Adiponectin-leptin ratio: a useful estimate of insulin resistance in patients with Type 2 diabetes.

AIMS: Adiponectin and leptin are adipocytokines associated with insulin resistance. The objective of this study was to evaluate the performance of the adiponectin-leptin ratio as a measure of insulin resistance in comparison with other surrogate measures of insulin resistance based on fasting insulin and glucose levels [homeostasis model assessment (HOMA), quantitative insulin sensitivity check index (QUICKI), fasting glucose/insulin ratio] and with measures based on fasting insulin and triglyceride levels (McAuley index) in Caucasian patients with Type 2 diabetes (T2D). METHODS: In 70 patients included in DEMAND (delapril and manidipine for nephroprotection in diabetes) study, fasting samples of plasma insulin and adiponectin were determined by a radioimmunoassay, whereas plasma leptin was determined by an enzyme-linked immunosorbent assay. Insulin resistance estimates were derived by the established equations and compared with the direct measurement of insulin resistance obtained with the euglycemic hyperinsulinemic clamp. Insulin resistance estimates and the clamp derived sensitivity index were compared by correlation analysis. RESULTS: The adiponectin-leptin ratio correlated best with the clamp derived sensitivity index (r=0.553, p<0.001) compared to other surrogate measures of insulin resistance. In multiple linear regression models including different surrogate measures of insulin resistance as independent predictors of the sensitivity index, the model with the adiponectin-leptin ratio accounted for the highest variability of the sensitivity index (r2=0.336, p<0.001). CONCLUSIONS: The adiponectin-leptin ratio is associated with insulin resistance, measured with the euglycemic hyperinsulinemic clamp, in Caucasians with T2D. The association with clamp derived sensitivity index is even stronger than that of HOMA, QUICKI, fasting glucose/insulin ratio or McAuley index and is independent of body mass index or glycemic control. The adiponectin-leptin ratio promises to become a new laboratory marker of insulin resistance in T2D.

Sequence variations in the osteoprotegerin gene promoter in patients with postmenopausal osteoporosis.

Osteoprotegerin (OPG) is a recently discovered member of the TNF receptor superfamily that acts as an important paracrine regulator of bone remodeling. OPG knockout mice develop severe osteoporosis, whereas administration of OPG can prevent ovariectomy-induced bone loss. These findings implicate a role for OPG in the development of osteoporosis. In the present study, we screened the OPG gene promoter for sequence variations and examined their association with bone mineral density (BMD) in 103 osteoporotic postmenopausal women. Single-strand conformation polymorphism analysis followed by DNA sequencing revealed a presence of four nucleotide substitutions: 209 G-->A, 245 T-->G, 889 C-->T, and 950 T-->C. The frequencies of genotypes were as follows: GG (89.3%), GA (10.7%) for 209 G-->A polymorphism; TT (89.3%), TG (10.7%) for 245 T-->G polymorphism; and TT (25.2%), TC (53.4%), CC (21.4%) for 950 T-->C polymorphism. Substitution 889 C-->T was found in only two patients. Statistically significant association of genotypes with BMD at the lumbar spine (P = 0.005) was observed for 209 G-->A and 245 T-->G polymorphisms. Haplotype GATG was associated with lower BMD as compared with GGTT haplotype. Our results suggest that 209 G-->A and 245 T-->G polymorphisms in the OPG gene promoter may contribute to the genetic regulation of BMD.

GH-IGF-I axis in non-obese women with functional hyperandrogenism.

The study was conducted to assess the GH-IGF-I axis in non-obese women with functional hyperandrogenism (FH). Eighteen FH women aged 18-35 yr with a body weight within 20% of ideal body weight and 10 weight-matched controls were included in the study. Basal serum GH, GH-binding protein (GHBP), IGF-I, IGF-binding protein-3 (IGFBP-3) levels were determined as well as GH levels during GHRH stimulation. In addition, basal serum androgens [free T (FT), delta4 and DHEAS], insulin and glucose levels were determined. The group of non-obese patients with FH differed from controls in GHBP (1.21+/-0.37 vs 0.93+/-0.25 nmol/l; p<0.05) and androgen levels (FT: 8.0+/-3.2 vs 1.9+/-1.2 pmol/l, p<0.001; delta4: 10.5+/-3.2 vs 5.9+/-2.1 nmol/l, p<0.001; DHEAS: 9.3+/-3.0 vs 5.1+/-1.8 micromol/l, p<0.001). GH (r=0.365; p<0.05) and IGF-I (r=0.508, p<0.01) serum levels were significantly correlated to serum DHEAS levels in a combined group of patients and controls. Our results support the suggestion that the GH-IGF-I axis plays an important role in the evolution of hormonal and metabolic derangement in non-obese FH women.

New steroid 5alpha-reductase type I (SRD5A1) homologous sequences on human chromosomes 6 and 8.

To date, two genes encoding 5alpha-reductase isoenzymes are known (type I, type II), and one type I pseudogene. The divergent localization of these genes and the still not fully understood function of the encoded enzymes as well as the perplexing results we obtained after sequencing PCR-amplified SRD5A1 gene fragments (out of genomic DNA), made us assume that, in addition to the known SRD5A1 gene, one or more different human 5alpha-reductase type I coding genes may exist. Our research provide the first evidence for the existence of two new SRD5A1 related, previously unidentified sequences in the human genome. These sequences which were localized to chromosomes 6 and 8 are highly homologous (> 99%) to SRD5A1, and also do not contain any deletions or insertions that are otherwise a characteristic of the SRD5API pseudogene. Our results imply that these sequences may be either coding parts of yet unknown, active SRD5A1 genes, and/or of previously unidentified pseudogenes. These findings additionally support data of Chen et al. who confirmed the existence of various SRD5A1 proteins in cultured human skin cells.

Codon 325 sequence polymorphism of the estrogen receptor alpha gene and bone mineral density in postmenopausal women.

Estrogen receptor alpha (ER alpha) encoding gene is one of the candidate genes to be involved in the development of osteoporosis. Until now correlation between three ER gene polymorphisms (identified with PvuII, XbaI and BstUI) and bone mineral density (BMD) have been investigated. The results of these studies are contradictory. Thus the aim of our work was to search for new, yet unknown, and probably more informative polymorphism(s) of the ER alpha gene. For detection of mutations the whole coding region of the ER alpha gene was screened systematically. In a group of 85 late postmenopausal women all of the eight exons were amplified by polymerase chain reaction (PCR) and fragments were further analyzed by single-stranded conformation polymorphism (SSCP) analysis. Mutations were confirmed by direct DNA sequencing. In the whole coding region of the ER alpha gene two silent mutations in codon 87 and 325, respectively, were found. The silent mutation in codon 85 of exon 1 (GCG-->GCC; A87A) was described previously, as BstUI polymorphism. On the other side, the silent mutation in codon 325 (CCC-->CCG; P325P), located in exon 4, has not been analyzed so far in correlation with BMD. According to the distribution of genotypes CC:CG:GG=49.4:41.2:9.4, we can affirm the existence of genetic polymorphism in codon 325 in our population of late postmenopausal women. The mean femoral neck BMD, but not the lumbar spine BMD, was significantly lower (P=0.029) in the homozygous GG-women with CCG/CCG codon 325 as compared to the homozygous CC-women with the normal codon CCC/CCC. Our results suggest that codon 325 sequence polymorphism of the ER alpha gene might be one of the factors associated with low femoral neck BMD.

Effect of vitamin D and calcium on bone mineral density in children with CP and epilepsy in full-time care.

Atraumatic fractures are often seen in children and adolescents with cerebral palsy (CP) and epilepsy in full-time care. Increased bone fragility was postulated to be due to osteopenia resulting from a combination of factors including immobilization and antiepileptic treatment. The aim of this study was to determine the effect of vitamin D and calcium substitution on bone mineral density (BMD) in a group of children with CP in full-time care. Twenty children with the most severe form of CP (spastic quadriplegia) who had been treated with antiepileptic drugs for a relatively long period of time were included in the study. Physical examination and laboratory analyses excluded other possible causes of osteopenia. BMD was measured by dual X-ray absorptiometry. Thirteen patients were treated for 9 months with 1,25-dihydroxy-cholecalciferol vitamin D (0.25 mcg daily) and with calcium (500 mg daily). Seven control children were used for observation only. BMD greatly increased in the treated group, while children with CP in full-time care who did not receive vitamin D and calcium substitution continued to lose their bone mass. It can be concluded that the addition of vitamin D and calcium increases BMD in children with the most severe form of CP, who are receiving antiepileptic drugs.

Association of vitamin D receptor gene polymorphism with bone mineral density in Slovenian postmenopausal women.

Osteoporosis is a common bone disease which affects one in three women after the 60th year of life and is a major cause of morbidity in older people. To identify patients with osteoporosis, measurement of bone mineral density (BMD) is recommended. The association of BMD with vitamin D receptor (VDR) genotype in Slovenian postmenopausal women was studied. We determined VDR genotype in 102 late postmenopausal women aged 47-77 years. BMD measurements were performed at the level of the lumbar spine (L2-L4) by dual X-ray absorptiometry. Our data show significantly lower BMD in BB women compared to those with bb genotype. The relative distribution of VDR genotypes and alleles in the Slovenian population was 18.6:57.8:23.6% for BB:Bb:bb, respectively. The results are consistent with those of a previous study which found an excellent correlation between BB VDR genotype and low BMD. The data were derived from a relatively small, but ethnically homogeneous population of the same socioeconomic status, with very similar dietary and physical activity habits. Dietary habits in particular seem to be important because of the relatively low calcium intake which may enhance the phenotypic expression of VDR gene polymorphisms.

Adrenal 21-hydroxylase gene mutations in Slovenian hyperandrogenic women: evaluation of corticotrophin stimulation and HLA polymorphisms in screening for carrier status.

OBJECTIVE: To study the incidence of 21-hydroxylase deficiency in Slovenian hyperandrogenic women, at the gene level. Previous endocrine studies indicated large differences in the incidence of 21-hydroxylase deficiency in hyperandrogenic women. The predictive values of the 17-hydroxyprogesterone (17-OHP) response to ACTH stimulation and of HLA typing in screening for carrier status were re-evaluated. DESIGN: Molecular analysis of CYP21 gene, ACTH stimulation and human leucocyte antigen (HLA) typing were performed in 83 consecutive Slovenian hyperandrogenic women. MEASUREMENTS: Cortisol and 17-OHP concentrations were measured at baseline and 60 min after ACTH stimulation. Basal adrenal androgen concentrations were also measured. RESULTS: None of 83 hyperandrogenic patients was affected with non-classical 21-hydroxylase deficiency, but 12 of 81 patients (14.8%) had high concentrations of 17-OHP after stimulation, indicative of carrier status. The increase in 17-OHP concentrations could be explained by a carrier status for CYP21 gene mutations in only three of 12 patients (25%), whereas seven of 69 patients (10. 1%) with normal concentrations of 17-OHP after stimulation were found to be carriers of CYP21 gene mutations, indicating low positive predictive values of ACTH stimulation as a screening test for carriers of 21-hydroxylase deficiency. In total, 11 carriers were identified among 83 patients: seven CYP21 gene deletions/conversions, two Gln(318)Stop and one Val(281)Leu mutation and one gene conversion extending from exon 4 to exon 7 were found. The association between Val(281)Leu mutation and HLA-B14 antigen was confirmed in this Slovenian population. CONCLUSIONS: Basal or ACTH-stimulated 17-OHP concentrations are not a good indicator of the carrier status for 21-hydroxylase deficiency among Slovenian hyperandrogenic patients. Reliable screening for carriers of 21-hydroxylase deficiency is possible only by molecular analysis of the CYP21 gene.

Growth hormone (GH) treatment reverses early atherosclerotic changes in GH-deficient adults.

Hypopituitary patients have increased mortality from vascular disease, and in these patients, early markers of atherosclerosis [increased carotid artery intima-media thickness (IMT) and reduced distensibility] are more prevalent. As GH replacement can reverse some risk factors of atherosclerosis, the present study examined the effect of GH treatment on morphological and functional changes in the carotid and brachial arteries of GH-deficient (GHD) adults. Eleven GHD hypopituitary men (24-49 yr old) were treated with recombinant human GH (0.018 U/kg BW x day) for 18 months. IMT of the common carotid artery (CCA) and the carotid bifurcation (CB), and flow-mediated endothelium-dependent dilation (EDD) of the brachial artery were measured by B mode ultrasound before and at 3, 6, 12, and 18 months of treatment, and values were compared with those in 12 age-matched control men. Serum concentrations of lipids, lipoprotein(a), insulin-like growth factor I (IGF-I), and IGF-binding protein-3 (IGFBP-3) were also measured. In GHD men before treatment the IMTs of the CCA [mean(SD), 0.67(0.05) mm] and CB [0.75(0.04) mm] were significantly greater (P < 0.001) than those in control men [0.52(0.07) and 0.65(0.07) mm, respectively]. GH treatment normalized the IMT of the CCA by 6 months [0.53(0.04) mm] and that of the CB by 3 months [0.68(0.05) mm]. The IMT of the carotid artery (CCA and CB) was negatively correlated with serum IGF-I (r = -0.53; P < 0.0001). There was a significant improvement in flow-mediated EDD of the brachial artery at 3 months, which was sustained at 6 and 18 months of GH treatment (P < 0.05). GH treatment increased high density lipoprotein cholesterol at 3 and 6 months, but did not reduce total or low density lipoprotein cholesterol and was without effect on lipoprotein(a). There was no correlation between plasma lipids and changes in IMT or EDD of the arteries examined. In conclusion, GH treatment of hypopituitary GHD men reverses early morphological and functional atherosclerotic changes in major arteries and, if maintained, may reduce vascular morbidity and mortality. GH seems to act via IGF-I, which is known to have important effects on endothelial cell function.

VDR genotype and response to etidronate therapy in late postmenopausal women.

Twenty-four late postmenopausal women with osteoporosis were studied. The patients were separated in three subgroups according to the BsmI polymorphism of the vitamin D receptor (VDR) gene: BB (n = 8), Bb (n = 10) and bb (n = 6). They did not differ in age (mean ages were 66.0 years, 65.9 years and 63.9 years, respectively), years after menopause (18.7 years, 18.1 years and 18.4 years) or body weight (64.9 kg, 65.3 kg and 63.8 kg), the variables known to be associated with bone mineral density (BMD). The results show that the response to antiresorptive bisphosphonate therapy in combination with calcium supplementation is modified by VDR genotype. The lumbar spine BMD increased significantly faster in the BB and Bb groups (7.3% and 7.0%, respectively) compared with the bb group (2.5%) during 1 year of cyclic etidronate therapy (400 mg/day) and calcium supplementation (1000 mg/day). The biochemical marker of bone resorption (urinary hydroxyproline excretion) as well as the bone formation marker (serum levels of osteocalcin) decreased during the treatment. With respect to VDR genotype, a significantly higher decrease in osteocalcin level was observed in bb as compared with BB subjects. We conclude that the VDR genotype is involved in an individual's response to cyclic etidronate therapy with calcium supplementation.

CYP17 gene analysis in hyperandrogenised women with and without exaggerated 17-hydroxyprogesterone response to ovarian stimulation.

Nine patients with polycystic ovary syndrome (PCOS) and exaggerated serum level of 17-hydroxyprogesterone (17-OHP) response to gonadotropin (GnRH) agonist stimulation, and seven patients with PCOS, but normal 17-OHP response have been analysed for possible linkage of PCOS with genetic defects on the 17 alpha-hydroxylase/17,20-lyase gene (CYP17). A portion of the regulatory and the entire coding domain for this enzyme have been analysed by PCR-SSCP analysis. Samples have been also screened for the previously reported -34 bp polymorphism, which creates a new SP1-type promoter site, but was excluded as the primary genetic defect. We have varied gel concentrations, reduced running temperatures, added glycerol to polyacrylamide gels and performed electrophoresis on longer gels in order to improve the resolving power of SSCP. Screening of the CYP17 gene revealed no mutations associated with the disease in the examined group of patients. Also, the -34 bp polymorphism proved to be equally distributed among patient and control samples, which in our case were non-related. The results indicate that, when germline mutations in question, CYP17 may be excluded as a candidate gene for these subtypes of PCOS.

Insulin-like growth factor binding protein-3 (IGFBP-3) serum concentrations and ovarian responsiveness in in-vitro fertilization.

The growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis seems to play an important role in ovarian responsiveness. Recently IGF binding protein-3 (IGFBP-3) serum concentrations have been reported to be a good marker of GH/IGF-I axis activity. In view of this finding, we measured IGFBP-3 serum concentrations in 29 women undergoing in-vitro fertilization. We found a significant correlation among IGFBP-3 serum concentrations and markers of ovarian stimulation including efficacy index, serum oestradiol concentrations and the number of follicles on the day of human chorionic gonadotrophin (HCG) administration. The results of our study add additional evidence to the importance of the GH/IGF-I system in regulating ovarian responsiveness to gonadotrophin stimulation.

Androstanediol glucuronide in patients with pancreatic cancer and in those with chronic pancreatitis.

The differential diagnosis between pancreatic cancer and chronic pancreatitis is extremely difficult. Beside CA19-9 level determinations, many tests have been tried with the aim to facilitate this distinction. Serum androgen levels have been used for this purpose. To further explore the value of androgen markers in differentiating pancreatic cancer from chronic pancreatitis we determined the serum levels of androstanediol glucuronide and of androgens in the two groups of patients and compared them with CA19-9 levels. A total of 25 males were entered into the study. Of these, 13 patients had pancreatic cancer and 12 chronic pancreatitis. They were comparable as to their body weight and age. Patients with pancreatic cancer had significantly lower serum testosterone, dihydrotestosterone and androstanediol glucuronide levels, but not testosterone/dihydrotestosterone ratios when compared to patients with chronic pancreatitis. Only androstanediol glucuronide and dihydrotestosterone serum concentrations had such a small overlap between the two groups that could be used for differentiation, their sensitivity and specificity being comparable to those of CA19-9 levels. The present study has shown for the first time that serum androstanediol glucuronide levels in male patients with pancreatic cancer are significantly lower than in those patients with chronic pancreatitis. Furthermore, the sensitivity and specificity of serum andorstanediol glucuronide levels which can be used to differentiate between pancreatic cancer and chronic pancreatitis are comparable to those of CA19-9.

Antiandrogen treatment with spironolactone and linestrenol decreases bone mineral density in eumenorrhoeic women with androgen excess.

Increased bone mineral density (BMD) has been reported in young women with androgen excess. To determine whether antiandrogen treatment in young women with androgen excess reduces BMD in these patients, the authors measured BMD before and a year after the beginning of antiandrogen therapy with spironolactone and linestrenol in 17 consecutive androgenized patients (median age 22 years). After a year's treatment BMD declined in 15 out of 17 patients, the mean decrease--0.032 g/cm2 (95% CI of the difference 0.016-0.048)--being highly significant (p < 0.001). Androstenedione decrease was the only hormonal variable significantly correlating with BMD decrease (r = 0.5; p = 0.037) according to simple linear regression. A decrease of BMD might become a key factor in deciding about the duration of antiandrogen treatment with spironolactone in functional hyperandrogenemia.

Bone mineral density in hyperandrogenic amenorrhoea.

The authors assessed bone mineral density in androgenized amenorrheic (group A; n = 9) and androgenized nonamenorrheic patients (group B; n = 30) and compared it with controls (n = 22). Bone mineral density of group A patients (1.023 +/- 0.045 g/cm2) did not differ from controls (1.047 +/- 0.83 g/cm2); both groups had significantly lower values than group B women (1.099 +/- 0.085 g/cm2). Of the hormonal variables explaining bone mineral density in androgenized women, only dehydroepiandrosterone sulfate had a significant negative correlation (r = -0.45). In contrast to other forms of amenorrhea, women with hyperandrogenic amenorrhea seem to be spared from osteopenia.

Parlodel LAR in the treatment of macroprolactinomas.

Eight patients with macroprolactinomas were treated with a long-acting injectable form of bromocriptine, depot-bromocriptine (Parlodel LAR). With the exception of one male patient who had partial and short-lasting suppression of PRL levels after two injections and who underwent a second adenomectomy, the patients were given Parlodel LAR injections at 28-day intervals for six months. In all patients, there was a significant fall in serum PRL levels after the first injection. PRL secretion was suppressed to within the normal range in 3 of 7 patients on long-term treatment. PRL was consistently within the normal range in 2 patients from the sixth week and in one, from the 14th week onwards. In the other 4 of 7 patients, a marked suppression of PRL secretion, resumption of menses, and normal libido and potency were recorded. In 3 of 8 patients, no adverse effects were noted. Two patients reported short-lasting nausea, one vomiting, one constipation and in 2 patients, orthostatic dizziness occurred after the first injection. Subsequent injections, however, were well tolerated systematically and locally. Five patients had CT scan evidence of tumour shrinkage. A very large tumour virtually disappeared after the first injection of 50 mg depot-bromocriptine in one patient. The decrease of serum PRL secretion within the first 12 hours after injection did not predict normalization of serum PRL levels during long-term treatment, whereas the fall of serum PRL levels to below 5% of the basal values within the first months of treatment could be a good indicator for the final outcome.

Dexamethasone and spironolactone in the treatment of non-tumorous hyperandrogenism.

To test the hypothesis that a combination therapy with dexamethasone and spironolactone in hirsute women with menstrual disorders due to non-tumorous hyperandrogenism might yield better results than monotherapy with spironolactone, we evaluated 25 women randomly assigned to dexamethasone-spironolactone (n = 15) and spironolactone (n = 10) groups. The Ferriman-Gallwey score and hormonal levels (LH, FSH, PRL, serum testosterone, androstenedione, dehydroepiandrosterone sulfate, estradiol, estrone and salivary testosterone) were determined before and after 6 months of therapy. There were comparable results, with a significant drop in the Ferriman-Gallwey score, in serum androstenedione and estrone concentrations and in salivary testosterone levels in both groups. The only difference between the two groups after therapy was a significant fall in serum dehydroepiandrosterone levels in patients treated with the combination therapy. The results indicate that the combination therapy with spironolactone and dexamethasone presents no real advantage over therapy with spironolactone alone, for the initial treatment of non-tumorous hyperandrogenism.

Salivary free testosterone in hirsutism.

A sensitive, specific and accurate direct radioimmunoassay of testosterone in human saliva is described. A single salivary testosterone result is shown to be of greater diagnostic use in hirsutism than any of the currently used serum androgen assays. Thus, of 50 hirsute patients, salivary testosterone (Sa-T) was elevated in 34 patients, sex hormone-binding globulin (SHBG) was decreased in 30 women, serum testosterone (S-T) elevated in 13, dehydroepiandrosterone sulphate (DHEA-S) was elevated in 14, and androstenedione in three of the investigated group.