated constructs. The pleckstrin homology (PH) domain of ORP3 binds the phosphoinositide-3-kinase (PI3K) products, PI(3,4)P2 and PI(3,4,5)P3. A functional PH domain and flanking sequences are crucial for the plasma membrane (PM) targeting of ORP3. The endoplasmic reticulum (ER) targeting of ORP3 is regulated the by a FFAT motif (EFFDAxE), which mediates interaction with VAMP-associated protein (VAP)-A. The targeting function of the FFAT motif dominates over that of the PH domain. In addition, the exon 10/11 region modulates interaction of ORP3 with the ER and the nuclear membrane. Analysis of a chimeric ORP3:OSBP protein suggests that ligand binding by the C-terminal domain of OSBP induces allosteric changes that activate the N-terminal targeting modules of ORP3. Notably, over-expression of ORP3 together with VAP-A induces stacked ER membrane structures also known as organized smooth ER (OSER). Moreover, lipid starvation promotes formation of dilated peripheral ER (DPER) structures dependent on the ORP3 protein. Based on the present data, we introduce a model for the inter-relationships of the functional domains of ORP3 in the membrane targeting of the protein.

The human OSBP related protein (ORP) family consists of 12 members, which can be divided into six subfamilies based on the genomic organization and amino acid homology. Here we performed basic characterization of subfamily III, which consists of three members: ORP3, ORP6, and ORP7. According to cDNA hybridization, the three genes are expressed in a tissue-specific manner. While ORP3 mRNA is most abundant in kidney, lymph nodes, and thymus, ORP6 shows highest expression in brain and skeletal muscle, and ORP7 in the gastrointestinal tract. Using monospecific peptide antibodies, we confirmed the presence of the three proteins in human and mouse tissues. ORP6 gene expression was induced upon differentiation of F9 embryonic carcinoma cells into parietal endoderm, while ORP3 and ORP7 mRNA levels were unchanged. In the F9 cells, endogenous ORP6 associated predominantly with the nuclear envelope. When expressed from the cDNA in cultured cells, the three proteins were distributed between the cytosol and endoplasmic reticulum (ER) membranes, with a minor portion found at the plasma membrane. Experiments with truncated constructs showed that the N-terminal portion of the proteins, containing a pleckstrin homology (PH) domain, has markedly strong plasma membrane targeting specificity, while the C-terminal half remains largely cytosolic. The expression data demonstrates that ORP3, -6, and -7 are not merely redundant gene products but show marked quantitative differences in tissue expression, suggesting tissue-specific aspects in their function. The dual targeting of the proteins indicates a putative role in communication between the ER and the plasma membrane.

Oxidized derivatives of cholesterol have been investigated actively for decades in the context of the oxidative hypothesis of atherosclerosis. Oxysterols arise in our tissues as a result of enzymatic or non-enzymatic oxidation reactions and are also obtained from dietary sources. Even though these compounds are found enriched in the atherosclerotic lesions in arterial walls, the plasma concentrations of oxysterols cannot, in the light of current knowledge, be regarded as a risk factor for atherosclerotic disease. However, oxysterols may still have important local effects in the arterial wall as factors that regulate the cellular lipid homeostasis and possibly the maturation of the lesions. Work during the past few years has revealed that oxysterols have a potential as signaling molecules that may play important roles in lipid metabolism, especially the reverse cholesterol transport process. This finding has recently moved oxysterols and the protein mediators of their biological effects, liver X receptors and cytosolic oxysterol binding proteins, into the center stage of atherosclerosis research.

Oxysterol binding protein (OSBP) homologs comprise a family of 12 proteins in humans ( Jaworski et al., 2001 ; Lehtoet al., 2001 ). Two variants of OSBP-related protein (ORP) 1 have been identified: a short one that consists of the carboxy-terminal ligand binding domain only (ORP1S, 437 aa) and a longer N-terminally extended form (ORP1L, 950 aa) encompassing three ankyrin repeats and a pleckstrin homology domain (PHD). We now report that the two mRNAs show marked differences in tissue expression. ORP1S predominates in skeletal muscle and heart, whereas ORP1L is the most abundant form in brain and lung. On differentiation of primary human monocytes into macrophages, both ORP1S and ORP1L mRNAs were induced, the up-regulation of ORP1L being >100-fold. The intracellular localization of the two ORP1 variants was found to be different. Whereas ORP1S is largely cytosolic, the ORP1L variant localizes to late endosomes. A significant amount of ORP1S but only little ORP1L was found in the nucleus. The ORP1L ankyrin repeat region (aa 1–237) was found to localize to late endosomes such as the full-length protein. This localization was even more pronounced for a fragment that additionally includes the PHD (aa 1–408). The amino-terminal region of ORP1L consisting of the ankyrin repeat and PHDs is therefore likely to be responsible for the targeting of ORP1L to late endosomes. Interestingly, overexpression of ORP1L was found to enhance the LXRα-mediated transactivation of a reporter gene, whereas ORP1S failed to influence this process. The results suggest that the two forms of ORP1 are functionally distinct and that ORP1L is involved in control of cellular lipid metabolism.

Proteins/genes showing high sequence homology to the mammalian oxysterol binding protein (OSBP) have been identified in a variety of eukaryotic organisms from yeast to man. The unifying feature of the gene products denoted as OSBP-related proteins (ORPs) is the presence of an OSBP-type ligand binding (LB) domain. The LB domains of OSBP and its closest homologue bind oxysterols, while data on certain other family members suggest interaction with phospholipids. Many ORPs also have a pleckstrin homology (PH) domain in the amino-terminal region. The PH domains of the family members studied in detail are known to interact with membrane phosphoinositides and play an important role in the intracellular targeting of the proteins. It is plausible that the ORPs constitute a regulatory apparatus that senses the status of specific lipid ligands in membranes, using the PH and/or LB domains, and mediates information to yet poorly known downstream machineries. Functional studies carried out on the ORP proteins in different organisms indicate roles of the gene family in diverse cellular processes including control of lipid metabolism, regulation of vesicle transport, and cell signalling events.

In an attempt to identify novel susceptibility genes predisposing to early-onset diabetes (EOD), we performed a genome-wide scan using 433 markers in 222 individuals (119 with diabetes) from 29 Scandinavian families with ≥2 members with onset of diabetes ≤45 years. The highest nonparametric linkage (NPL) score, 2.7 (P < 0.01), was observed on chromosome 1p (D1S473/D1S438). Six other regions on chromosomes 3p, 7q, 11q, 18q, 20q, and 21q showed a nominal P value

Circulating lipopolysaccharides (LPSs), associated with both infection and inflammation, may arise from the gastrointestinal tract microbiota, and the levels may be affected by daily nutrition. We investigated whether nutrient intake affects the association of serum LPS activity with prevalent obesity, metabolic syndrome (MetS), diabetes, and coronary heart disease (CHD) and with the risk of incident CHD events. The nutrition cohort (n = 2,452, mean age ± SD, 52.2 ± 10.1 years) of the FINRISK 1997 Study was followed up for 10 years. Information on macronutrient intake at baseline was collected from 24-h dietary recall. Serum endotoxin activities were determined by the Limulus amebocyte lysate assay. LPS activity was associated directly with the total energy intake and indirectly with carbohydrate intake in lean, healthy subjects. High LPS was significantly associated with prevalent obesity, MetS, diabetes, and CHD events, independently of established risk factors, CRP, and total energy or nutrient intake. The ORs (95 % CI) were 1.49 (1.21–1.85, p

Diabetic kidney disease (DKD) is the most common cause of ESRD in the United States. Podocyte injury is an important feature of DKD that is likely to be caused by circulating factors other than glucose. Soluble urokinase plasminogen activator receptor (suPAR) is a circulating factor found to be elevated in the serum of patients with FSGS and causes podocyte αVβ3 integrin-dependent migration in vitro. Furthermore, αVβ3 integrin activation occurs in association with decreased podocyte-specific expression of acid sphingomyelinase-like phosphodiesterase 3b (SMPDL3b) in kidney biopsy specimens from patients with FSGS. However, whether suPAR-dependent αVβ3 integrin activation occurs in diseases other than FSGS and whether there is a direct link between circulating suPAR levels and SMPDL3b expression in podocytes remain to be established. Our data indicate that serum suPAR levels are also elevated in patients with DKD. However, unlike in FSGS, SMPDL3b expression was increased in glomeruli from patients with DKD and DKD sera-treated human podocytes, where it prevented αVβ3 integrin activation by its interaction with suPAR and led to increased RhoA activity, rendering podocytes more susceptible to apoptosis. In vivo, inhibition of acid sphingomyelinase reduced proteinuria in experimental DKD but not FSGS, indicating that SMPDL3b expression levels determined the podocyte injury phenotype. These observations suggest that SMPDL3b may be an important modulator of podocyte function by shifting suPAR-mediated podocyte injury from a migratory phenotype to an apoptotic phenotype and that it represents a novel therapeutic glomerular disease target.

Dietary fats have been shown to promote the translocation of bacterial endotoxins from the gut into circulation, which may induce systemic inflammation and modulate the inflammatory response of circulating immune cells. The aim of this study was to determine the effect of the postprandial milieu on inflammation and the inflammatory response of antigen presenting cells in the context of type 1 diabetes (T1D). Eleven patients with T1D and eleven nondiabetic controls were recruited as part of the FinnDiane study and given two fatty meals during 1 day. Cytokine responses in monocytes and myeloid dendritic cells (mDCs) as well as serum lipopolysaccharide activity levels, triglyceride concentrations and cytokine concentrations were measured from fasting and postprandial blood samples. Postprandially, patients with T1D and controls showed significant increases in eight inflammatory cytokines (IL-6, TNF-α, IL-1β, IFN-α, IL-10, IFN-γ, IL-12 and MIP-1β) without concomitant increase in serum LPS activity. Serum cytokine production was similar in both groups. No postprandial change was seen in the IL-6, TNF-α or IL-1β production of mDCs or monocytes. At fasting, diabetic mDCs exhibited higher LPS-induced IL-6 and IL-1β production than controls. Acute high-fat meals increase circulating cytokines but have no effect on serum lipopolysaccharide activity levels or cytokine production in circulating mDCs or monocytes. Our results suggest that postprandial increase in serum cytokine levels is neither mediated by circulating endotoxins nor the activation of circulating innate cells. The production of high-fat meal-induced inflammatory markers is most likely regulated at the tissue level.

Oxysterols are oxygenated derivatives of cholesterol that have a number of biological effects and play a key role in the maintenance of the body cholesterol balance. In this study, we describe the cDNA sequences and genomic structures of the recently identified human oxysterol-binding protein (OSBP)-related protein (ORP) family (Laitinen, S. et al. 1999. J. Lipid Res. 40: 2204–2211). The family now includes 12 genes/proteins, which can be divided into six distinct subfamilies. The ORP have two major structural features: a highly conserved OSBP-type sterol-binding domain in the C-terminal half and a pleckstrin homology domain present in the N-terminal region of most family members. Several ORP genes are present in S. cerevisiae, D. melanogaster, and C. elegans, suggesting that the protein family has functions of fundamental importance in the eukaryotic kingdom. Analysis of ORP mRNA levels in unloaded or acetylated LDL-loaded human macrophages revealed that the expression of ORP genes was not significantly affected by the loading, with the exception of ORP6, which was up-regulated 2-fold. The present study summarizes the basic characteristics of the OSBP-related gene/protein family in humans, and provides tools for functional analysis of the encoded proteins. —Lehto, M., S. Laitinen, G. Chinetti, M. Johansson, C. Ehnholm, B. Staels, E. Ikonen, and V. M. Olkkonen. The OSBP-related protein family in humans.

Protein families related to OSBP (oxysterol-binding protein) are present in eukaryotes from yeast to human. The functions of the ORPs (OSBP-related proteins) have remained largely enigmatic. Even though they have been implicated in the function of ERJs (endoplasmic reticulum junctions), it is evident that any single model for their mechanism of action is insufficient. The existing evidence points in many different directions, such as integration of sterol and sphingomyelin metabolism, regulation of neutral lipid metabolism, control of signalling cascades, regulation of secretory vesicle generation, and function in the microtubule-based motility of endo/lysosomes. Some of these functions could involve ERJ and non-vesicular transport of lipids, but this is unlikely to be the unifying feature. We believe, rather, that the common denominator for ORP function is acting as sterol sensors that relay information to a spectrum of cellular processes.

Maturity-onset diabetes of the young 3 (MODY3) is a type of NIDDM caused by mutations in the transcription factor hepatocyte nuclear factor-la (HNF-la) located on chromosome 12q. We have identified four novel HNF-la missense mutations in M0DY3 families. In four additional and unrelated families, we observed an identical insertion mutation that had occurred in a polycytidine tract in exon 4. Among those families, one exhibited a de novo mutation at this location. We propose that instability of this sequence represents a general mutational mechanism in M0DY3. We observed no HNFla mutations among 86 unrelated late-onset diabetic patients with relative insulin deficiency. Hence mutations in this gene appear to be most strongly associated with early-onset diabetes. Diabetes 46:1081-1086,1997 MODY. The MODY1 locus, on chromosome 20q, was shown to be linked in a single extended family (2). M0DY1 was recently shown to be caused by mutations in the transcription factor hepatocyte nuclear factor-la (HNF4a) (3). MODY2, located on chromosome 7p (4), is caused by mutations in the glucokinase gene (5). MODY3 was mapped to chromosome 12q (6) and is known to be caused by mutations in another transcription factor, HNF-la (7). Although the relative prevalence of these MODY subtypes varies regionally (8-10), families linked to chromosome 12q are the most frequently described. The glucokinase defects in MODY2 interfere with glucose uptake, resulting in hyperglycemia (5). Although the mechanism of hyperglycemia in MODY1 and MODY3 is not yet understood, phenotypic characterization of these families has shown a deficient insulin secretion response to glucose (11-13). We report here the characterization of the MODY3 gene

Hypoxemia is common in diabetes, and reflex responses to hypoxia are blunted. These abnormalities could lead to cardiovascular/renal complications. Interval hypoxia (IH) (5–6 short periods of hypoxia each day over 1–3 weeks) was successfully used to improve the adaptation to hypoxia in patients with chronic obstructive pulmonary disease. We tested whether IH over 1 day could initiate a long-lasting response potentially leading to better adaptation to hypoxia. In 15 patients with type 1 diabetes, we measured hypoxic and hypercapnic ventilatory responses (HCVRs), ventilatory recruitment threshold (VRT-CO2), baroreflex sensitivity (BRS), blood pressure, and blood lactate before and after 0, 3, and 6 h of a 1-h single bout of IH. All measurements were repeated on a placebo day (single-blind protocol, randomized sequence). After IH (immediately and after 3 h), hypoxic and HCVR increased, whereas the VRT-CO2 dropped. No such changes were observed on the placebo day. Systolic and diastolic blood pressure increased, whereas blood lactate decreased after IH. Despite exposure to hypoxia, BRS remained unchanged. Repeated exposures to hypoxia over 1 day induced an initial adaptation to hypoxia, with improvement in respiratory reflexes. Prolonging the exposure to IH (>2 weeks) in type 1 diabetic patients will be a matter for further studies.

Ammonia concentrations in exhaled breath (eNH(3)) and skin gas of 20 healthy subjects were measured on-line with a commercial cavity ring-down spectrometer and compared to saliva pH and plasma ammonium ion (NH4+), urea and creatinine concentrations. Special attention was given to mouth, nose and skin sampling procedures and the accurate quantification of ammonia in humid gas samples. The obtained median concentrations were 688 parts per billion by volume (ppbv) for mouth-eNH(3), 34 ppbv for nose-eNH3, and 21 ppbv for both mouth-and nose-eNH(3) after an acidic mouth wash (MW). The median ammonia emission rate from the lower forearm was 0.3 ng cm(-2) min(-1). Statistically significant (p < 0.05) correlations between the breath, skin and plasma ammonia/ammonium concentrations were not found. However, mouth-eNH(3) strongly (p < 0.001) correlated with saliva pH. This dependence was also observed in detailed measurements of the diurnal variation and the response of eNH(3) to the acidic MW. It is concluded that eNH(3) as such does not reflect plasma but saliva and airway mucus NH4+ concentrations and is affected by saliva and airway mucus pH. After normalization with saliva pH using the Henderson-Hasselbalch equation, mouth-eNH(3) correlated with plasma NH4+, which points to saliva and plasma NH4+ being linked via hydrolysis of salivary urea.

In muscle, hexokinase II (HK2) regulates phosphorylation of glucose to glucose 6-phosphate, which has been reported to be impaired in patients with non-insulin-dependent diabetes mellitus (NIDDM). Here we report decreased HK2 enzyme activity in skeletal muscle biopsies from patients with impaired glucose tolerance compared with healthy control subjects (2.7 +/- 0.9 vs 4.9 +/- 1.1 nmol.min-1.mg protein-1). Therefore, mutations in the HK2 gene could contribute to skeletal muscle insulin resistance in NIDDM. To address this question, we first determined the exon-intron structure of the human HK2 gene and using this information, we screened all 18 exons with single-strand conformation polymorphism technique in 80 Finnish NIDDM patients. Nine nucleotide substitutions were found, one of which was a missense mutation (Gln142-His142) in exon 4. In human muscle, a single HK2 mRNA transcript with a size of approximately 5500 nucleotides was detected with Northern blot analysis. We also describe an HK2 pseudogene (HK2P1), which was mapped to chromosome 4, band q26, by fluorescence in situ hybridization to metaphase chromosomes. The clinical characteristics and HK2 enzyme activities of the subjects with either Gln or His at residue 142 did not differ from each other. Instead, HK2 activity correlated inversely with fasting blood glucose levels, suggesting that changes in HK2 activity could be secondary to other metabolic abnormalities (r = 0.55; p0.0003; n = 39). In conclusion; the data suggest that impaired HK2 activity in prediabetic individuals is a consequence of impaired glucose tolerance rather than of a genetic abnormality. The data thus seem to rule out mutations in the HK2 gene as a major cause of inherited insulin resistance in NIDDM.

Bacterial endotoxins have been associated with chronic inflammation and the development and progression of diabetic nephropathy. We hypothesized that subjects with high serum lipopolysaccharide activity also carry remains of bacterial DNA in their system. Serum-derived bacterial DNA clones were isolated and identified from 10 healthy controls and 14 patients with type 1 diabetes (T1D) using universal primers targeted to bacterial 16S rDNA. A total of 240 clones representing 35 unique bacterial species were isolated and identified. A significant proportion of the isolated bacteria could be assigned to our living environment. Proteobacteria was by far the most prevalent phylum among the samples. Notably, the patients had significantly higher frequencies of Stenotrophomonas maltophilia clones in their sera compared to the healthy controls. Real-time PCR analysis of S. maltophilia and Pseudomonas aeruginosa flagellin gene copy number in the human leukocyte DNA fraction revealed that the overall Pseudomonal bacterial load was higher in older patients with T1D. Serum IgA- and IgG-antibody levels against Pseudomonal bacteria Delftia acidovorans, P. aeruginosa, and S. maltophilia were also determined in 200 healthy controls and 200 patients with T1D. The patients had significantly higher serum levels of IgA antibodies against all three Pseudomonal bacteria. Additionally, the IgA antibodies against Pseudomonal bacteria correlated significantly with serum C-reactive protein. These findings indicate that recurrent or chronic Pseudomonal exposure may increase susceptibility to chronic inflammation in patients with T1D.

Diabetic kidney disease, or diabetic nephropathy (DN), is a major complication of diabetes and the leading cause of end-stage renal disease (ESRD) that requires dialysis treatment or kidney transplantation. In addition to the decrease in the quality of life, DN accounts for a large proportion of the excess mortality associated with type 1 diabetes (T1D). Whereas the degree of glycemia plays a pivotal role in DN, a subset of individuals with poorly controlled T1D do not develop DN. Furthermore, strong familial aggregation supports genetic susceptibility to DN. However, the genes and the molecular mechanisms behind the disease remain poorly understood, and current therapeutic strategies rarely result in reversal of DN. In the GEnetics of Nephropathy: an International Effort (GENIE) consortium, we have undertaken a meta-analysis of genome-wide association studies (GWAS) of T1D DN comprising ∼2.4 million single nucleotide polymorphisms (SNPs) imputed in 6,691 individuals. After additional genotyping of 41 top ranked SNPs representing 24 independent signals in 5,873 individuals, combined meta-analysis revealed association of two SNPs with ESRD: rs7583877 in the AFF3 gene (P = 1.2×10−8) and an intergenic SNP on chromosome 15q26 between the genes RGMA and MCTP2, rs12437854 (P = 2.0×10−9). Functional data suggest that AFF3 influences renal tubule fibrosis via the transforming growth factor-beta (TGF-β1) pathway. The strongest association with DN as a primary phenotype was seen for an intronic SNP in the ERBB4 gene (rs7588550, P = 2.1×10−7), a gene with type 2 diabetes DN differential expression and in the same intron as a variant with cis-eQTL expression of ERBB4. All these detected associations represent new signals in the pathogenesis of DN., Author Summary The global prevalence of diabetes has reached epidemic proportions, constituting a major health care problem worldwide. Diabetic kidney disease, or diabetic nephropathy (DN)—the major long term microvascular complication of diabetes—is associated with excess mortality among patients with type 1 diabetes. Even though DN has been shown to cluster in families, the underlying genetic and molecular pathways remain poorly defined. We have undertaken the largest genome-wide association study and meta-analysis to date on DN and on its most severe form of kidney disease, end-stage renal disease (ESRD). We identified new loci significantly associated with diabetic ESRD: AFF3 and an intergenic locus on chromosome 15q26 residing between RGMA and MCTP2. Our functional analyses suggest that AFF3 influences renal tubule fibrosis, a pathological hallmark of severe DN. Another locus in ERBB4 was suggestively associated with DN and resides in the same intronic region as a variant affecting the expression of ERBB4. Subsequent pathway analysis of the genes co-expressed with ERBB4 indicated involvement of fibrosis.

Activation of the receptor for AGE (RAGE) is implicated in the development and progression of vascular complications of diabetes. In this study, we explore factors and mortality outcomes associated with soluble RAGE (sRAGE) in a multicentre nationwide cohort of Finnish adults with type 1 diabetes. Baseline sRAGE concentrations were estimated in 3,100 adults with type 1 diabetes. Clinical and biological variables independently associated with sRAGE were identified using multivariate regression analysis. Independent predictors of mortality were determined using Cox and Fine–Gray proportional-hazards models. The main independent determinants of sRAGE concentrations were estimated glomerular filtration rate, albuminuria, body mass index, age, duration of diabetes, HbA1c and insulin dose (all p

OBJECTIVE The aim of the study was to investigate whether serum lipopolysaccharide (LPS) activities are associated with the progression of kidney disease in patients with type 1 diabetes. RESEARCH DESIGN AND METHODS For this prospective study, we chose 477 Finnish patients with type 1 diabetes, who were followed for 6 years. At the baseline visit, 239 patients had a normal albumin excretion rate (normoalbuminuria) and 238 patients had macroalbuminuria. Patients were further divided into nonprogressors and progressors based on their albumin excretion rate at follow-up. Eighty normoalbuminuric patients had developed microalbuminuria, and 79 macroalbuminuric patients had progressed to end-stage renal disease. Serum LPS activity was determined with the Limulus amoebocyte lysate chromogenic end point assay. RESULTS Serum LPS activity was significantly higher in the macroalbuminuric group than in the normoalbuminuric group (P < 0.001). Notably, normoalbuminuric progressor patients had a significantly higher LPS activity at baseline than normoalbuminuric nonprogressor patients (median 49 [interquartile range 34–87] vs. 39 [29–54] EU/ml; P = 0.001). The normoalbuminuric progressor patients exhibited features of the metabolic syndrome with higher triglyceride concentrations and lower estimated glucose disposal rate. A high LPS-to-HDL ratio was associated with the progression of kidney disease in both groups. Insulin resistance (P < 0.001) and serum LPS activity (P = 0.026) were independent risk factors of disease development, when A1C was removed from the regression analysis. CONCLUSIONS High serum LPS activity is associated with the development of diabetic nephropathy in Finnish patients with type 1 diabetes.

Oxysterol-binding protein (OSBP)-related protein 3 (ORP3) is highly expressed in epithelial, neuronal and hematopoietic cells, as well as in certain forms of cancer. We assessed the function of ORP3 in HEK293 cells and in human macrophages. We show that ORP3 interacts with R-Ras, a small GTPase regulating cell adhesion, spreading and migration. Gene silencing of ORP3 in HEK293 cells results in altered organization of the actin cytoskeleton, impaired cell-cell adhesion, enhanced cell spreading and an increase of β1 integrin activity–effects similar to those of constitutively active R-Ras(38V). Overexpression of ORP3 leads to formation of polarized cell-surface protrusions, impaired cell spreading and decreased β1 integrin activity. In primary macrophages, overexpression of ORP3 leads to the disappearance of podosomal structures and decreased phagocytotic uptake of latex beads, consistent with a role in actin regulation. ORP3 is phosphorylated when cells lose adhesive contacts, suggesting that it is subject to regulation by outside-in signals mediated by adhesion receptors. The present findings demonstrate a new function of ORP3 as part of the machinery that controls the actin cytoskeleton, cell polarity and cell adhesion.

OSBP (oxysterol-binding protein) homologues, ORPs (OSBP-related proteins), constitute a 12-member family in mammals. We employed an in vitro [3H]25OH (25-hydroxycholesterol)-binding assay with purified recombinant proteins as well as live cell photo-cross-linking with [3H]photo-25OH and [3H]photoCH (photo-cholesterol), to investigate sterol binding by the mammalian ORPs. ORP1 and ORP2 [a short ORP consisting of an ORD (OSBP-related ligand-binding domain) only] were in vitro shown to bind 25OH. GST (glutathione S-transferase) fusions of the ORP1L [long variant with an N-terminal extension that carries ankyrin repeats and a PH domain (pleckstrin homology domain)] and ORP1S (short variant consisting of an ORD only) variants bound 25OH with similar affinity (ORP1L, Kd=9.7×10−8 M; ORP1S, Kd=8.4 ×10−8 M), while the affinity of GST–ORP2 for 25OH was lower (Kd=3.9×10−6 M). Molecular modelling suggested that ORP2 has a sterol-binding pocket similar to that of Saccharomyces cerevisiae Osh4p. This was confirmed by site-directed mutagenesis of residues in proximity of the bound sterol in the structural model. Substitution of Ile249 by tryptophan or Lys150 by alanine markedly inhibited 25OH binding by ORP2. In agreement with the in vitro data, ORP1L, ORP1S, and ORP2 were cross-linked with photo-25OH in live COS7 cells. Furthermore, in experiments with either truncated cDNAs encoding the OSBP-related ligand-binding domains of the ORPs or the full-length proteins, photo-25OH was bound to OSBP, ORP3, ORP4, ORP5, ORP6, ORP7, ORP8, ORP10 and ORP11. In addition, the ORP1L variant and ORP3, ORP5, and ORP8 were cross-linked with photoCH. The present study identifies ORP1 and ORP2 as OSBPs and suggests that most of the mammalian ORPs are able to bind sterols.

Background— Oxysterol binding protein (OSBP) has previously been implicated as a sterol sensor that regulates sphingomyelin synthesis and the activity of extracellular signal-regulated kinases (ERK). Methods and Results— We determined the effects of adenovirus-mediated hepatic overexpression of OSBP and its homologues ORP1L and ORP3 on mouse serum lipids. Whereas ORP1L and ORP3 had no effect on serum lipids, OSBP induced a marked increase of VLDL triglycerides (TG). Also, the liver tissue TG were elevated in the AdOSBP-injected mice, and their TG secretion rate was increased by 70%. The messenger RNAs for enzymes of fatty acid synthesis and their transcriptional regulator, SREBP-1c, as well as the Insig-1 mRNA, were upregulated two-fold in the OSBP-expressing livers. No change occurred in the messages of liver X receptor target genes ABCA1 , ABCG5 , and CYP7A1 , and the Insig-2a mRNA was reduced. The phosphorylation of ERK was decreased in AdOSBP-infected liver and cultured hepatocytes. Importantly, silencing of OSBP in hepatocytes suppressed the induction of SREBP1-c by insulin and resulted in a reduction of TG synthesis. Conclusion— Our results demonstrate that OSBP regulates hepatic TG metabolism and suggest the involvement of OSBP in the insulin signaling pathways that control hepatic lipogenesis.

High serum lipopolysaccharide (LPS) activity in normoalbuminuric patients with type 1 diabetes (T1D) predicts the progression of diabetic nephropathy (DN), but the mechanisms behind this remain unclear. We observed that treatment of cultured human podocytes with sera from normoalbuminuric T1D patients with high LPS activity downregulated 3-phosphoinositide-dependent kinase-1 (PDK1), an activator of the Akt cell survival pathway, and induced apoptosis. Knockdown of PDK1 in cultured human podocytes inhibited antiapoptotic Akt pathway, stimulated proapoptotic p38 MAPK pathway, and increased apoptosis demonstrating an antiapoptotic role for PDK1 in podocytes. Interestingly, PDK1 was downregulated in the glomeruli of diabetic rats and patients with type 2 diabetes before the onset of proteinuria, further suggesting that reduced expression of PDK1 associates with podocyte injury and development of DN. Treatment of podocytes in vitro and mice in vivo with LPS reduced PDK1 expression and induced apoptosis, which were prevented by inhibiting the Toll-like receptor (TLR) signaling pathway with the immunomodulatory agent GIT27. Our data show that LPS downregulates the cell survival factor PDK1 and induces podocyte apoptosis, and that blocking the TLR pathway with GIT27 may provide a non-nephrotoxic means to prevent the progression of DN.

Objective This study explored the annual occurrence/incidence of bacterial infections, and their association with chronic hyperglycemia and diabetic nephropathy, in patients with type 1 diabetes. Design In a register-based follow-up study, we investigated the frequency of bacterial infections in patients with type 1 diabetes (n=4748) and age-matched and sex-matched non-diabetic control (NDC) subjects (n=12 954) using nationwide register data on antibiotic drug prescription purchases and hospital discharge diagnoses, collected between 1996 and 2009. Diabetic nephropathy was classified based on the urinary albumin excretion rate (AER). Results The hospitalization rate due to bacterial infections was higher in patients with diabetes compared with NDCs (rate ratio (RR) 2.30 (95% CI 2.11 to 2.51)). The rate correlated with the severity of diabetic nephropathy: RR for microalbuminuria was 1.23 (0.94 to 1.60), 1.97 (1.49 to 2.61) for macroalbuminuria, 11.2 (8.1 to 15.5) for dialysis, and 6.72 (4.92 to 9.18) for kidney transplant as compared to patients with diabetes and normal AER. The annual number of antibiotic purchases was higher in patients with diabetes (1.00 (1.00 to 1.01)) as compared with NDCs (0.47 (0.46 to 0.47)), RR=1.71 (1.65 to 1.77). Annual antibiotic purchases were 1.18-fold more frequent in patients with microalbuminuria, 1.29-fold with macroalbuminuria, 2.43-fold with dialysis, and 2.74-fold with kidney transplant as compared to patients with normal AER. Each unit of increase in glycated hemoglobin was associated with a 6–10% increase in the number of annual antibiotic purchases. Conclusions The incidence of bacterial infections was significantly higher in patients with type 1 diabetes compared with age-matched and sex-matched NDC subjects, and correlated with the severity of diabetic nephropathy in inpatient and outpatient settings.

This article deals with empty spacetime and the question of its physical reality. By "empty spacetime" we mean a collection of bare spacetime points, the remains of ridding spacetime of all matter and fields. We ask whether these geometric objects--themselves intrinsic to the concept of field--might be observable through some physical test. By taking quantum-mechanical notions into account, we challenge the negative conclusion drawn from the diffeomorphism invariance postulate of general relativity, and we propose new foundational ideas regarding the possible observation--as well as conceptual overthrow--of this geometric ether., 21 pages, Latex. v2: Section 5 expanded

This article explores the overall geometric manner in which human beings make sense of the world around them by means of their physical theories; in particular, in what are nowadays called pregeometric pictures of Nature. In these, the pseudo-Riemannian manifold of general relativity is considered a flawed description of spacetime and it is attempted to replace it by theoretical constructs of a different character, ontologically prior to it. However, despite its claims to the contrary, pregeometry is found to surreptitiously and unavoidably fall prey to the very mode of description it endeavours to evade, as evidenced in its all-pervading geometric understanding of the world. The question remains as to the deeper reasons for this human, geometric predilection--present, as a matter of fact, in all of physics--and as to whether it might need to be superseded in order to achieve the goals that frontier theoretical physics sets itself at the dawn of a new century: a sounder comprehension of the physical meaning of empty spacetime., Comment: 41 pages, Latex. v2: Date added. v3: Main arguments refined, secondary discussions abridged; expands on the published version

Oxysterol binding protein (OSBP) related proteins (ORPs) constitute a family that has at least 12 members in humans. In the present study we characterize one of the novel OSBP homologs, ORP2, which we show to be expressed ubiquitously in mammalian tissues. The ORP2 cDNA encodes a deduced 55 kDa protein that lacks a pleckstrin homology (PH) domain, a feature found in the other family members. Sucrose gradient centrifugation analysis of Chinese hamster ovary (CHO) cell post-nuclear supernatant demonstrated that ORP2 is distributed in soluble and membrane-bound fractions. Immunofluorescence microscopy of the endogenous and overexpressed ORP2 in CHO cells suggested that the membrane-bound fraction of the protein localizes to the Golgi apparatus. Stably transfected CHO cells that overexpress ORP2 showed an increase in [14C]cholesterol efflux to serum, apolipoprotein A-I (apoA-I), and phosphatidyl choline vesicles. The proportion of cellular [14C]cholesterol that is esterified and the ACAT activity measured as [14C]oleyl-CoA conversion into cholesteryl [14C]oleate by the cellular membranes, were markedly decreased in the ORP2 expressing cells. Transient high level overexpression of ORP2 interfered with the clearance of a secretory pathway protein marker from the Golgi complex. The results implicate ORP2 as a novel regulator of cellular sterol homeostasis and intracellular membrane trafficking. —Laitinen, S., M. Lehto, S. Lehtonen, K. Hyvarinen, S. Heino, E. Lehtonen, C. Ehnholm, E. Ikonen, and V. M. Olkkonen. ORP2, a homolog of oxysterol binding protein, regulates cellular cholesterol metabolism.

OBJECTIVE—To investigate whether genetic susceptibility to type 1 diabetes or maturity-onset diabetes of the young (MODY) increases susceptibility to gestational diabetes mellitus (GDM). RESEARCH DESIGN AND METHODS—We studied mutations in MODY1–4 genes, the presence of GAD antibodies, and HLA DQB1 risk genotypes in 66 Swedish women with GDM and a family history of diabetes. An oral glucose tolerance test was repeated in 46 women at 1 year postpartum. RESULTS—There was no increase in type 1 diabetes–associated HLA-DQB1 alleles or GAD antibodies when compared with a group of type 2 diabetic patients (n = 82) or healthy control subjects (n = 86). Mutations in known MODY genes were identified in 3 of the 66 subjects (1 MODY2, 1 MODY3, and 1 MODY4). Of the 46 GDM subjects, 2 had diabetes (4%) and 17 had impaired glucose tolerance (IGT) (37%) at 1 year postpartum. Of the two subjects who developed manifest diabetes, one carried a MODY3 mutation (A203H in the hepatocyte nuclear factor-1α gene). There was no increase in high-risk HLA alleles or GAD antibodies in the women who had manifest diabetes or IGT at 1 year postpartum. CONCLUSIONS—MODY mutations but not autoimmunity contribute to GDM in Swedish women with a family history of diabetes and increase the risk of subsequent diabetes.

We thank Koduru et al. (1) for highlighting their findings regarding the effects of diet composition on endotoxemia and inflammation. Based on their results, it is evident that dietary factors may modulate the state of endotoxemia in humans. We, however, believe that the issue is much more complex. Pathogen-associated microbial compounds may arise from various sources: diet, bacterial infections, and commensal microbiota. Different dietary food products contain various amounts of microbial compounds, the biological activity of which might be modulated by nutrient composition and …

We earlier identified OSBP-related protein 8 (ORP8) as an endoplasmic reticulum oxysterol-binding protein implicated in cellular lipid homeostasis. We now investigated its action in hepatic cells in vivo and in vitro. Adenoviral overexpression of ORP8 in mouse liver induced a decrease of cholesterol, phospholipids, and triglycerides in serum (-34%, -26%, -37%, respectively) and liver tissue (-40%, -12%, -24%), coinciding with reduction of nuclear (n)SREBP-1 and -2 and mRNA levels of their target genes. Consistently, excess ORP8 reduced nSREBPs in HuH7 cells, and ORP8 overexpression or silencing by RNA interference moderately suppressed or induced the expression of SREBP-1 and SREBP-2 target genes, respectively. In accordance, cholesterol biosynthesis was reduced by ORP8 overexpression and enhanced by ORP8 silencing in [(3)H]acetate pulse-labeling experiments. ORP8, previously shown to bind 25-hydroxycholesterol, was now shown to bind also cholesterol in vitro. Yeast two-hybrid, bimolecular fluorescence complementation (BiFC), and co-immunoprecipitation analyses revealed the nuclear pore component Nup62 as an interaction partner of ORP8. Co-localization of ORP8 and Nup62 at the nuclear envelope was demonstrated by BiFC and confocal immunofluorescence microscopy. Furthermore, the impact of overexpressed ORP8 on nSREBPs and their target mRNAs was inhibited in cells depleted of Nup62. Our results reveal that ORP8 has the capacity to modulate lipid homeostasis and SREBP activity, probably through an indirect mechanism, and provide clues of an entirely new mode of ORP action.

Three largepT jet production ine+e− initiated photon-photon collisions is studied in perturbative Quantum Chromodynamics. In this process bothe+ ande− emit a photon followed by photon-photon collision into a quark, an antiquark and a gluon. Those final state jets that lie in the plane transverse toe+e− collision axis are investigated using the infrared insensitive variable thrustT. Experimental tests are discussed.

Infrared behavior of QCD at finite temperature is investigated using Schwinger-Dyson equations for the gluon propagator. It is found that at very high temperatures heavy quarks are free due to the screening effect.

Maturity-onset diabetes of the young (MODY) is a model for genetic studies of non-insulin-dependent diabetes mellitus. We have identified 15 MODY families in which diabetes is not the result of mutations in the glucokinase gene. This cohort of families will be useful for identifying other diabetes-susceptibility genes. Nine other candidate genes potentially implicated in insulin secretion or insulin action have been tested for linkage with MODY in these families, including glucokinase regulatory protein, hexokinase II, insulin receptor substrate 1, fatty acid–binding protein 2, glucagon-like peptide-1 receptor, apolipoprotein C-II, glycogen synthase, adenosine deaminase (a marker for the MODY gene on chromosome 20), and phosphoenolpyruvate carboxykinase. None of these loci showed evidence for linkage with MODY, implying that mutations in these genes do not make a major genetic contribution to the development of MODY. In addition to these linkage analyses, one or two affected subjects from each family were screened for the presence of the A to G mutation at nucleotide 3,243 of the mitochondrial tRNALeu(UUR) gene. This mutation was not found in any of these subjects. Finally, we report the localization of the gene encoding the regulatory protein of glucokinase to chromosome 2, band p22.3 and the identification of a restriction fragment length polymorphism at this locus.