[Lipid-lowering therapy in the elderly : Who profits from which target values?] / Lipidsenkende Therapie im Alter : Wer profitiert von welchem Zielwert?

Lowering low-density lipoprotein (LDL) cholesterol levels has been proven to reduce the incidence of cardiovascular and cerebrovascular events and mortality. So far recommendations have not provided information as to a meaningful duration of cholesterol-lowering therapy and were largely guided by economic constraints and limited therapeutic options. In light of the decline in the price of statins, the essential therapeutic agent and the increased efficacy of therapeutic options, treatment can nowadays be geared to target values that can be expected to have an optimal effect even in old age. The most favorable level of LDL-cholesterol for primary prevention is around and below 100 mg/dl, provided continuous adherence to these low levels from adolescence onwards. With later onset of cholesterol reduction the existence of initial atheromatous deposits must be expected. Therefore, with age and the manifestation of other risk factors the optimal treatment targets increasingly converge to those for which experience has been gained from secondary prevention. Both measurements of the effect of cholesterol lowering on the volume of atheromatous plaques and of the incidence of vascular events indicate a target for LDL-cholesterol well below 70 mg/dl and in the range 50-60 mg/dl. At the onset of cholesterol lowering in advanced age, a smaller effect has to be expected but due to the increasing incidence rate of vascular events a higher number of events may be avoided; thus, the efficiency does not necessarily decrease; however, long-term studies indicate that earlier cholesterol lowering provides an advantage for more than a decade, in terms of preventing vascular disease, which tends to increase. Therefore, optimal cardiovascular prevention involves moderate measures to maintain the LDL-cholesterol below 100 mg/dl lifelong from childhood on.

[New AHA and ACC guidelines on the treatment of blood cholesterol to reduce atherosclerotic cardiovascular risk : Statement of the D•A•CH Society for Prevention of Cardiovascular Diseases, the Austrian Atherosclerosis Society and the Working Group on Lipids and Atherosclerosis (AGLA) of the Swiss Society for Cardiology]. / Neue AHA- und ACC-Leitlinie zur Risikoreduktion von Herz-Kreislauf-Erkrankungen durch Cholesterinsenkung : Stellungnahme der D•A•CH-Gesellschaft Prävention von Herz-Kreislauf-Erkrankungen e. V., der Österreichischen Atherosklerose Gesellschaft und der Arbeitsgruppe Lipide und Atherosklerose (AGLA) der Schweizer Gesellschaft für Kardiologie.

Guidelines for the reduction of cholesterol to prevent atherosclerotic vascular events were recently released by the American Heart Association and the American College of Cardiology. The authors claim to refer entirely to evidence from randomized controlled trials, thereby confining their guidelines to statins as the primary therapeutic option. The guidelines derived from these trials do not specify treatment goals, but refer to the percentage of cholesterol reduction by statin medication with low, moderate, and high intensity. However, these targets are just as little tested in randomized trials as are the cholesterol goals derived from clinical experience. The same applies to the guidelines of the four patient groups which are defined by vascular risk. No major statin trial has included patients on the basis of their global risk; thus the allocation criteria are also arbitrarily chosen. These would actually lead to a significant increase in the number of patients to be treated with high or maximum dosages of statins. Also, adhering to dosage regulations instead of cholesterol goals contradicts the principles of individualized patient care. The option of the new risk score to calculate lifetime risk up to the age of 80 years in addition to the 10-year risk can be appreciated. Unfortunately it is not considered in the therapeutic recommendations provided, despite evidence from population and genetic studies showing that even a moderate lifetime reduction of low-density lipoprotein (LDL) cholesterol or non-HDL cholesterol has a much stronger effect than an aggressive treatment at an advanced age. In respect to secondary prevention, the new American guidelines broadly match the European guidelines. Thus, the involved societies from Germany, Austria and Switzerland recommend continuing according to established standards, such as the EAS/ESC guidelines.

[Familial hypercholesterolemia]. / Familiäre Hypercholesterinämie.

Familial hypercholesterolemia is one of the most common hereditary metabolic disorders, untreated with grave cardiovascular consequences. A general practitioner will see at least one affected individual each month, but will rarely be aware of the diagnosis, though it is easily suspected: an LDL-cholesterol ≥ 190 mg/dl, a family history of premature cardiovascular disease, or clinical signs as arcus lipoides, tendinous xanthomata, or a thickened Achilles' tendon must draw the attention to familial hypercholesterolemia. Because of the burden of high cholesterol levels from childhood on therapy should be initiated early enough, which has become greatly ameliorated since the introduction of statins. In conjunction with additional risk factors, notably low HDL-cholesterol or elevated lipoprotein(a) the cardiovascular sequelae can be dramatic and may call for more intense therapies. However, often the routine of successful cholesterol lowering covers the diagnosis nowadays, so that a heritable metabolic disorder is not suspected, which, however, prevents an effective prevention in relatives, particularly the children of the patient.

Resequencing the apolipoprotein A5 (APOA5) gene in patients with various forms of hypertriglyceridemia.

OBJECTIVE: Genomewide association studies (GWAS), conventional association studies and the characterization of families with ApoA5 deficiency have shown that variation in the apolipoprotein A5 (APOA5) gene is associated with plasma triglyceride levels. The aim of this study was to determine the frequency of rare variants in the APOA5 gene in patients with various forms of hypertriglyceridemia. METHODS: The DNA sequence of the exons plus exon/intron boundaries of the APOA5 gene of 291 patients with triglycerides above the 95th percentile for age and sex (98 of whom had triglycerides above 875 mg/dl), 111 patients with APOE2/2 genotype of whom 100 had Type III Hyperlipidemia and 108 probands with triglycerides below the 25th percentile for age and sex was determined. RESULTS: Twenty four variants were detected of which eight have been previously reported. There were nine patients with triglycerides above 875 mg/dl and nine patients with moderately elevated triglycerides who were carriers of at least one deleterious mutation in the APOA5 gene. Of the patients with Type III HLP, three (3%) were carriers of rare variants and there was a single rare variant detected in the group of probands with triglycerides below the 25th percentile for age and sex. CONCLUSION: Rare mutations in the APOA5 gene are more frequent in patients with elevated triglycerides than in those with Type III HLP.

Cerebrovascular atherosclerosis in type III hyperlipidemia is modulated by variation in the apolipoprotein A5 gene.

OBJECTIVE: Type III Hyperlipoproteinemia is a rare lipid disorder with a frequency of 1-5 in 5000. It is characterized by the accumulation of triglyceride rich lipoproteins and patients are at increased risk of developping atherosclerosis. Type III HLP is strongly associated with the homozygous presence of the ε2 allele of the APOE gene. However only about 10% of subjects with APOE2/2 genotype develop hyperlipidemia and it is therefore assumed that further genetic and environmental factors are necessary for the expression of disease. It has recently been shown that variation in the APOA5 gene is one of these co-factors. The aim of this study is to investigate the development of cerebrovascular athero?sclerosis in patients with Type III hyperlipopro?teinemia (Type III HLP) and the role of variation in the APOA5 gene as a risk factor. METHODS: 60 patients with type III hyperlipidemia and ApoE2/2 genotype were included in the study after informed consent. The presence of cerebrovascular atherosclerosis was investigated using B-mode ultrasonography of the carotid artery. Serum lipid levels were measured by standard procedures.The APOE genotype and the 1131T>C and S19W SNPs in the APOA5 gene and the APOC3 sstI SNP were determined by restriction isotyping. Allele frequencies were determined by gene counting and compared using Fisher's exact test. Continuous variables were compared using the Mann Whitney test. A p value of 0.05 or below was considered statistically significant. Analysis was performed using Statistica 7 software. RESULTS: The incidence of the APOA5 SNPs, -1131T>C and S19W and the APOC3 sstI SNP were determined as a potential risk modifier. After correction for conventional risk factors, the C allele of the -1131T>C SNP in the APOA5 gene was associated with an increased risk for the development of carotid plaque in patients with Type III HLP with an odds ratio of 3.69. Evaluation of the genotype distribution was compatible with an independent effect of APOA5. CONCLUSIONS: The development of atherosclerosis in patients with Type III HLP is modulated by variation in the APOA5 gene.

Rare variants in the lipoprotein lipase (LPL) gene are common in hypertriglyceridemia but rare in Type III hyperlipidemia.

OBJECTIVE: Genomewide association studies (GWAS) have shown that variation in the lipoprotein lipase gene (LPL) is associated with plasma triglyceride levels but that common variants account for only 1.25% of the variance. The aim of this study was to determine the frequency of rare variants in the LPL gene in patients with various forms of hypertriglyceridemia. METHODS: The DNA sequence of the exons plus exon/intron boundaries of the LPL gene of 313 patients with triglycerides above the 95th percentile for age and sex (107 of whom had triglycerides above 875 mg/dl) and 121 patients with Type III hyperlipidemia was determined. RESULTS: Twenty rare variants were detected of which seven have been previously reported. All of the rare variants were present as heterozygotes. Sixteen were missense mutations, two were short deletion mutants and there were single nonsense and insertion mutations. Fifteen of the missense mutations resulted in an amino acid change. There were 13 patients (12.1%) with triglycerides above 875 mg/dl and 10 patients (4.9%) with moderately elevated triglycerides, who were carriers of at least one rare, non-synonymous mutation in the LPL gene. Of the patients with Type III HLP, two were carriers of rare variants. CONCLUSION: Rare mutations in the LPL gene are frequent in patients with elevated triglycerides.

Screening for atherosclerotic plaques in the abdominal aorta in high-risk patients with multicontrast-weighted MRI: a prospective study at 3.0 and 1.5 tesla.

OBJECTIVE: This prospective study compares MRI of atherosclerotic plaque in the abdominal aorta at 3 T with that at 1.5 T in patients suffering from hereditary hyperlipidaemia, a major risk factor for atherosclerosis. METHODS: MRI of the abdominal aorta at 1.5 and 3 T was performed in 21 patients (mean age 58 years). The study protocol consisted of proton density (PD), T(1), T(2) and fat-saturated T(2) weighted black blood images of the abdominal aorta in corresponding orientation. Two independent radiologists performed image rating. First, image quality was rated on a five-point scale. Second, atherosclerotic plaques were scored according to the modified American Heart Association (AHA) classification and analysed for field strength-related differences. Weighted κ statistics were calculated to assess interobserver agreement. RESULTS: Interobserver agreement was substantial for nearly all categories. MRI at 3 T offered superior image quality in all contrast weightings, most significantly in T(1) and T(2) weighted techniques. Plaque burden in the study collective was unexpectedly moderate. The majority of plaques were classified as AHA III lesions; no lesions were classified above AHA V. There was no significant influence of the field strength regarding the AHA classification. CONCLUSION: Abdominal aortal plaque screening is basically feasible at both field strengths, whereas the image quality is rated superior at 3 T. However, the role of the method in clinical practice remains uncertain, since substantial findings in the high-risk collective were scarce.

Current strategies and recent advances in the therapy of hypercholesterolemia.

Lipid therapy is an option for preventing atherosclerotic vascular disease that has been intensively studied and proved to be effective independent of the underlying risk factors. Since the optimal LDL-cholesterol appears to lie well below 100 mg/dl most potent lipid lowering drugs and adjunctive HDL-raising therapeutics are mandatory. Inhibition of cholesterol synthesis and absorption is currently the preferred measure. However, new developments may substantially increase the efficacy of lipid therapy. One is add-on colesevelam, a synthetic bile-acid sequestrant with increased binding affinity which allows smaller dosages for better tolerability. Alternatively HDL-cholesterol may be increased by 25% using niacin with improved tolerability due to the combination with laropiprant, an inhibitor of the receptor for prostaglandin D2-receptor, which minimizes flushing close to placebo level. Mipomersen, a specific oligonucleotide capable to reduce apolipoprotein B-100 up to 70%, is certainly the most advanced approach to challenge even apheresis as the most effective measure to lower exceptionally elevated cholesterol levels.

Genetic variation in two proteins of the endocannabinoid system and their influence on body mass index and metabolism under low fat diet.

The endocannabinoid system (ECS) plays an important and not yet fully understood role in hypothalamic and peripheral regulation of food intake, obesity, and metabolism. Two frequent single nucleotide polymorphisms (snp) have been identified in members of the ECS: the 1359 G/A variant in the cannabinoid receptor 1 ( CB1) and the P129T polymorphism in fatty acid amide hydrolase ( FAAH), a key degradation enzyme of endocannabinoids. -While for the 1359 G/A variant an association has been shown only with psychiatric diseases such as drug-abusing schizophrenia, the P129T polymorphism has recently been proved to be correlated to a higher body mass index (BMI) in a group of black and white Americans. However, no knowledge exists as to whether these variants affect the outcome of a low fat diet in obese subjects. Therefore, we genotyped a group of 451 obese and dyslipidaemic participants and observed the biometric and metabolic outcome of a 6 week low fat diet. While no significance was seen for the 1359 G/A variant, carriers of the P129T mutation in FAAH had a significantly greater decrease in triglycerides and total cholesterol as compared to wild type. The reason for our findings remains to be elucidated, however, a hepatic downregulation of endocannabinoid tone may contribute to the observed outcome in studied subjects.

TNF-alpha alters visfatin and adiponectin levels in human fat.

Adiponectin and visfatin are newly discovered adipokines that are strongly expressed in human visceral adipose tissue. To identify new regulatory mechanisms in fat, the effect of TNF-alpha (TNF) on adiponectin, on its two receptors, and on visfatin was investigated by incubating human visceral adipose tissue from patients without diabetes mellitus with TNF for 24, 48 and 72 hours. The mRNA expression of visfatin, adiponectin, and its two receptors, as well as the protein expression of adiponectin were determined. A decrease of adiponectin mRNA expression of 97% after incubation with TNF (5.75 nmol/l) for 24 hours, a decrease of 91% after 48 hours, and a decrease of 96% after 72 hours were measured. The reduction of protein expression was measured to be 42% after 24 hours, 28% after 48 hours, and 39% after 72 hours of incubation with TNF (5.75 nmol/l). The mRNA level of adiponectin receptor 1 (AdipoR1) was elevated about 72% after 48 hours of incubation and 67% after 72 hours of incubation, whereas the mRNA expression of adiponectin receptor 2 (AdipoR2) was not altered significantly. The visfatin mRNA level was found to be highly increased by 255% after 24 hours and 335% after 48 hours and 341% after 72 hours of incubation with TNF (5.75 nmol/l). Our results support the concept of visceral adipose tissue as an endocrine organ. We demonstrate that TNF has regulatory functions on adiponectin, AdipoR1 and on visfatin in human visceral adipose tissue. TNF levels are elevated in states of obesity and insulin resistance. Due to this fact TNF could be the reason that there is a decrease in the level of adiponectin, whereas there is an increase in the level of visfatin in states of obesity and insulin resistance.

Association of peroxisome proliferator-activated receptor delta +294T/C with body mass index and interaction with peroxisome proliferator-activated receptor alpha L162V.

OBJECTIVE: To investigate the association of a polymorphism at position 294 (+294T/C) in the Peroxisome Proliferator-activated Receptor delta (PPARdelta) with body mass index (BMI) and the additional role of a gene-to-gene interaction between PPARdelta, PPARalpha and PPARgamma. DESIGN: An association between genetic variations in PPARdelta, PPARalpha and PPARgamma and indices of obesity and metabolism. SUBJECTS: A group of 462 moderately obese (mean BMI 28.9+/-7.7) and dyslipidemic, middle-aged (mean age 43.9+/-13.7), Caucasion men and women. MEASUREMENTS: The three most frequent single-nucleotide-polymorphisms (snp) in PPARdelta (+294T/C), PPARalpha (L162V) and PPARgamma (P12A) were genotyped and associated with clinical parameters. RESULTS: The C allele in PPARdelta was significantly associated with a lower body mass index. Moreover an interaction between the polymorphisms in PPARalpha and PPARdelta on body weight could be demonstrated. CONCLUSION: Our data provide further evidence for an involvement of PPARdelta in the regulation of BMI.

Polymorphisms in the apolipoprotein A5 (APOA5) gene and type III hyperlipidemia.

The great majority of patients with type III hyperlipidemia (type III HLP) are homozygous for the epsilon2 allele of the APOE gene. However, only about 10% of epsilon2 homozygotes develop type III HLP, and it has been proposed that additional genetic factors are required for the development of the condition. The frequency of two polymorphisms in the APOA5 gene, -1131T>C and S19W, has been determined in 72 hyperlipidemic patients with APOE2/2 genotype attending a lipid clinic. The frequency of both polymorphisms was significantly higher in APOE2/2 patients than in the normal population. Fifty-three percent of APOE2/2 patients were carriers of one of the polymorphisms compared to 19.7% of controls. Thus, genetic variation in the APOA5 gene is an important cofactor in the development of type III HLP.

A polymorphism in the apolipoprotein A5 gene is associated with weight loss after short-term diet.

Apolipoprotein A5 is a recently discovered apolipoprotein involved primarily in triglyceride metabolism. Several single-nucleotide polymorphisms have been investigated since the initial report. The -1131T>C polymorphism has been associated with higher triglyceride levels and a decreased high-density lipoprotein cholesterol as well as with susceptibility to coronary heart disease. However, no study has so far emphasized on the association of a dietary intervention with apolipoprotein A5 polymorphisms. In a group of 606 hyperlipaemic and overweight men, we investigated how a short-term fat restriction affects lipid traits and body mass index (BMI) in wildtype and carriers of the -1131T>C polymorphism. Our result was that the reduction of BMI was significantly higher in C allele carriers (p=0.0021). Since the -1131T>C polymorphism predisposes to coronary heart disease, a restriction diet is an important therapeutic approach in -1131T>C carriers.

Vitamin B6 modulates glucocorticoid-dependent gene transcription in a promoter- and cell type-specific manner.

Due to their immunosuppressive effects, glucocorticoids (GC) are widely used in the treatment of inflammatory and autoimmune states. However, long-term GC treatment is associated with severe side effects. To increase the ratio of wanted and unwanted GC effects, is, therefore, a desirable goal, which could be achieved by either developing new "dissociating" GC or by combining conventional GC therapy with substances that selectively interfere with glucocorticoid receptor (GR) function. Vitamin B6 was previously shown to inhibit GR transactivation in non-immune cells. In the present study, we tested whether vitamin B6 would also interfere with GR function in immune cells and/or with transrepression in non-immune cells. Normal human lymphocytes and Jurkat T lymphoma cells were transfected with luciferase reporter constructs under the control of the interleukin-2 (IL-2) and the leukemia inhibitory factor (LIF) promoter, respectively. Cells were stimulated with phorbol ester, ionomycin, and different concentrations of dexamethasone, either in the absence (a vitamin B6-free medium was especially prepared for this study) or presence of vitamin B6. Both promoters were strongly induced in response to phorbol ester and ionomycin. Dexamethasone inhibited this effect in a dose-dependent manner both in the presence and absence of vitamin B6. Similar results were obtained at the protein level (IL-2- and LIF-specific ELISAs). Induction of a glucocorticoid response element (GRE)-driven promoter construct by dexamethasone in lymphoid cells was only marginally reduced by vitamin B6. In contrast, GR-mediated transactivation was strongly inhibited by vitamin B6 in HeLa cells, while GR-mediated transrepression of a matrix metalloproteinase 9 (MMP9) promoter construct was not affected. Our data indicate that vitamin B6 does not interfere with GC action in immune cells (wanted GC effects) while selectively inhibiting GR-dependent transactivation in non-immune cells (unwanted GC effects). Combination of GC treatment with supraphysiological doses of vitamin B6 may, thus, reduce the side effects of this type of immunosuppressive therapy, provided that the observed effects can be reproduced at subtoxic vitamin B6 concentrations in vivo.

[Primary prevention of cardiovascular diseases. Stepchild of internal medicine]. / Primärprävention von Herz-Kreislauf-Erkrankungen. Ein Stiefkind der Inneren Medizin.

Preventive medicine has not been adequately established in our health care system. Despite growing in-sight into the causes underlying arteriosclerotic cardiovascular disease, half of the population dies and even more suffer from it. Generally the correction of risk factors is regarded as causal therapy. Modification of the lipid and carbohydrate metabolism or the blood pressure are certainly effective, however, intervention trials have also demonstrated the limitations. Mostly an unhealthy lifestyle underlies these risk factors so that correction of the lifestyle is the causal therapy in the true sense. That is the principle basis for primary prevention, while pharmacotherapy can only be an adjunct. Inadequate nutrition, physical inactivity and smoking are considered the true major risk factors in our society. Changes in nutrition in favor of plant products, some physical activity and refraining from smoking can serve an effective contribution to health. In the future not only medical, but also economic requirements will increasingly force us to establish prevention on the basis of lifestyle changes as a mainstay of medicine.

[Treatment of sudden hearing loss through Fibrinogen/LDL-apheresis. A prospective, randomized multicenter trial]. / Behandlung des Hörsturzes durch Fibrinogen-LDL-Apherese. Eine prospektive randomisierte Multizenterstudie.

INTRODUCTION: Sudden sensorineural hearing loss (SSHL) is thought to be of various origins. Disturbances of microcirculation, autoimmune pathology and viral infection are among the most likely causes. Acute reduction of plasma fibrinogen and serum LDL positively influences hemorheology and endothelial function and might thus be an effective therapy for SSHL. OBJECTIVE: To test the hypothesis that fibrinogen/LDL-apheresis is as effective or superior to conventional therapy with plasma expanders and prednisolone in the treatment of SSHL. DESIGN: controlled, prospective, randomized, multicenter trial. SETTING AND PATIENTS: 201 patients were recruited from 01/2000 to 6/2001 at the University Clinics of Munich, Berlin, Hamburg and Bochum. Inclusion criteria was sudden sensorineural hearing loss of unknown origin within 6 days of onset. INTERVENTIONS: Single fibrinogen/ LDL-apheresis infusion of prednisolone (250 mg, tapered by 25 mg daily), hydroxyethyl starch (500 ml, 6%) and pentoxifylin (400 mg/day). MAIN OUTCOMES: Improvement of pure tone thresholds 48 h after onset of therapy. RESULTS: Over all improvement of pure tone thresholds in the fibrinogen/ LDL-apheresis treated patients is slightly but not significantly better than in the standard therapy group. After 48 h, 50% speech perception in the fibrinogen/ LDL-apheresis group (21.6+/-20.1 dB) is significantly (p<0.034) better than in the standard group (29.3+/-29.4 dB). Patients with plasma fibrinogen levels of more than 295 mg/dl have a substantial and significantly (p<0.005) better improvement of speech perception (15.3+/-17.3 dB) than standard treated patients (6.1+/-10.4 dB). CONCLUSIONS: Fibrinogen/LDLapheresis is at least equally effective compared to prednisolone treatment in sudden hearing loss. Selected patients with plasma fibrinogen of more than 295 mg/dl improve significantly better when treated with fibrinogen/LDLapheresis.

The single nucleotide polymorphism -1131T>C in the apolipoprotein A5 (APOA5) gene is associated with elevated triglycerides in patients with hyperlipidemia.

The -1131T>C polymorphism in the newly identified apolipoprotein A5 (APOA5) gene has been associated with elevated plasma triglycerides. We determined its incidence in 915 patients attending a lipid outpatient clinic. The frequency of the C allele was significantly higher in patients with triglycerides above the 90th percentile and patients with type III hyperlipidemia compared to those with hypercholesterolemia. The C allele was associated with increased plasma triglycerides and decreased plasma HDL cholesterol, conditions associated with an increased risk of coronary heart disease. The effects on plasma lipids were only observed in overweight (BMI>25) patients and were greater in patients who were also carriers of a least one epsilon4 allele in the APOE gene. Thus additional genetic and/or metabolic factors are required in order for the triglyceride raising and HDL lowering effect of the -1131T>C polymorphism in APOA5 to be expressed.

Genomic structure and transcriptional regulation of the human growth hormone secretagogue receptor.

Synthetic GH secretagogues stimulate GH release through binding to a recently cloned specific GH secretagogue receptor (GHS-R). The endogenous ligand of this receptor may be part of a new endocrine pathway controlling GH secretion. Two different receptor variants, type 1a and 1b, have been described that differ in their 3'-terminal amino acids. We investigated the genomic structure and transcriptional regulation of the human GHS-R. An 18-kb genomic clone including sequences encoding for the two GHS-R variants was isolated. Sequencing revealed that the two variants originate from specific RNA processing of a single gene that spans approximately 4.1 kb. The transcription start site was defined by 5'-inverse PCR analysis at position -227. RT-PCR analysis points to differential transcriptional initiation and processing. Type 1a is encoded by two exons; 2152 bp of intronic sequence are removed by splicing at position 796/797 relative to the translation start site. Type 1b is encoded by a single exon. A putative polyadenylation signal consensus motif was identified at position +4118; 2.7 kb of the 5'-flanking region were sequenced, and putative transcription factor binding sites were identified. Transcriptional regulation was investigated by transient transfections using promoter fragments ranging in size from 168-1745 bp; 1745 bp of the GHS-R promoter directed significant levels of luciferase expression in GH(4) rat pituitary cells, whereas no activity was detected in monkey kidney COS-7 cells, human endometrium Skut-1B cells, mouse hypothalamic LHRH neuronal GT1-7 cells, or mouse corticotroph pituitary AtT20 cells. A minimal 309-bp promoter allowed pituitary-specific expression. Its activity in COS-7 cells was enhanced by cotransfection of the pituitary-specific transcription factor Pit-1. We did not find any regulation of the GHS-R promoter by forskolin, somatostatin, insulin-like growth factor I, or 12-O-tetraphorbol 12-myristate 13-acetate. Thyroid hormone and estrogen lead to a significant stimulation; glucocorticoids lead to a significant inhibition. Further mapping suggests a thyroid hormone-responsive element, an estrogen-responsive element, and a glucocorticoid-responsive element located between -309 and the translation start codon. These studies demonstrate the nature of the human GHS-R gene and identify its 5'-flanking region. Furthermore, pituitary-specific activity of the promoter and regulation by various hormones are demonstrated.

Expression of leukemia inhibitory factor (LIF) and LIF receptor (LIF-R) in the human adrenal cortex: implications for steroidogenesis.

It is well established that steroidogenesis in the adrenal cortex is regulated by extraadrenal factors, such as ACTH and angiotensin II. However, over the last years, it has become increasingly clear that paracrine and autocrine mechanisms are also important for steroid synthesis in the adrenal gland. The current study was designed to analyze whether the pleiotropic cytokine leukemia inhibitory factor (LIF) and/or its receptor (LIF-R) are expressed in the normal human adrenal cortex, and whether they may play a role in regulating steroidogenesis. Using LIF- and LIF-R-specific primers, we show by RT-PCR that both mRNAs are expressed in this tissue, as well as in the NCI-H295 adrenal carcinoma cell line. The correct sequences of the PCR products were verified by restriction enzyme analysis and DNA sequencing. Immunohistochemistry, employing specific antibodies against LIF and LIF-R, reveals expression of both proteins in the normal human adrenal cortex. Finally, we show that LIF can significantly enhance basal and ACTH-induced production of cortisol and aldosterone in NCI-H295 cells. In summary, we show for the first time that LIF and its receptor are expressed in the normal human adrenal cortex. Our stimulation experiments indicate that the intraadrenal LIF/LIF-R system may participate in regulating adrenal steroidogenesis.

Absence of somatostatin receptor type 2 A mutations and gip oncogene in pituitary somatotroph adenomas.

OBJECTIVE: Somatostatin, acting via specific receptors in the anterior pituitary, tonically inhibits pituitary growth hormone secretion and somatotroph proliferation. Reduction of growth hormone secretion and tumour regression in GH-secreting pituitary adenomas treated with long-acting somatostatin analogues varies widely. In 30-40% of these tumours dominant somatic mutations of the Gsalpha gene (gsp) have been demonstrated leading to constitutive adenylyl cyclase induction. A relationship between somatostatin sensitivity and tumour pathogenesis in some tumours has been suggested. Changes in the function of the somatostatin receptor or intracellular signal elements may be of relevance. Somatostatin receptor type 2 A (sst2A) and Gi2 are proposed to mediate selectively the inhibition of GH release in the somatotroph. We therefore investigated the presence of sst2A mutations and gip oncogene in somatotrophic pituitary adenomas. DESIGN: Tumour samples from 15 patients with pituitary somatotroph adenomas were obtained. RNA was isolated and used for reverse transcription and subsequent polymerase chain reaction. All samples were screened for the presence of sst2A mutations and of the gip oncogene by SSCP analysis and sequencing. For comparison, the gsp oncogene was examined. The relationship between clinical data and molecular analysis results was investigated. RESULTS: Seven of the tumours harboured a gsp mutation. No mutations affecting the sst2A protein were found in any of the tumours analysed. Furthermore, gip oncogene was absent in all tumours. CONCLUSION: Mutations of the somatostatin receptor type 2 A and the gip oncogene are unlikely to be involved in the pathogenesis of acromegaly.