Suppression of plasma free fatty acids reduces myocardial lipid content and systolic function in type 2 diabetes.

BACKGROUND AND AIM: Type 2 diabetes (T2DM) is closely associated with the development of heart failure, which might be related with impaired substrate metabolism and accumulation of myocardial lipids (MYCL). The aim of this study was to investigate the impact of an acute pharmacological inhibition of adipose tissue lipolysis leading to reduced availability of circulating FFA on MYCL and heart function in T2DM. METHODS AND RESULTS: 8 patients with T2DM (Age: 56 ± 11; BMI: 28 ± 3.5 kg/m(2); HbA1c: 7.29 ± 0.88%) were investigated on two study days in random order. Following administration of Acipimox or Placebo MYCL and heart function were measured by (1)H-magnetic-resonance-spectroscopy and tomography at baseline, at 2 and at 6 h. Acipimox reduced circulating FFA by -69% (p < 0.001), MYCL by -39 ± 41% (p < 0.001) as well as systolic heart function (Ejection Fraction (EF): -13 ± 8%, p = 0.025; Cardiac Index: -16 ± 15%, p = 0.063 compared to baseline). Changes in plasma FFA concentrations strongly correlated with changes in MYCL (r = 0.707; p = 0.002) and EF (r = 0.651; p = 0.006). Diastolic heart function remained unchanged. CONCLUSIONS: Our results indicate, that inhibition of adipose tissue lipolysis is associated with a rapid depletion of MYCL-stores and reduced systolic heart function in T2DM. These changes were comparable to those previously found in insulin sensitive controls. MYCL thus likely serve as a readily available energy source to cope with short-time changes in FFA availability.

Fasting and postprandial liver glycogen content in patients with type 1 diabetes mellitus after successful pancreas-kidney transplantation with systemic venous insulin delivery.

BACKGROUND: In patients with type 1 diabetes mellitus (T1DM), insulin is usually replaced systemically (subcutaneously) and not via the physiological portal route. According to previous studies, the liver's capacity to store glycogen is reduced in T1DM patients, but it remains unclear whether this is due to hyperglycaemia, or whether the route of insulin supply could contribute to this phenomenon. T1DM patients after successful pancreas-kidney transplantation with systemic venous drainage (T1DM-PKT) represent a suitable human model to further investigate this question, because they are normoglycaemic, but their liver receives insulin from the pancreas transplant via the systemic route. MATERIALS AND METHODS: In nine T1DM-PKT, nine controls without diabetes (CON) and seven patients with T1DM (T1DM), liver glycogen content was measured at fasting and after two standardized meals employing (13) C-nuclear-magnetic-resonance-spectroscopy. Circulating glucose and glucoregulatory hormones were measured repeatedly throughout the study day. RESULTS: The mean and fasting concentrations of peripheral plasma glucose, insulin, glucagon and C-peptide were comparable between T1DM-PKT and CON, whereas T1DM were hyperglycaemic and hyperinsulinaemic (P < 0·05 vs T1DM-PKT and CON). Total liver glycogen content at fasting and after breakfast did not differ in the three groups. After lunch, T1DM-PKT and T1DM had a 14% and 21% lower total liver glycogen content than CON (P < 0·02). CONCLUSION: In spite of normalized glycaemic control, postprandial liver glycogen content was reduced in T1DM-PKT with systemic venous drainage. Thus, not even optimized systemic insulin substitution is able to resolve the defect in postprandial liver glycogen storage seen in T1DM patients.

Reduction of both number and proliferative activity of human endothelial progenitor cells in obesity.

OBJECTIVE: Circulating endothelial progenitor cells (EPCs), responsible for neoangiogenesis and vascular repair, negatively correlate with vascular dysfunction and atherosclerotic risk factors. Because obesity may have a crucial role in the development of endothelial dysfunction, this study evaluated the number and proliferative activity of circulating human EPCs in obese (body mass index (BMI)=48+/-9, n=45) compared with lean (23+/-2, n=45) volunteers. METHODS: EPCs were quantified after isolation of peripheral blood mononuclear cells (PBMCs) using fluorescence-activated cell sorting analyses. In addition, plated PBMCs developed colony-forming units (CFUs) from which 'outgrowth' endothelial cells (OECs) sprouted and differentiated into mature endothelial cells. Growth rates were monitored by periodical microscopic evaluation. Cell-cycle protein expression was determined by western blot analyses. RESULTS: BMI negatively correlated (P<0.01) with the number of CD34(+)/CD133(+)/KDR(+) (r=-0.442), CD34(+)/KDR(+) (r=-0.500) and CD133(+)/KDR(+) (r=-0.282) EPCs. Insulin, leptin, HbA(1c), high-sensitivity C-reactive protein and hypertension, as well as diminished high-density lipoprotein and apolipoprotein A1, were not only associated with obesity but also with significantly reduced EPC levels. Applying selective culture conditions, EPC-CFUs differentiated into OECs that proliferated more slowly when derived from obese compared with lean subjects (obese: 19.9+/-2.2% vs lean: 30.9+/-3.2% grown area per week, P<0.01). The reduced proliferation was reflected by decreased (P<0.05, n=24 for each group) expression of cell-cycle-promoting cyclins and E2F-1, by hypophosphorylation of retinoblastoma protein and by increased (P<0.05, n=24 for each group) expression of the cell-cycle inhibitor p21(WAF-1/Cip1). CONCLUSIONS: Reduced numbers of EPCs along with their premature senescence, as shown in this study, could function as early contributors to the development and progression of vascular dysfunction in obesity.

Cloning and molecular characterization of Dashurin encoded by C20orf116, a PCI-domain containing protein.

BACKGROUND: Characterization of gene products originating from undefined open reading frames and delineation of biological functions has become the task after the human genome has been decoded. METHODS: We cloned the human C20orf 116 and defined its transcript in liver, kidney and various brain regions by Northern analysis. Antibodies against recombinant protein used for immunofluorescence and immunoblots confirmed its expression in these tissues. With the focus on kidney, its tubular expression and presence in glomerula were shown. RESULTS: A 28 aa long signal peptide predicted by in silico analysis is reflected by visualization of size variants of approximately 3kDa difference suggesting a signal peptidase cleavage of the proform. Cell compartment separation confirmed the presence of Dashurin in peroxisomes/mitochondria, microsomes, cytosol and nucleus. This is in line with green fluorescent protein (GFP)-Dashurin fusion protein shuttling between cytosol and nucleus. Luciferase reporter studies revealed a 2-3 fold increase of promoter activities upon over-expression. Bioinformatic analysis identified a PCI-domain at the C-terminus providing protein-protein interaction capabilities. CONCLUSION: Our present findings suggest the involvement of Dashurin in gene transcription or mRNA translation. GENERAL SIGNIFICANCE: Dashurin shares the PCI-domain with three multisubunit protein complexes (26S proteasome, COP9 signalosome and eIF3 translation initiation factor).

Duplications of the functional CYP21A2 gene are primarily restricted to Q318X alleles: evidence for a founder effect.

CONTEXT: Rare haplotypes with Q318X mutations and duplicated CYP21A2 genes have been reported to occur in different populations to a varying extent. Discrimination between a normal (Q318X mutation on one of the duplicated CYP21A2 genes) and a congenital adrenal hyperplasia (CAH, Q318X mutation without duplicated functional gene) allele is of importance, particularly for prenatal diagnosis and the respective genetic counseling. Although methods to differentiate between such alleles have been published only recently, it remains unclear with which frequency Q318X mutations are associated with duplicated CYP21A2 genes and whether these haplotypes have a common ancestry. SUBJECTS AND METHODS: Human leukocyte antigen (HLA) typing has been performed in 38 unrelated individuals and in 11 family members detected to carry a Q318X mutation in the course of CYP21 genotyping using sequence, multiplex ligation-dependent probe amplification, and Southern blot analyses. RESULTS: The majority (n = 32, 84.2%) of the 38 unrelated individuals carrying the Q318X mutation had the trimodular RCCX haplotype, carrying the Q318X mutation on a duplicated CYP21A2 gene. Twenty-two individuals of these 32 (68.8%) were of the rare HLA-B*50-Cw*06 haplotype, suggesting a common ancestry of this haplotype. In five (13.2%) of the 38 subjects, the Q318X mutation was not associated with a duplicated CYP21A2 gene and thus represents a CAH allele. None of these five patients had the above mentioned HLA haplotype. CONCLUSION: The majority of individuals in whom Q318X mutations are detected carry a duplicated functional CYP21A2 gene and the rare HLA-B*50-Cw*06 haplotype.

Insulin does not regulate glucose transport and metabolism in human endothelium.

BACKGROUND: Although endothelial cells express insulin receptors, it is controversially discussed whether the endothelium represents an insulin-responsive tissue. Since available data are primarily restricted to animal endothelial cells, this study tested (i) whether insulin affects glucose metabolism in human endothelium; (ii) whether insulin sensitivity is different in micro- versus macrovascular endothelial cells; and (iii) whether glucose concentration in the incubation medium affects the cells' response to insulin. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVECs), human adult saphenous vein endothelial cells (HAVECs), human aortic endothelial cells (HAEC), and human retinal endothelial cells (HRECs) as well as human smooth muscle cells were incubated with/without insulin (0.3 nmol L(-1) or 1 micromol L(-1)). Glucose transport, glycogen synthesis, glycogen content, lactate release, and expression of phospho-Akt, Akt, and endothelial nitric oxide synthase (eNOS) were determined. RESULTS: In HUVECs and HRECs, insulin (1 micromol L(-1)) increased (P < 0.05) eNOS expression by ~70% and doubled Akt phosphorylation, but the latter was by far more pronounced in human smooth muscle cells (+1093 +/- 500%, P < 0.05). In human smooth muscle cells, insulin (1 micromol L(-1)) stimulated glycogen synthesis by 67 +/- 11% (P < 0.01). In human micro- (HRECs) and macrovascular endothelial cells (HUVECs, HAVECs and HAECs), insulin, however, failed to stimulate glucose transport, glycogen synthesis, glycogen content, or lactate release under various conditions, i.e. after glucose deprivation or in medium with normal (5.5 mmol L(-1)) or high glucose (30 mmol L(-1)). CONCLUSIONS: Insulin stimulated glycogen synthesis and Akt phosphorylation in human smooth muscle cells. In human micro- and macrovascular endothelial cells, insulin, however, failed to affect glucose uptake and metabolism under all experimental conditions applied, whereas it increased Akt phosphorylation and eNOS expression.

Predisposition for de novo gene aberrations in the offspring of mothers with a duplicated CYP21A2 gene.

CONTEXT: Although CYP21A2 de novo mutations are assumed to account for 1 to 2% of congenital adrenal hyperplasia (CAH) alleles and CYP21 genotyping has been done worldwide, there are only a few well-documented cases of CYP21A2 de novo mutations. The majority of these are deletions resulting from unequal crossings over owing to misalignment of homologous chromosomes during meiosis. Whereas so far, only heterozygous deletions of the CYP21A1P pseudogene were seen as premutations for de novo aberrations, the present report addresses such a predisposing role for parental duplicated CYP21A2 genes. SUBJECTS AND METHODS: As part of routine diagnostic procedures, CYP21 genotyping has been performed in two unrelated female CAH index patients and in their clinically asymptomatic parents and siblings. RESULTS: Both patients have inherited the paternal Intron2splice mutation and have harbored a de novo gene aberration (large deletion and I271N/exon 4) on their maternal haplotype. Surprisingly, both mothers were carriers of rare duplicated CYP21A2 haplotypes carrying CAH alleles, which were not detected in the daughters. Among 133 CAH alleles that were detected in patients and that could be traced to the respective family members by genotyping, these two de novo aberrations (representing 1.5% of 133 traced CAH alleles) were the only ones identified. CONCLUSION: Because both de novo CYP21A2 gene aberrations so far identified in our laboratory occurred in the gametes of mothers carrying rare duplicated CYP21A2 haplotypes, we hypothesize that duplicated CYP21A2 genes could predispose for de novo mutations in the offspring, which is of relevance for prenatal CYP21 genotyping and genetic counseling.

Free fatty acids do not acutely increase asymmetrical dimethylarginine concentrations.

Concentrations of asymmetrical dimethylarginine (ADMA) and free fatty acids (FFAs) are elevated in insulin resistance which is associated with impaired vascular function. We hypothesized that FFAs could alter vascular tone by affecting ADMA concentrations. Plasma FFA levels were increased in seventeen healthy male volunteers by Intralipid/heparin infusion; hemodynamic and biochemical parameters were measured after 90 minutes. Plasma collected before and during Intralipid/heparin or equivalent synthetic FFAs was incubated with human umbilical vein endothelial cells (HUVECs) in vitro. Intralipid/heparin infusion resulted in an approximately seven-fold increase in plasma FFA levels to 1861 +/- 139 micromol/l, which was paralleled by increased systemic blood pressure and forearm blood flow. Intralipid/heparin did not affect ADMA (baseline mean 0.59 [95 % confidence interval [CI]: 0.54; 0.64] and 0.56 [CI: 0.51; 0.59] after 90 minutes), but slightly decreased SDMA (from 0.76, [CI: 0.70; 0.83] to 0.71 [CI: 0.64; 0.74], p < 0.05), and had no effect on ADMA/SDMA ratio. There was no correlation between ADMA and FFA concentrations or forearm blood flow. Incubation of HUVECs with FFA-rich plasma or synthetic FFAs induced an ADMA release after 24 hours, but not after 90 minutes. Acutely increased FFA levels caused hemodynamic effects but did not affect ADMA. Prolonged elevation of FFA levels might influence vascular function by increasing ADMA levels.

Early diagnosis and curative therapy of medullary thyroid carcinoma by routine measurement of serum calcitonin in patients with thyroid disorders.

To identify patients with medullary thyroid carcinoma (MTC) at a potentially curable stage of the disease, serum concentrations of calcitonin (hCT) were determined in 14,000 patients (including 10,158 patients with thyroid nodules) referred to a thyroid outpatient clinic. Excluding patients in whom elevated basal hCT concentrations had already been known at the time of their referral, 507 patients with thyroid nodules presented basal concentrations of hCT of more than 10 pg/ml. Following stimulation by IV pentagastrin (0.5 microg/kg BW), hCT concentrations of more than 100 pg/ml were seen in 103 patients. This group included 32 new cases of MTC (29 patients with sporadic MTC and 3 new index cases of the familial form) and 43 patients with C cell hyperplasia (CCH). Among the 3,843 patients without thyroid nodules, 2 were found to harbor sporadic MTC while 4 had CCH. As compared to 1.1 cases of MTC per 1,000 patients with nodular thyroid diseases diagnosed in our institution before hCT screening was begun, 3.2 cases of MTC per 1,000 patients were identified when hCT was determined in all patients with thyroid nodules. The determination of hCT in all patients with thyroid nodular disease facilitates the timely diagnosis of MTC, thus providing the chance of curative surgery.

Primary hyperparathyroidism as the leading symptom in a patient with a Y791F RET mutation.

Primary hyperparathyroidism (PHP; serum calcium 2.75 mmol/L, PTH 226 pg/ml) had been the first clinical manifestation of MEN-2A in a female patient (aged 55 years) with a mutation (Y791F, TAT-->TTT) in exon 13 of the RET proto-oncogene. The patient has a pentagastrin-induced rise in serum calcitonin (up to 57 pg/ml) considered normal for noncarriers but abnormal in family members of MEN-2 patients. This is the first case of MEN-2 due to this specific mutation with primary hyperparathyroidism as the first manifestation of the disease. In addition, the patient harbored, within the Menin gene, a polymorphism (D418D) reportedly associated with sporadic primary hyperparathyroidism. This case report indicates that molecular biological tests in MEN- 2 may only suggest a certain phenotype but cannot predict it with certainty. It may also suggest that genetic screening for MEN-2 may be advisable in patients with primary hyperparathyroidism and a borderline-high pentagastrin stimulation test, even in the absence of a positive family history.

Polymorphisms in exon 13 and intron 14 of the RET protooncogene: genetic modifiers of medullary thyroid carcinoma?

CONTEXT: Single-nucleotide polymorphisms (SNPs) of the RET protooncogene (RET) could modify disease susceptibility and clinical phenotype in patients with sporadic or familial medullary thyroid carcinoma (FMTC). OBJECTIVE/DESIGN OF THE STUDY: Because frequencies of RET SNPs have not yet been evaluated in patients with elevated serum concentrations of calcitonin (hCt), a biochemical marker for medullary thyroid carcinoma (MTC), we studied RET SNPs in patients with FMTC (n = 22), patients with sporadic MTC (n = 45), and 71 subjects presenting with moderately elevated hCt concentrations (basal, >10 pg/ml; pentagastrin stimulated, > 50 < 100 pg/ml) in comparison with an age- and gender-matched control group (n = 79) with basal hCt concentrations in the normal range (<5 pg/ml). METHODS: After DNA extraction from citrated whole blood, RET exons 10, 11, 13, 14, 15, and 16 and exon/intron boundaries were analyzed by PCR-based cycle sequencing for RET germ line mutations, exonic (G691S, L769L, S836S, S904S) and intronic (IVS13+158; NCBI rs2472737 = IVS14-24) SNPs. RESULTS: In FMTC patients, the F791Y mutation was found to be associated (P = 0.001) with the L769L SNP. The exonic SNPs (G691S, L769L, S836S, and S904S) were not different among the four groups. The intron 14 SNP (IVS14-24), however, was more frequent in individuals with elevated hCt serum concentrations (P = 0.016) and patients with sporadic MTC (P < 0.001) when compared with the control group. CONCLUSIONS: These data suggest that the exon 13 (L769L) and the intron 14 (IVS14-24) SNPs could act as genetic modifiers in the development of some forms of hereditary and sporadic MTC, respectively.

Mutational spectrum of steroid 21-hydroxylase and the genotype-phenotype association in Middle European patients with congenital adrenal hyperplasia.

OBJECTIVE: To analyze the mutational spectrum of steroid 21-hydroxylase (CYP21) and the genotype- phenotype correlation in patients with congenital adrenal hyperplasia (CAH) registered in the Middle European Society for Pediatric Endocrinology CAH database, and to design a reliable and rational approach for CYP21 mutation detection in Middle European populations. DESIGN AND METHODS: Molecular analysis of the CYP21 gene was performed in 432 CAH patients and 298 family members. Low-resolution genotyping was performed to detect the eight most common point mutations. High-resolution genotyping, including Southern blotting and sequencing was performed to detect CYP21 gene deletions, conversions, point mutations or other sequence changes. RESULTS: CYP21 gene deletion and In2 and Ile172Asn mutation accounted for 72.7% of the affected alleles in the whole study group. A good genotype-phenotype correlation was observed, with the exception of Ile172Asn and Pro30Leu mutations. In 37% of patients low resolution genotyping could not identify the causative mutation or distinguish homozygosity from hemizygosity. Using high-resolution genotyping, the causative mutations could be identified in 341 out of 348 analyzed patients. A novel mutation Gln315Stop was found in one simple virilising CAH (SV-CAH) patient from Austria. In the remaining seven patients polymorphisms were identified as the leading sequence alteration. The presence of elevated basal and ACTH-stimulated 17-hydroxyprogesterone, premature pubarche, advanced bone age and clitoral hypertrophy directly implicated Asn493Ser polymorphism in the manifestation of nonclassical- (NC) and even SV-CAH. CONCLUSIONS: By genotyping for the most common point mutations, CYP21 gene deletion/conversion and the 8 bp deletion in exon 3, it should be possible to identify the mutation in 94-99% of the diseased alleles in any investigated Middle European population. In patients with a mild form of the disease and no detectable mutation CYP21 gene polymorphisms should be considered as a plausible disease-causing mutation.

Thiazolidinediones inhibit proliferation of microvascular and macrovascular cells by a PPARgamma-independent mechanism.

AIMS/HYPOTHESIS: This study evaluated the hypothesis that peroxisome proliferator-activated receptor-gamma (PPARgamma) agonists, including thiazolidinediones (TZDs) and the rexinoid LG100268 (LG), directly affect human vascular cell function (proliferation, cell cycle, protein expression, lactate release) independently of (1) their PPARgamma-activating potential and (2) the cells' vascular origin. METHODS: Human umbilical vein endothelial cells (HUVECs), human adult vein endothelial cells (HAVECs), human retinal endothelial cells (HRECs) and human retinal pericytes (HRPYCs) were incubated (48 h) with 2-50 micromol/l rosiglitazone (RSG), RWJ241947 (RWJ), pioglitazone (PIO), troglitazone (TRO), 15-deoxy-Delta(12,14)-prostaglandin J2 (PGJ2) and LG. Proliferation, cell cycle distribution, protein expression, peroxisome proliferator-activated receptor responsive element (PPRE) transcriptional activity and mitochondrial effects were determined by [3H]thymidine incorporation, FACS analyses, western blots, reporter assays and lactate release respectively. RESULTS: In HUVECs, RSG, RWJ, PIO, TRO, PGJ2 and LG reduced (p<0.01) proliferation (due to a G0/G1 cell cycle arrest) by up to 23%, 36%, 38%, 86%, 99% and 93% respectively. The antiproliferative response was similar in HRPYCs and HAVECs, but was attenuated in HRECs. Whereas p21WAF-1/Cip1 and p27Kip were differently affected in HUVECs, all agents reduced (p<0.05) expression of cyclins (D3, A, E, B), cyclin-dependent kinase-2 and hyperphosphorylated retinoblastoma protein. The rank order of the antiproliferative effects of TZDs in HUVECs (RSG approximately PIO approximately RWJ

Carrier frequency of congenital adrenal hyperplasia (21-hydroxylase deficiency) in a middle European population.

Based on newborn screening data, the carrier frequency of congenital adrenal hyperplasia (CAH) in the general population has been estimated to be 1:55. The higher CAH frequency (particularly of milder forms of the disease) reported for certain populations including Yugoslavs (1.6%) relates to population genetic and hormonal data. However, so far, true carrier frequency for CAH due to 21-OH deficiency has not been determined by comprehensive mutation analysis of the 21-OH gene (CYP21A2) in an unselected European population. This study used CYP21A2 genotyping (sequence/Southern blot analysis) to determine CAH carrier frequency in a middle European (Austrian) population. The study included 100 migrants from the former Yugoslavia and 100 individuals of non-Yugoslavian origin. None of these individuals showed clinical hyperandrogenism or had a family history of CAH. Genotyping 400 unrelated alleles from 200 clinically unaffected individuals, this study revealed a carrier frequency of 9.5%, including so-called "classic" (5.5%) and "nonclassic" (4%) CYP21A2-gene aberrations. The observed heterozygosity for CAH in Yugoslavs was not different (P = 0.8095) from that in non-Yugoslavs. In conclusion, the observed CAH carrier frequency of 9.5% suggests a higher prevalence of CAH heterozygosity in a middle European population than hitherto estimated independently of the individuals' Yugoslav or non-Yugoslav origin.

Frequency of RET proto-oncogene mutations in patients with normal and with moderately elevated pentagastrin-stimulated serum concentrations of calcitonin.

The possibility of germline mutations of the RET proto-oncogene (exons 10, 11, 13, 14, and 16) was investigated in 75 patients (57 men, 18 women) with a negative family history for medullary thyroid carcinoma (MTC), elevated (> 10 pg/mL) basal serum concentrations of human calcitonin (hCT) and a pentagastrin (PG)-stimulated serum hCT ranging from 50-100 pg/mL. Seventy patients (50 men, 20 women) with basal serum calcitonin concentrations in the normal range served as controls. Among the 75 patients with elevated basal serum hCT concentrations we identified 1 man with the mutation S649L and 2 patients (1 man and 1 woman) with the mutation Y791F. Among the 70 individuals with normal basal calcitonin 1 man and 1 woman presented with the mutation Y791F. No other mutations (such as those in codons 618 or 634, considered to be of greater clinical relevance) were identified in either group. On the other hand, the RET proto-oncogene mutation Y791F, characterized by a low penetrance, occurs comparatively frequently among patients with normal serum calcitonin concentrations. To preselect patients for RET screening by moderately (50-100 pg/mL) pentagastrin stimulation hCT concentrations does not increase the number of identified cases of familial MTC.

A rare duplicated 21-hydroxylase haplotype and a de novo mutation: a family analysis.

21-Hydroxylase (21-OH) genotyping was performed in clinically unaffected family members of a congenital adrenal hyperplasia (CAH) index patient (Prader stage 3), who is a compound heterozygous carrier of the I172N (exon 4) and the intron2 splicing mutations. Whereas the latter mutation could be traced to the father, the exon 4 aberration represents a de novo mutation (accounting for 1% of CAH alleles) harbored on an unaffected allele, which was inherited from the mother. Although clinically and biochemically unaffected, the patient's brother was found to be compound heterozygous for intron2splice (paternal allele) and Q318X in exon 8 (maternal allele). As shown by PCR-based sequence and Southern blot analysis, the maternal haplotype, inherited by the brother, has a duplicated CYP21B (functional) gene, one of which carries a Q318X mutation. This duplicated Q318X-affected haplotype is the first of its kind among 800 alleles screened for 21-OH deficiency in our laboratory and has to date been reported only in three Swedish CAH patients, all of them bearing an intron2splice and a Q318X mutation. This family analysis highlights the complexity of the CYP21/CYP21P(pseudogene) loci and the difficulties of 21-OH genotyping.

Increased prevalence of heterozygous 21-OH germline mutations in patients with adrenal incidentalomas.

OBJECTIVE: As a result of the widespread use and the enhanced quality of high-resolution radiological techniques [computed tomography (CT), magnetic resonance imaging (MRI)] a high frequency (4-10%) of adrenal incidentalomas has been detected in the general population. It is still debated whether undiagnosed 21-hydroxylase (21-OH) deficiency, accounting for more than 90% of congenital adrenal hyperplasia (CAH) cases, predisposes for adrenal tumours. We therefore performed an analysis of the prevalence of 21-OH germline mutations in patients with non-functional adrenal incidentalomas. SUBJECTS AND METHODS: Fifty Austrian patients with non-functional adrenal adenomas detected by CT for unrelated reasons were screened by PCR-based sequencing for the most common point mutations and by Southern blot analysis for large gene deletion/conversion events of the 21-OH gene. RESULTS: Heterozygosity for large gene conversions was shown in 5 (10%), for Q318 point mutations in 2 (4%) and for the Intron2splice mutation in 1 (2%) of the 50 patients with adrenal adenomas. One (2%) patient (70 years of age), identified to have a chimeric CYP21AB gene with a junction site before Intron 2 on one and a large (30 kb) deletion on the other allele, was diagnosed to be affected by CAH. CONCLUSION: 21-OH mutation screening indicates a higher frequency of classic CAH carriers (16%) and of manifest CAH (2%) due to 21-OH-deficiency among patients with adrenal adenomas than in the general population (1-2% carrier frequency for classic CAH).

Modulation by leptin of proliferation and apoptosis in vascular endothelial cells.

AIM: Plasma leptin concentrations not only correlate with body fat mass, but also with the degree of hypertensive retinopathy. The present study was designed to further examine, whether leptin's proliferative, proangiogenic activity relates to a yet uncovered anti-apoptotic effect. RESULTS: Leptin (10-50 nmol/l) concentration-dependently reduced apoptosis in HUVECs (human umbilical vein endothelial cells), HAVECs (human adult vein endothelial cells) and HMECs (human microvascular endothelial cells) by 20% (P < or = 0.05). These findings were supported by increased expression of the apoptosis inhibitor bcl-2 (+55%, P < or = 0.05) as well as by differential modulation of the respective cell cycle checkpoint genes/proteins p53 (-20%, P < or = 0.01), p21(WAF-1/Cip1) (-23%, P < or = 0.05) and the Retinoblastoma protein (+123%, P < or = 0.01). CONCLUSION: bcl-2 dependent anti-apoptotic action might contribute to leptin's proangiogenic activity and thereby promote the development of vascular proliferative disease in obesity.

Mutational spectrum of the steroid 21-hydroxylase gene in Austria: identification of a novel missense mutation.

This study attempted an analysis of the mutational spectrum of 21-hydroxylase deficiency in 79 unrelated Austrian patients with classical and nonclassical forms of congenital adrenal hyperplasia and their respective 112 family members. Apparent large gene deletions/conversions were present in 31% of the 158 unrelated congenital adrenal hyperplasia alleles, whereas the most frequent point mutations were intron 2 splice (22.8%), I172N (15.8%), V281L (12%), and P30L (7.6%), in line with the frequencies reported for other countries. In 5 of the 12 congenital adrenal hyperplasia alleles carrying a P30L mutation the aberration is based on a single base substitution, whereas the remaining 7 represent part of a CYP21B conversion (1 allele) or CYP21B/21A hybrid gene (6 alleles), the latter characterized by a junction site before intron 2 as indicated by Southern blot, PCR, and sequence analyses. Previously described mutations were not present in 1.2% of unrelated congenital adrenal hyperplasia alleles, including one female patient presenting with severe genital virilization. Sequence analysis of the complete functional 21-hydroxylase gene revealed an as yet undescribed mutation in exon 10-Arg(426)His, which has not yet been described to represent a common pseudogene sequence. In vitro expression experiments showed the Arg(426)His mutant to exhibit only low enzyme activity toward the natural substrate 17-hydroxyprogesterone corresponding to the degree of disease manifestation in the patient in whom it was found.

The endothelium as a metabolic and endocrine organ: its relation with insulin resistance.

The vascular endothelium, building the inner layer of capillaries and blood vessels of all sorts, represents a highly active metabolic and endocrine organ producing a multitude of different molecules, including vasoactive peptide hormones, growth factors, coagulation factors and adhesion molecules. In addition, it expresses many of the respective endocrine, paracrine and cytokine/growth factor receptors. It thereby regulates the delicate balance between vasoconstriction and vasodilation, between coagulation and fibrinolysis, proliferation and apoptosis, as well as between transient adhesion and diapedesis of blood borne leukocytes. This minireview addresses the potential interactions of these important functions in the states of diabetes and of insulin resistance including obesity, hypertension and dyslipidaemia, all of which characterized by endothelial dysfunction.