Prevalence of Retinopathy in Adult Patients with GCK-MODY and HNF1A-MODY.

We aimed to assess the prevalence of diabetic retinopathy (DR) in adult patients with GCK-MODY and HNF1A-MODY in Poland and to identify biochemical and clinical risk factors associated with its occurrence.We examined 74 GCK mutation carriers, 51 with diabetes and 23 with prediabetes, respectively, and 63 patients with HNF1A-MODY. Retinal photographs, 12 for each patient, were done by a fundus camera. Signs of DR were graded according to the DR disease severity scale. Statistical tests were performed to assess differences between the groups and logistic regression was done for the association with DR.The mean age at examination was 34.5±14.8 and 39.9±15.2 in the GCK-MODY and HNF1A-MODY groups, respectively. Mild nonproliferative DR (NPDR) was found in one patient with the GCK mutation and likely concomitant type 1 diabetes, whereas DR was diagnosed in 15 HNF1A-MODY patients: 9 with proliferative, 3 with moderate NPDR and 2 with mild NPDR. In univariate logistic regression analysis in the HNF1A-MODY group, significant results were found for diabetes duration, fasting glycemia, HbA1c, arterial hypertension, age at the examination, and eGFR. The strongest independent predictors of DR in HNF1A-MODY were markers of glucose control: HbA1c (OR: 2.05, CL%95: 1.2-3.83, p=0.01) and glucose (p=0.006, OR: 1.40, CL%95: 1.12-1.83) analyzed in 2 separated models. Additionally, arterial hypertension independently predicted DR (OR: 9.06, CL%95: 1.19-98.99, p=0.04) in the model with HbA1c as glycaemic control marker.In conclusion, DR of any degree was not present in our GCK-MODY group, while in spite of young age almost every fourth subject with HNF1A-MODY showed signs of this complication.

Characterisation of Atherogenic Effects of Low Carbohydrate, High Protein Diet (LCHP) in ApoE/LDLR-/- Mice.

INTRODUCTION: Low Carbohydrate High Protein diet represents a popular strategy to achieve weight loss. OBJECTIVE: The aim of this study was to characterize effects of low carbohydrate, high protein diet (LCHP) on atherosclerotic plaque development in brachiocephalic artery (BCA) in apoE/LDLR-/- mice and to elucidate mechanisms of proatherogenic effects of LCHP diet. MATERIALS AND METHODS: Atherosclerosis plaques in brachiocephalic artery (BCA) as well as in aortic roots, lipoprotein profile, inflammation biomarkers, expression of SREBP-1 in the liver as well as mortality were analyzed in Control diet (AIN-93G) or LCHP (Low Carbohydrate High Protein) diet fed mice. RESULTS: Area of atherosclerotic plaques in aortic roots or BCA from LCHP diet fed mice was substantially increased as compared to mice fed control diet and was characterized by increased lipids and cholesterol contents (ORO staining, FT-IR analysis), increased macrophage infiltration (MOMA-2) and activity of MMPs (zymography). Pro-atherogenic phenotype of LCHP fed apoE/LDLR-/- mice was associated with increased plasma total cholesterol concentration, and in LDL and VLDL fractions, increased TG contents in VLDL, and a modest increase in plasma urea. LCHP diet increased SCD-1 index, activated SREBP-1 transcription factor in the liver and triggered acute phase response as evidence by an increased plasma concentration of haptoglobin, CRP or AGP. Finally, in long-term experiment survival of apoE/LDLR-/- mice fed LCHP diet was substantially reduced as compared to their counterparts fed control diet suggesting overall detrimental effects of LCHP diet on health. CONCLUSIONS: The pro-atherogenic effect of LCHP diet in apoE/LDLR-/- mice is associated with profound increase in LDL and VLDL cholesterol, VLDL triglicerides, liver SREBP-1 upregulation, and systemic inflammation.

Pleiotropic effects of TCF7L2 gene variants and its modulation in the metabolic syndrome: from the LIPGENE study.

AIMS/HYPOTHESIS: Variants of the TCF7L2 gene predict the development of type 2 diabetes mellitus (T2DM). We investigated the associations between gene variants of TCF7L2 and clinical features of the metabolic syndrome (MetS) (an entity often preceding T2DM), and their interaction with non-genetic factors, including plasma saturated fatty acids (SFA) concentration and insulin resistance (IR). METHODS: Fasting lipid profiles, insulin sensitivity, insulin secretion, anthropometrics, blood pressure and 10 gene variations of the TCF7L2 gene were determined in 450 subjects with MetS. RESULTS: Several single nucleotide polymorphisms (SNP) showed phenotypic associations independent of SFA or IR. Carriers of the rare T allele of rs7903146, and of three other SNPs in linkage disequilibrium with rs7903146, had lower blood pressure and insulin secretion. High IR and the presence of the T-allele of rs7903146 acted synergistically to define those with reduced insulin secretion. Carriers of the minor allele of rs290481 exhibited an altered lipid profile, with increased plasma levels of apolipoprotein B, non-esterified fatty acids, cholesterol and apolipoprotein B in triglyceride rich lipoproteins, and LDL cholesterol. Carriers of the minor allele of rs11196224 that had higher plasma SFA levels showed elevated procoagulant/proinflammatory biomarkers, impaired insulin secretion and increased IR, whereas carriers of the minor allele of rs17685538 with high plasma SFA levels exhibited higher blood pressure. CONCLUSIONS/INTERPRETATION: SNP in the TCF7L2 gene are associated with differences in insulin secretion, blood pressure, blood lipids and coagulation in MetS patients, and may be modulated by SFA in plasma or IR.

The adipose tissue gene expression in mice with different nitric oxide availability.

Mice with the knockout of endothelial nitric oxide synthase (eNOS ko) demonstrate symptoms resembling the human metabolic syndrome. NO has been recently demonstrated to be deeply involved in regulation of not only blood flow and angiogenesis, but also in modulation of mammalian basal energy substrate metabolism. Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of NOS. The enzyme dimethylarginine dimethylaminohydrolase (DDAH) catabolizes ADMA, what leads to increase of endogenous NO bioavailability. This study was aimed to compare the brown (BAT) and white (WAT) adipose tissue gene expression of age matched mice with decreased (eNOS ko) and increased (overexpressing DDAH) endogenous NO generation. The 19 week old eNOS ko mice demonstrated significantly lower weight, higher circulating glucose, insulin, leptin and cholesterol concentrations. The adiponectin as well as fasting triglyceride concentrations were not significantly altered. Animals with DDAH knock in, presented significantly increased angiogenic activity than eNOS ko mice. The microarray analysis pointed to activation of adipogenesis-related genes in eNOS ko mice in WAT, what was in contrast with the inhibition observed in the DDAH overexpressing mice. The angiogenesis related gene expression was down-regulated in both models in comparison to WT animals. This study support the multipotential role of endogenous NO in maintaining homeostasis of energy substrate catabolism.

Correlation of the -3826A >G polymorphism in the promoter of the uncoupling protein 1 gene with obesity and metabolic disorders in obese families from southern Poland.

The aim of the study was to examine the allelic frequency of the -3826A > G mutation of UPC1 in patients with familiar obesity and to investigate putative association of this polymorphism with metabolic disorders. One hundred and eighteen overweight /obese patients participated in the study. The UCP1 polymorphism was determined by RFLP. Glucose, lipid, insulin and leptin levels were measured both during OGTT and OLTT. The majority of patients had a homozygous A/A genotype (51,38%), while 14,68% had a G/G genotype. We found no significant association of the G allele with either BMI or glucose tolerance. Patients with the homozygous G/G genotype had significantly higher fasting levels of TG (p < 0.04) and decreased levels of HDL-cholesterol (p = 0,004). They also had an increased concentration of FFA and the rise of TG levels during the OLTT compared to controls was significant (p = 0,058). In addition, the carriers of the G/G genotype had the lowest insulin levels both during OGTT and OLTT. In our study we have demonstrated that the -3826A > G polymorphism of UCP1 does not play a major role in the development of obesity and/or disturbances of glucose metabolism. However, the increased levels of TG and FFA and decreased levels of HDL observed in carriers of the G allele suggest FFA-induced impairment of the HDL turnover and disturbance of the beta-cell function, both of which are risk factors for endothelial injury.

[Is atherosclerosis an autoimmunological process?]. / Miazdzyca procesem autoimmunologicznym?

The theories formulated to explain atherogenesis evolved from simple vessel wall lipid accumulation assumption to endothelial dysfunction with adverse vascular wall remodelling hypothesis. The theory that has been accepted lately integrates the former hypotheses and allows for introducing the local immunological activation concept. This immunological activation is initiated by negatively charged and oxidatively modified lipids (e.g. oxPAPC) and their complexes with proteins (like beta 2-GP I). Antibodies and cellular response against chaperonins: HSP 60 and their analogues from bacterial pathogens such as HSP 65, GroEi etc.) as well as release of cytokines, adhesion molecules and inflammatory mediators (CD 40/CD 40-L, IL 15, IFN gamma, IL 1 beta, TNF alpha) also take part in the process. Another important element of atherogenesis is the pathological angiogenic response within the plaque connected with the expression of angiogenic growth factors (such as VEGF, bFGF and PDGF), metallo-proteinases and local hemostasis regulators. This complex activation of local inflammatory and immunological process initiates such phenomena as development of unstable plaque, vascular remodelling, vessel lumen constriction and ischemic, thromboembolic complications of atherosclerosis.

Oxidized low density lipoprotein inhibits inducible nitric oxide synthase, GTP cyclohydrolase I and transforming growth factor beta gene expression in rat macrophages.

Several studies have already demonstrated that oxidized- LDL decreases nitric oxide (NO) generation by cytokine-stimulated macrophages. However, the mechanisms of such an inhibition have not been yet elucidated. NO generation by inducible nitric oxide synthase (iNOS) is dependent on the presence of cofactors for NO generation, tetrathydrobiopterin (BH4) among them. The NO generation by these cells is also regulated by some endogenous inhibitors, like TGF-beta. Therefore, the aim of our recent study was to investigate the influence of ox-LDL on the expression of iNOS and GTP cyclohydrolase I (GTP-CH I), the key enzyme involved in the BH4 synthesis as well as the ox-LDL effect on TGF-beta expression in rat macrophages stimulated with IFNgamma (250 U/ml) and LPS (500 ng/ml). Macrophages, activated in this way, express iNOS, GTP-CH I, and TGF-beta mRNA. This expression was inhibited when the macrophages were preincubated for 24 hours with ox-LDL (100 microg/ml). Quantitative PCR revealed about 10-fold inhibition of iNOS gene expression by ox-LDL. As a consequence of down-regulation of iNOS and GTP-CH I genes, almost 3-fold diminished generation of NO2- by rat macrophages was observed. An inhibition of the TGFbeta mRNA expression was also found. Our studies indicate that decreased NO generation by ox-LDL treated macrophages may be the result of the diminished expression of both iNOS and GTP-CH I genes. This effect may be mediated by the activity of certain endogenous inhibitors of gene expression, however, our studies exclude the TGF-beta as a candidate for this activity.

Expression of beta-galactosidase gene and endothelial nitric oxide synthase gene in rat vascular smooth muscle cells after in vitro lipotransfection.

The aim of this study was to optimize the conditions for in vitro lipotransfection of rat vascular smooth muscle cells (VSMC) with bacterial beta-galactosidase gene and bovine endothelial nitric oxide synthase (ecNOS) gene. Transfection efficiency of four liposomes: Transfectam, Lipofectin, Unifectin-10, and Maxifectin was compared. The best results (efficiency 1-5%) were obtained with Maxifectin, when transfections were performed in VSMC cultures being at 50% confluency, with 1 microg DNA and 10 microl liposome per well, and when the liposome/DNA complexes were coincubated with the cells for 24 h. This method allowed detection of the transgene activity 12 h after the beginning of the transfection, with maximum values between the second and fourth days. The expression of the potentially therapeutic ecNOS gene was evidenced by confirmation of ecNOS mRNA generation, indirect detection of active ecNOS protein and by measurement of nitrite ion accumulation in the medium from the transfected cell cultures.

Nitric oxide mediates the mitogenic effects of insulin and vascular endothelial growth factor but not of leptin in endothelial cells.

The regulation of vascular wall homeostasis by nitric oxide (NO) generated by endothelium is being intensively studied. In the present paper, the involvement of NO in the vascular endothelial growth factor (VEGF), insulin or leptin-stimulated proliferation of human endothelial cells (HUVEC) was measured by [3H]thymidine or bromodeoxyuridine incorporation. VEGF and insulin, but not leptin, increased NO generation in HUVEC, as detected with ISO-NO electrode. Proliferation of HUVEC induced by leptin was not changed or was higher in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME) a nitric oxide synthase (NOS) inhibitor. In contrast, L-NAME blunted the proproliferative effect of VEGF and insulin. Furthermore, we demonstrated that, in human arterial smooth muscle cells (hASMC) transfected with endothelial NOS (eNOS) gene, the generation of biologically active VEGF protein was NO-dependent. Inhibition of NO generation by L-NAME decreased the synthesis of VEGF protein and attenuated HUVEC proliferation induced by conditioned media from transfected hASMC. Endothelium-derived NO seems to participate in VEGF and insulin, but not leptin, mitogenic activity. Additionally, the small amounts of NO released from endothelial cells, as mimicked by eNOS transfection into hASMC, may activate generation of VEGF in sub-endothelial smooth muscle cells, leading to increased synthesis of VEGF protein necessary for turnover and restitution of endothelial cells.

Influence of insulin on cholesterol removal from macrophages and cholesterol ester uptake by HepG2 cells.

The fact that an increased blood insulin level is observed in patients with coronary artery disease (CAD) confirms the hypothesis that insulin promotes the development of atherosclerosis. The low high-density lipoprotein (HDL) concentration observed in such patients may contribute to alteration in reverse cholesterol transport and promote the accumulation of sterols in vascular tissue. We examined the effect of insulin (20-1000 microU mL-1) on cholesterol efflux into HDL3 particles from human blood monocyte/macrophages and rat peritoneal macrophages preloaded with labelled cholesterol esters, and the influence of insulin on the accumulation of sterols by rat liver cells and HepG2 cell line in vitro models. Insulin at concentrations up to 250 microU mL-1 inhibited the efflux of cholesterol from rat macrophages and promoted high uptake of sterols by both types of hepatic cells. Pharmacological concentrations higher than 250 microU mL-1 exerted the opposite effect. In the case of human macrophages, an insulin concentration of 20 microU mL-1 increased cholesterol removal, whereas 100-200 microU mL-1 insulin inhibited cholesterol removal from cells, and very high concentrations (> 350 microU mL-1) again increased cholesterol removal. We have shown that insulin excess counteracts the beneficial effects of HDL in removing cellular cholesterol and, therefore, may promote development of atherogenesis.

Heterogeneity of high-density lipoprotein particles and insulin output during oral glucose tolerance test in men with coronary artery disease.

We compared the high-density lipoprotein (HDL) composition and particle heterogeneity in 60 nonobese (normal body mass index, BMI) men suffering from coronary artery disease (CAD) with normolipemia and normoinsulinemia with lower and higher insulin output during the oral glucose tolerance test (silent hyperinsulinemia). The apolipoprotein apoAI, apoAII, and apoE levels were higher in the high insulin response (HI) group than in low insulin response (LI) group. The ratio of apoAI versus total protein and the ratio of apoAI versus total cholesterol were increased in HI compared with LI. The lipid components in HDL were higher in LI than in HI, while for HDL2 they were higher in HI. The fractioning of HDL by gradient gel electrophoresis revealed a different pattern of HDL particles in both groups. The larger particles, HDL2b and HDL2a (mean particle diameters 10.6 and 9.2 nm, respectively), occur more frequently in HI patients (up to 60%) than in LI patients, whereas the smaller particles, HDL3a and HDL3b (mean particle diameters 8.6 and 7.8 nm, respectively), predominate in LI patients. Our results demonstrate that even in the normoglycemic, normocholesterolemic CAD patients, a high insulin output observed during the oral glucose tolerance test may be connected with a different HDL particle pattern, which suggests changes in the reverse cholesterol transport.

[The importance of nitric oxide (NO) in the development of Helicobacter pylori infection]. / Znaczenie tlenku azotu (NO) w rozwoju zakazenia Helicobacter pylori.

In this work we discussed selected questions about the Helicobacter pylori pathogenesis linked with the components of cell-walls as well as with toxins and enzymes externally secreted by this bacteria. We also paid attention to some theories which try to imperf the damaging effects of the bacteria on the mucosa of the upper part of the alimentary canal, specifically underlining the possible link between the Helicobacter pylori infection and the increase of the concentration of cytotoxically acting nitric oxide (NO).

Influence of SIN-1 and sodium nitroprusside (NANP) on ox-LDL metabolism in macrophages.

Effects of NO-donors (3-morpholinosydnonimine-SIN-1 and sodium nitroprusside NaNP) on the accumulation and degradation of oxidized LDL (ox-LDL) by macrophages were studied. Ox-LDL, but not native-LDL (n-LDL) suppressed the LPS-stimulated biosynthesis of NO by macrophages. SIN-1 at low concentrations < 100 microM was without any effect while SIN-1 at high concentration (300 microM) and NaNP (30-300 microM) stimulated the accumulation and degradation of ox-LDL by macrophages. The pretreatment of macrophages with NG-monomethyl-L-arginine (L-NMMA, 3 microM) for 24 hours had the same stimulatory effect. The inhibition of endogenous formation of NO, by L-NMMA profoundly changed the pattern of action of NO-donors on ox-LDL catabolism by macrophages; the stimulatory action of SIN-1 was transformed to the inhibitory action on the accumulation and degradation of ox-LDL whereas NaNP lost its stimulatory action entirely. Our interpretation of this unexpected interactions between SIN-1, NaNP and L-NMMA is as follows. Endogenous NO in macrophages inhibits the accumulation of ox-LDL and therefore, the stimulatory effect of L-NMMA has been overcome by exogenous NO from SIN-1. However, NO at high concentrations promotes lipid accumulation in macrophages and thereby, in the absence of L-NMMA, SIN-1 at high concentrations and NaNP produced a paradoxical stimulatory effect in macrophages. NaNP is not a proper NO-donor and its mode of action differed from that of SIN-1. In conclusion, NO at low physiological concentrations keeps scavenger receptors of macrophages downregulated and hence endogenous NO may show anti-atherogenic properties.

Modulation of LDL catabolism by sodium nitroprusside and PGE2 in human peripheral blood lymphocytes.

The effect of nitric oxide donor--sodium nitroprusside (NaNP) and PGE2 on the LDL-receptor activity and LDL cellular accumulation by isolated human blood lymphocytes was investigated. Preincubation of lymphocytes with lipoprotein deficient medium (LPDS) resulted in the increase of the LDL-receptor activity and the LDL-cellular accumulation. NaNP (30-300 microM) dose-dependently prevented the increase of the LDL-receptor activity as well as the accumulation of LDL by lymphocytes. However, in "starwing" cells (cells with high LDL-receptor activity) the effect of NaNP on the receptor activity was biphasic. At concentration up to 100 microM NaNP inhibited, while at a concentration of 300 microM, it activated the LDL-receptor activity. PGE2 (3-30 microM) inhibited LDL catabolism, however, this effect was hardly concentration-dependent.

[Decrease in the level of free cholesterol fraction HDL as a risk factor for threatened atherosclerosis in idiopathic hyperinsulinemia]. / Zmniejszenie stezenia wolnego cholesterolu frakcji HDL czynnikiem zagrozenia miazdzyca w samoistnej hiperinsulinemii.

Inversive association between concentration of plasma high density lipoprotein (HDL) components and endogenous insulin response to oral glucose uptake was observed in 89 healthy, normolipidaemic, non obese, non diabetic male subjects. Inversive correlation between sum of insulin released during oral glucose tolerance test and HDL free and esterified cholesterol as well as apolipoprotein A1 concentration were confirmed. Coexistence of increased insulin secretion and low ratio of free cholesterol concentration to esterified cholesterol and to proteins in HDL was also observed. In separate group of subjects with normal glucose tolerance in high insulin response subgroup the cholesterol (free and esterified) and protein concentrations were lower than in subgroup with low insulin response. The free cholesterol/esterified cholesterol and free cholesterol/protein (apoA1) ratios were decreased in high insulin response subgroup. It is concluded that low concentration of free cholesterol is a most significant change of HDL composition in hyperinsulinemic, healthy men.

Interaction of reconstituted high density lipoprotein discs containing human apolipoprotein A-I (ApoA-I) variants with murine adipocytes and macrophages. Evidence for reduced cholesterol efflux promotion by apoA-I(Pro165-->Arg).

Interaction of cells with both native and reconstituted high density lipoproteins (rHDL) containing apolipoprotein (apo) A-I mediates efflux of cellular cholesterol. To characterize structural requirements for this activity in apoA-I, six different genetic apoA-I variants and the corresponding normal allele products were isolated from heterozygous carriers, reconstituted with dimyristoylphosphatidylcholine (DMPC) into discoidal HDL and compared with regard to their ability to release intracellular cholesterol from murine adipocytes and peritoneal macrophages. In previous studies we determined the amino acid changes of these variants: Pro3-->Arg, Pro4-->Arg, Lys107-->0, Lys107-->Met, Pro165-->Arg, and Glu198-->Lys (von Eckardstein, A., Funke, H., Walter, M., Altland, K., Benninghoven, A., and Assmann, G. (1990) J. Biol. Chem. 265, 8610-8617) and demonstrated that all apoA-I variants except apoA-I(Lys107-->0) form discoidal HDL particles with phosphatidylcholine analogues indistinguishable from normal apoA-I (Jonas, A., von Eckardstein, A., Kezdy, K. E., Steinmetz, A., and Assmann, G. (1991) J. Lipid Res. 32, 95-106). In the present study, all apoA-I.DMPC complexes except those containing apoA-I(Pro165-->Arg) promoted cholesterol efflux as effectively as those containing normal apoA-I. Cholesterol efflux from adipocytes obtained after 180 min of incubation with apoA-I(Pro165-->Arg).DMPC complexes was decreased by 30% although this variant was bound to adipocytes with normal affinity. By contrast, apoA-I(Lys107-->Met).DMPC complexes were decreased in their binding affinity compared to normal apoA-I.DMPC complexes but normally promoted cholesterol efflux. Incubation of mouse peritoneal macrophages with apoA-I(Pro165-->Arg).DMPC complexes did also result in a 30% decreased efflux of radiolabeled cholesterol if compared to rHDL with the normal allele product from the same donor. Together the observations suggest that the substitution of proline at residue 165 interferes with the formation of a structural domain in apoA-I that is crucial for cellular cholesterol efflux stimulation. Furthermore, our results suggest that binding of HDL to adipocytes and cholesterol efflux promotion by HDL requires different structural domains.

[Beclobrate (Turec) in the treatment of primary hyperlipoproteinemia. I. Effect on cholesterol, lipid and apoprotein levels]. / Beklobrat (Turec) w leczeniu pierwotnej hiperlipoproteinemii. Czesc I: Wplyw na stezenie cholesterolu, lipidy i apoproteiny.

The effects of a new fibric acid derivative--beclobrate (Turec, Zyma) on serum lipid and apoprotein concentrations in 63 patients with primary hyperlipoproteinemia were examined. Beclobrate was given in the evening, 100 mg, once daily. After 3 months of beclobrate treatment mean total cholesterol concentration in serum decreased from 9.35 to 7.73 mmol/l (17.3%), mean LDL-cholesterol concentration from 6.32 to 5.38 mmol/l (14.9%), mean HDL-cholesterol concentration increased by 0.21 mmol/l (15.3% of initial value). The greatest decrease was observed in triglyceride concentration--by 50% of the initial value. Apoprotein B concentration decreased by 19.7%, apoprotein A1 and A2 concentration increased by 20.3% and 26.8% respectively. Higher initial values of total cholesterol and triglyceride concentration in serum were associated with greater concentration decrease after beclobrate treatment.

Glycine and serine rich peptides isolated from rat liver preparations enriched with smooth endoplasmic membranes.

Three electrochromatographic homogeneous peptides belonging to acidic, basic and neutral electrophoretic glycopeptide fractions isolated from rat liver preparations enriched with smooth endoplasmic membranes contain a very high level of glycine and serine and only a slight level of phenylalanine but do not contain tyrosine. Their amino acid compositions differ chiefly in the contents of basic and acidic amino acids.