Aino Soro-Paavonen

Familial combined hyperlipidemia is associated with upstream transcription factor 1 ( USF1 )

Familial combined hyperlipidemia (FCHL), characterized by elevated levels of serum total cholesterol, triglycerides or both, is observed in about 20% of individuals with premature coronary heart disease. We previously identified a locus linked to FCHL on 1q21–q23 in Finnish families with the disease. This region has also been linked to FCHL in families from other populations as well as to type 2 diabetes mellitus. These clinical entities have several overlapping phenotypic features, raising the possibility that the same gene may underlie the obtained linkage results. Here, we show that the human gene encoding thioredoxin interacting protein (TXNIP) on 1q, which underlies combined hyperlipidemia in mice, is not associated with FCHL. We show that FCHL is linked and associated with the gene encoding upstream transcription factor 1 (USF1) in 60 extended families with FCHL, including 721 genotyped individuals (P = 0.00002), especially in males with high triglycerides (P = 0.0000009). Expression profiles in fat biopsy samples from individuals with FCHL seemed to differ depending on their carrier status for the associated USF1 haplotype. USF1 encodes a transcription factor known to regulate several genes of glucose and lipid metabolism.

Receptor for Advanced Glycation End Products (RAGE) Deficiency Attenuates the Development of Atherosclerosis in Diabetes

OBJECTIVE—Activation of the receptor for advanced glycation end products (RAGE) in diabetic vasculature is considered to be a key mediator of atherogenesis. This study examines the effects of deletion of RAGE on the development of atherosclerosis in the diabetic apoE−/− model of accelerated atherosclerosis. RESEARCH DESIGN AND METHODS—ApoE−/− and RAGE−/−/apoE−/− double knockout mice were rendered diabetic with streptozotocin and followed for 20 weeks, at which time plaque accumulation was assessed by en face analysis. RESULTS—Although diabetic apoE−/− mice showed increased plaque accumulation (14.9 ± 1.7%), diabetic RAGE−/−/apoE−/− mice had significantly reduced atherosclerotic plaque area (4.9 ± 0.4%) to levels not significantly different from control apoE−/− mice (4.3 ± 0.4%). These beneficial effects on the vasculature were associated with attenuation of leukocyte recruitment; decreased expression of proinflammatory mediators, including the nuclear factor-κB subunit p65, VCAM-1, and MCP-1; and reduced oxidative stress, as reflected by staining for nitrotyrosine and reduced expression of various NADPH oxidase subunits, gp91phox, p47phox, and rac-1. Both RAGE and RAGE ligands, including S100A8/A9, high mobility group box 1 (HMGB1), and the advanced glycation end product (AGE) carboxymethyllysine were increased in plaques from diabetic apoE−/− mice. Furthermore, the accumulation of AGEs and other ligands to RAGE was reduced in diabetic RAGE−/−/apoE−/− mice. CONCLUSIONS—This study provides evidence for RAGE playing a central role in the development of accelerated atherosclerosis associated with diabetes. These findings emphasize the potential utility of strategies targeting RAGE activation in the prevention and treatment of diabetic macrovascular complications.

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.

RAGE biology, atherosclerosis and diabetes.

Diabetes is characterized by accelerated atherosclerosis with widely distributed vascular lesions. An important mechanism by which hyperglycaemia contributes to vascular injury is through the extensive intracellular and extracellular formation of AGEs (advanced glycation end products). AGEs represent a heterogeneous group of proteins, lipids and nucleic acids, irreversibly cross-linked with reducing sugars. AGEs are implicated in the atherosclerotic process, either directly or via receptor-mediated mechanisms, the most extensively studied receptor being RAGE (receptor for AGEs). The AGE-RAGE interaction alters cellular signalling, promotes gene expression and enhances the release of pro-inflammatory molecules. It elicits the generation of oxidative stress in numerous cell types. The importance of the AGE-RAGE interaction and downstream pathways leading to injurious effects as a result of chronic hyperglycaemia in the development, progression and instability of diabetic atherosclerotic lesions has been amply demonstrated in animal studies. Moreover, the deleterious link of AGEs with diabetic vascular complications has been suggested in many human studies. In the present review, our current understanding of their role as an important mediator of vascular injury through the various stages of atherosclerosis in diabetes will be reviewed and critically assessed.

Role of insulin as a negative regulator of plasma endocannabinoid levels in obese and nonobese subjects

OBJECTIVE Endocannabinoids (ECs) control metabolism via cannabinoid receptors type 1 (CB1). Their plasma levels are elevated in overweight type 2 diabetes (T2D) and in obese patients, and decrease postprandially in normoweight individuals. We investigated in two different cohorts of nonobese or obese volunteers whether oral glucose in glucose tolerance tests (OGTT) or acute insulin infusion during euglycemic hyperinsulinemic clamp affect plasma EC levels. DESIGN AND METHODS OGTT was performed in ten obese hyperinsulinemic patients (body mass index (BMI)=35.8 kg/m2, fasting insulin=14.83 mU/l), and ten normoweight normoinsulinemic volunteers (BMI=21.9 kg/m2, fasting insulin=7.2 mU/l). Insulin clamp was performed in 19 mostly nonobese men (BMI=25.8 kg/m2) with varying degrees of liver fat and plasma triglycerides (TGs), with (n=7) or without T2D. Plasma levels of ECs (anandamide and 2-arachidonoylglycerol (2-AG)) were measured by liquid chromatography-mass spectrometry, before and 60 and 180 min after OGTT, and before and 240 and 480 min after insulin or saline infusion. RESULTS Oral glucose load decreased anandamide plasma levels to an extent inversely correlated with BMI, waist circumference, subcutaneous fat, fasting insulin and total glucose, and insulin areas under the curve during the OGTT, and nonsignificantly in obese volunteers. Insulin infusion decreased anandamide levels to an extent that weakly, but significantly, correlated negatively with TGs, liver fat and fasting insulin, and positively with high density lipoprotein cholesterol. OGTT decreased 2-AG levels to a lower extent and in a way weakly inversely correlated with fasting insulin. CONCLUSIONS We suggest that insulin reduces EC levels in a way inversely related to anthropometric and metabolic predictors of insulin resistance and dyslipidemia.

Decreased High-Density Lipoprotein (HDL) Particle Size, Preβ-, and Large HDL Subspecies Concentration in Finnish Low-HDL Families Relationship With Intima-Media Thickness

Objective—High-density lipoprotein (HDL) cholesterol correlates inversely with the risk of coronary heart disease (CHD). The precise antiatherogenic mechanisms of HDL subspecies are not thoroughly elucidated. We studied the relationship between carotid intima-media thickness (IMT) and HDL subspecies distribution in Finnish families with low HDL cholesterol and premature CHD. Methods and Results—Altogether, 148 members of Finnish low-HDL families and 133 healthy control subjects participated in our study. HDL particle size was significantly smaller in affected family members (HDL ≤10th Finnish age-sex specific percentile) compared with unaffected family members and control subjects (9.1±0.04 nm versus 9.5±0.05 nm, P<0.0001, versus 9.8±0.03 nm, P<0.0001 [mean±SE]). Large HDL2b particles as well as pre&bgr;-HDL concentration were significantly decreased among the affected family members. Mean IMT was significantly higher in the affected family members than in the control subjects (0.85±0.01 mm versus 0.79±0.01 mm; P<0.0001). Age, HDL2b, systolic blood pressure, and pre&bgr;-HDL were significant independent determinants of mean IMT. Conclusions—The decreased levels of HDL2b and pre&bgr;-HDL reflect the potentially efflux-deficient HDL subspecies profile in the affected low-HDL family members. Decreased HDL particle size caused by the decrease of plasma concentration of HDL2b and decreased pre&bgr;-HDL levels correlate with increased IMT.

Advanced glycation end-products induce vascular dysfunction via resistance to nitric oxide and suppression of endothelial nitric oxide synthase.

Objective A number of factors contribute to diabetes-associated vascular dysfunction. In the present study, we tested whether exposure to advanced glycation end-products (AGEs) impairs vascular reactivity independently of hyperglycemia and examined the potential mechanisms responsible for diabetes and AGE-associated vascular dysfunction. Methods Vasodilator function was studied using infusion of exogenous AGEs into Sprague–Dawley rats as compared with control and streptozotocin-induced diabetic rats all followed for 16 weeks (n = 10 per group). The level of arginine metabolites and expression of endothelial nitric oxide synthase (eNOS) and downstream mediators of nitric oxide-dependent signaling were examined. To further explore these mechanisms, cultured bovine aortic endothelial cells (BAECs) were exposed to AGEs. Results Both diabetic and animals infused with AGE-modified rat serum albumin (AGE–RSA) had significantly impaired vasodilatory response to acetylcholine. Unlike diabetes-associated endothelial dysfunction, AGE infusion was not associated with changes in plasma arginine metabolites, asymmetric dimethyl-L-arginine levels or eNOS expression. However, expression of the downstream mediator cGMP-dependent protein kinase 1 (PKG-1) was significantly reduced by both AGE exposure and diabetes. AGEs also augmented hyperglycemia-associated depletion in endothelial nitric oxide production and eNOS protein expression in vitro, and the novel AGE inhibitor, alagebrium chloride, partly restored these parameters. Conclusion We demonstrate that AGEs represent a potentially important cause of vascular dysfunction, linked to the induction of nitric oxide resistance. These findings also emphasize the deleterious and potentially additive effects of AGEs and hyperglycemia in diabetic vasculature.

Circulating ACE2 activity is increased in patients with type 1 diabetes and vascular complications.

Objective: Angiotensin-converting enzyme 2 (ACE2) is a homolog of ACE that counterbalances the actions of angiotensin (AT)II and promotes vasodilatation. Circulating ACE2 activity is increased in diabetes in experimental models. The role of ACE2 in human pathophysiology is unknown. We examined whether ACE2 activity is altered in patients with type 1 diabetes (T1D), with and without diabetic nephropathy. Methods: Quantitative ACE2 activity in serum was measured by a fluorometric assay in 859 patients with T1D in the Finnish Diabetic Nephropathy (FinnDiane) study and in 204 healthy controls. Pulse-wave analysis with augmentation index (AIx) measurement was performed in 319 patients with T1D and 114 controls. Results: ACE2 activity was increased in men with T1D and microalbuminuria (30.2 ± 1.5 ngE/ml) when compared to patients without albuminuria (27.0 ± 0.5 ngE/ml, P < 0.05) or controls (25.6 ± 0.8 ngE/ml, P < 0.05). ACE2 activity was increased in male and female patients who were on ACE inhibitor (ACEi) treatment, also independently of albuminuria. Male and female patients with coronary heart disease (CHD) had significantly increased ACE2 activity (35.5 ± 2.5 vs. 27.0 ± 0.5 ngE/ml, P < 0.001 among male T1D patients vs. male controls). ACE2 activity correlated positively with systolic blood pressure (rs = 0.175, P < 0.001), AIx (rs = 0.191, P = 0.010) and diabetes duration (rs = 0.198, P < 0.001), and negatively with estimated glomerular filtration rate (rs = −0.109, P = 0.016) among male T1D patients. Conclusions: ACE2 activity increases with increasing vascular tone and when the patient with T1D has microvascular or macrovascular disease, indicating that ACE2 may participate as a compensatory mechanism in the regulation of vascular and renal function in patients with T1D.

Alagebrium Reduces Glomerular Fibrogenesis and Inflammation Beyond Preventing RAGE Activation in Diabetic Apolipoprotein E Knockout Mice

Advanced glycation end products (AGEs) are important mediators of diabetic nephropathy that act through the receptor for AGEs (RAGE), as well as other mechanisms, to promote renal inflammation and glomerulosclerosis. The relative contribution of RAGE-dependent and RAGE-independent signaling pathways has not been previously studied in vivo. In this study, diabetic RAGE apoE double-knockout (KO) mice with streptozotocin-induced diabetes were treated with the AGE inhibitor, alagebrium (1 mg/kg/day), or the ACE inhibitor, quinapril (30 mg/kg/day), for 20 weeks, and renal parameters were assessed. RAGE deletion attenuated mesangial expansion, glomerular matrix accumulation, and renal oxidative stress associated with 20 weeks of diabetes. By contrast, inflammation and AGE accumulation associated with diabetes was not prevented. However, treatment with alagebrium in diabetic RAGE apoE KO mice reduced renal AGE levels and further reduced glomerular matrix accumulation. In addition, even in the absence of RAGE expression, alagebrium attenuated cortical inflammation, as denoted by the reduced expression of monocyte chemoattractant protein-1, intracellular adhesion molecule-1, and the macrophage marker cluster of differentiation molecule 11b. These novel findings confirm the presence of important RAGE-independent as well as RAGE-dependent signaling pathways that may be activated in the kidney by AGEs. This has important implications for the design of optimal therapeutic strategies for the prevention of diabetic nephropathy.

Common ABCA1 variants, HDL levels, and cellular cholesterol efflux in subjects with familial low HDL.

HDL promotes cholesterol efflux from peripheral cells via ABCA1 in the first step of reverse cholesterol transport (RCT). We investigated whether the early steps of RCT were disturbed in subjects with familial low HDL and an increased risk for early atherosclerosis. Cholesterol efflux from monocyte-derived macrophages to lipid-free apolipoprotein A-I (apoA-I; %) was measured in 22 patients with familial low HDL without Tangier disease mutations and in 21 healthy controls. In addition, we defined the different alleles of ABCA1 using single-nucleotide polymorphism haplotypes and measured ABCA1 and ABCG1 mRNA transcript levels in cholesterol-loaded macrophages. Similar ABCA1-mediated cholesterol efflux levels were observed for macrophages derived from control subjects and from low-HDL subjects. However, when efflux of cholesterol was estimated as cholesterol efflux to apoA-I (%)/relative ABCA1 mRNA expression level, cholesterol removal was significantly (P = 0.001) lower in the low-HDL group. Cholesterol-loaded macrophages from low-HDL subjects showed significantly increased levels of ABCA1 mRNA but not of ABCG1 mRNA and were more often carriers of the rare ABCA1 alleles L158 and R219K. These results suggest that defective ABCA1 function in cholesterol-loaded macrophages is one potential contributor to the impaired RCT process and the increased coronary heart disease risk in subjects with familial low HDL.