Markku Lehto

Endotoxemia Is Associated With an Increased Risk of Incident Diabetes

OBJECTIVE Diabetes is accompanied with a chronic low-grade inflammation, which may in part be mediated by endotoxins derived from Gram-negative bacteria. RESEARCH DESIGN AND METHODS We investigated in a population-based cohort whether endotoxemia is associated with clinically incident diabetes. The serum endotoxin activity was measured by limulus assay from the FINRISK97 cohort comprising 7,169 subjects aged 25–74 years and followed up for 10 years. RESULTS Both the subjects with prevalent diabetes (n = 537) and those with incident diabetes (n = 462) had higher endotoxin activity than the nondiabetic individuals (P < 0.001). The endotoxin activity was significantly associated with increased risk for incident diabetes with a hazard ratio 1.004 (95% CI 1.001–1.007; P = 0.019) per unit increase resulting in a 52% increased risk (P = 0.013) in the highest quartile compared with the lowest one. The association was independent of diabetes risk factors: serum lipids, γ-glutamyl transferase, C-reactive protein, BMI, and blood glucose. Furthermore, the association of endotoxemia with an increased risk of incident diabetes was independent of the metabolic syndrome as defined either by the National Cholesterol Educational Program-Adult Treatment Panel III or the International Diabetes Federation. Endotoxin activity was linearly related (P < 0.001) to the number of components of the metabolic syndrome. CONCLUSIONS Both prevalent and incident diabetes were associated with endotoxemia, which may link metabolic disorders to inflammation. The results suggest that microbes play a role in the pathogenesis of diabetes.

Characterization of the MODY3 phenotype. Early-onset diabetes caused by an insulin secretion defect.

Maturity-onset diabetes of the young (MODY) type 3 is a dominantly inherited form of diabetes, which is often misdiagnosed as non-insulin-dependent diabetes mellitus (NIDDM) or insulin-dependent diabetes mellitus (IDDM). Phenotypic analysis of members from four large Finnish MODY3 kindreds (linked to chromosome 12q with a maximum lod score of 15) revealed a severe impairment in insulin secretion, which was present also in those normoglycemic family members who had inherited the MODY3 gene. In contrast to patients with NIDDM, MODY3 patients did not show any features of the insulin resistance syndrome. They could be discriminated from patients with IDDM by lack of glutamic acid decarboxylase antibodies (GAD-Ab). Taken together with our recent findings of linkage between this region on chromosome 12 and an insulin-deficient form of NIDDM (NIDDM2), the data suggest that mutations at the MODY3/NIDDM2 gene(s) result in a reduced insulin secretory response, that subsequently progresses to diabetes and underlines the importance of subphenotypic classification in studies of diabetes.

New susceptibility loci associated with kidney disease in Type 1 diabetes

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.

The OSBP-related proteins: a novel protein family involved in vesicle transport, cellular lipid metabolism, and cell signalling

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.

The mammalian oxysterol-binding protein-related proteins (ORPs) bind 25-hydroxycholesterol in an evolutionarily conserved pocket

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, K(d)=9.7x10(-8) M; ORP1S, K(d)=8.4 x10(-8) M), while the affinity of GST-ORP2 for 25OH was lower (K(d)=3.9x10(-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.

OSBP-related Protein 8 (ORP8) Suppresses ABCA1 Expression and Cholesterol Efflux from Macrophages

ORP8 is a previously unexplored member of the family of oxysterol-binding protein-related proteins (ORP). We now report the expression pattern, the subcellular distribution, and data on the ligand binding properties and the physiological function of ORP8. ORP8 is localized in the endoplasmic reticulum (ER) via its C-terminal transmembrane span and binds 25-hydroxycholesterol, identifying it as a new ER oxysterol-binding protein. ORP8 is expressed at highest levels in macrophages, liver, spleen, kidney, and brain. Immunohistochemical analysis revealed ORP8 in the shoulder regions of human coronary atherosclerotic lesions, where it is present in CD68(+) macrophages. In advanced lesions the ORP8 mRNA was up-regulated 2.7-fold as compared with healthy coronary artery wall. Silencing of ORP8 by RNA interference in THP-1 macrophages increased the expression of ATP binding cassette transporter A1 (ABCA1) and concomitantly cholesterol efflux to lipid-free apolipoprotein A-I but had no significant effect on ABCG1 expression or cholesterol efflux to spherical high density lipoprotein HDL2. Experiments employing an ABCA1 promoter-luciferase reporter confirmed that ORP8 silencing enhances ABCA1 transcription. The silencing effect was partially attenuated by mutation of the DR4 element in the ABCA1 promoter and synergized with that of the liver X receptor agonist T0901317. Furthermore, inactivation of the E-box in the promoter synergized with ORP8 silencing, suggesting that the suppressive effect of ORP8 involves both the liver X receptor and the E-box functions. Our data identify ORP8 as a negative regulator of ABCA1 expression and macrophage cholesterol efflux. ORP8 may, thus, modulate the development of atherosclerosis.

Novel mutations and a mutational hotspot in the MODY3 gene

Maturity-onset diabetes of the young 3 (MODY3) is a type of NIDDM caused by mutations in the transcription factor hepatocyte nuclear factor-1a (HNF-1α) located on chromosome 12q. We have identified four novel HNF-1α 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 HNF-1α 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.

Oxysterols and oxysterol binding proteins: role in lipid metabolism and atherosclerosis

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 Induces Upregulation of SREBP-1c and Enhances Hepatic Lipogenesis

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.

The R-Ras interaction partner ORP3 regulates cell adhesion

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.