Stanniocalcin-2 overexpression reduces atherosclerosis in hypercholesterolemic mice.

BACKGROUND AND AIM: The metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) has been suggested as a proatherogenic molecule by its ability to locally increase insulin-like growth factor signaling. Stanniocalcin-2 (STC2) was recently discovered to be a potent inhibitor of PAPP-A activity, but has not previously been implicated in vascular disease. The aim of this study was to substantiate the interaction between PAPP-A and STC2 as a potential local regulatory mechanism in the artery wall. METHODS AND RESULTS: We found that PAPP-A is secreted from cultured primary smooth muscle cells obtained from human aortas as a covalent complex with STC2, devoid of proteolytic activity. Extracts of human carotid atherosclerotic plaques contain both complexed and uncomplexed PAPP-A, and we show by immunohistochemistry that PAPP-A and STC2 are present in the tissue throughout early human lesion development. We then used adeno-associated virus-mediated expression of STC2 to increase the fraction of PAPP-A present in the inhibited state and found that it decreased the development of atherosclerosis by 47% (P = 0.0005) in apolipoprotein E-deficient mice challenged with a Western type diet compared to controls. CONCLUSIONS: This study is the first to suggest the involvement of STC2 in regulating PAPP-A activity during the development of atherosclerosis. Furthermore, we demonstrate that lesion development can be inhibited in an experimental model by driving the balance towards inhibited PAPP-A.

Bone morphogenetic proteins regulate osteoprotegerin and its ligands in human vascular smooth muscle cells.

The bone-related protein osteoprotegerin (OPG) may be involved in the development of vascular calcifications, especially in diabetes, where it has been found in increased amounts in the arterial wall. Experimental studies suggest that members of the TGF-superfamily are involved in the transformation of human vascular smooth muscle cells (HVSMC) to osteoblast-like cells. In this study, we evaluated the effect of BMP-2, BMP-7 and transforming growth factor beta (TGF-beta1) on the secretion and mRNA expression of OPG and its ligands receptor activator of nuclear factor-kappabeta ligand (RANKL) and TNF-related apoptosis-inducing ligand (TRAIL) in HVSMC. All three growth factors decreased OPG protein production significantly; these results were paralleled by reduced OPG mRNA expression. TRAIL mRNA levels were also decreased. RANKL mRNA expression declined when treated with TGF-beta1 but were increased by both BMPs. Members of the TGF-superfamily, i.e. TGF-beta1, BMP-2 and BMP-7 exert effects on OPG and its ligands, indicating that these peptides may be involved in the development of vascular calcifications. The downregulation of OPG by these peptides does, however, not suggest that these factors are directly involved in OPG accumulation in diabetes.

Diminished NO release in chronic hypoxic human endothelial cells.

The present study addressed whether chronic hypoxia is associated with reduced nitric oxide (NO) release due to decreased activation of endothelial NO synthase (eNOS). Primary cultures of endothelial cells from human umbilical veins (HUVECs) were used and exposed to different oxygen levels for 24 h, after which NO release, intracellular calcium, and eNOS activity and phosphorylation were measured after 24 h. Direct measurements using a NO microsensor showed that in contrast to 1-h exposure to 5% and 1% oxygen (acute hypoxia), histamine-evoked (10 microM) NO release from endothelial cells exposed to 5% and 1% oxygen for 24 h (chronic hypoxia) was reduced by, respectively, 58% and 40%. Furthermore, chronic hypoxia also lowered the amount and activity of eNOS enzyme. The decrease in activity could be accounted for by reduced intracellular calcium and altered eNOS phosphorylation. eNOS Ser(1177) and eNOS Thr(495) phosphorylations were reduced and increased, respectively, consistent with lowered enzyme activity. Akt kinase, which can phosphorylate eNOS Ser(1177), was also decreased by hypoxia, regarding both total protein content and the phosphorylated (active) form. Moreover, the protein content of beta- actin, which is known to influence the activity of eNOS, was almost halved by hypoxia, further supporting the fall in eNOS activity. In conclusion, chronic hypoxia in HUVECs reduces histamine-induced NO release as well as eNOS expression and activity. The decreased activity is most likely due to changed eNOS phosphorylation, which is supported by decreases in Akt expression and phosphorylation. By reducing NO, chronic hypoxia may accentuate endothelial dysfunction in cardiovascular disease.

The effect of activated protein C on plasma cytokine levels in a porcine model of acute endotoxemia.

OBJECTIVE: To assess the anti-inflammatory effects of recombinant human activated protein C (rhAPC) in a porcine model of acute endotoxemia. DESIGN AND SETTING: Animal randomized controlled study at the Laboratory of Clinical Institute, Aarhus University Hospital. SUBJECTS: Eighteen female landrace pigs (30 kg). INTERVENTIONS: By pairwise randomization, pigs were given either LPS or LPS and rhAPC. Both groups received a stepwise increasing LPS infusion for 30[Symbol: see text]min; whereafter the infusion continued at a lower rate (300 min LPS in both groups). The LPS+rhAPC group received rhAPC (100 microg/kg per hour) 15 min before the LPS infusion began and throughout the trial period. RESULTS: While rhAPC showed no modifying effects on peak plasma levels of pro- or anti-inflammatory cytokines (TNF-alpha, IL-6, IL-8, IL-10), TNF-alpha and IL-10 peaked significantly later in the rhAPC-treated animals. The profibrinolytic effects of rhAPC were confirmed by decreased plasminogen activator inhibitor 1 levels, while no differences were found in other coagulation markers, hemodynamic, metabolic, or leukocyte data between the two groups. CONCLUSIONS: We found no significant effect of rhAPC on plasma levels of either pro- or anti-inflammatory cytokines in this porcine model of acute endotoxemia. However, TNF-alpha and IL-10 peaked significantly later in the rhAPC-treated animals.

Calcification of human vascular smooth muscle cells: associations with osteoprotegerin expression and acceleration by high-dose insulin.

Arterial medial calcifications occur often in diabetic individuals as part of the diabetic macroangiopathy. The pathogenesis is unknown, but the presence of calcifications predicts risk of cardiovascular events. We examined the effects of insulin on calcifying smooth muscle cells in vitro and measured the expression of the bone-related molecule osteoprotegerin (OPG). Human vascular smooth muscle cells (VSMCs) were grown from aorta from kidney donors. Induction of calcification was performed with beta-glycerophosphate. The influence of insulin (200 microU/ml or 1,000 microU/ml) on calcification was judged by measuring calcium content in the cell layer and by von Kossa staining. OPG was measured in the medium by ELISA. Histochemistry was used for determination of alkaline phosphatase (ALP). Bone sialoprotein (BSP) and OPG mRNA expressions were done by RT-PCR. beta-Glycerophosphate was able to induce calcification in human smooth muscle cells from a series of donors after variable time in culture. Decreased OPG amounts were observed from the cells during the accelerated calcification phase. High dose of insulin (1,000 microU/ml) accelerated the calcification, whereas lower concentrations (200 microU/ml) did not. Calcified cells expressed ALP and BSP activity in high levels. In conclusion, high concentration of insulin enhances in vitro-induced calcification in VSMCs. Altered OPG levels during the calcification raise the possibility that OPG may have a potent function in regulating the calcification process or it may represent a consequence of mineralization. Effects of insulin and modulations by OPG on the calcification process in arterial cells may play a role in the development of calcifications as part of the diabetic macroangiopathy.

Clinical and epidemiological description of aortic dissection in Turner's syndrome.

BACKGROUND: Women with Turner's syndrome have an increased risk of congenital cardiac malformations, ischaemic heart disease, hypertension and stroke. Aortic dissection seems to occur with increased frequency. AIM: To describe in more detail aortic dissection as encountered in Turner's syndrome, giving attention to clinical, histological and epidemiological aspects. MATERIALS AND METHODS: Based on a retrospective study, we describe the clinical, karyotypic, and epidemiological aspects of aortic dissection as encountered in cases of Turner's syndrome seen in Denmark and Sweden. RESULTS: The median age at onset of aortic dissection in 18 women was 35 years, ranging from 18 to 61 years. Fourteen of 18 women had a 45,X karyotype, while 2 patients had 45,X/45,XY, and 2 had the 45,X/46,X+r(X) complement, respectively. Echocardiography was performed in 10 of 18 patients before their acute illness, and showed signs of congenital cardiac disease, with either bifoliate aortic valves, dilation of the aortic root, or previous aortic coarctation evident in most patients. In 5 patients evidence of a bifoliate aortic valve was conclusive. Hypertension was present in 5 of 18 patients, while 10 of the patients died from aortic dissection, of so-called type A in 6, type B in 3, while in the final case the origin of dissection could not be determined. Biochemical analysis showed altered ratio between type I and type III collagen. Histology showed cystic medial necrosis in 3 of 7 cases. We estimated an incidence of dissection of 36 per 100,000 Turner's syndrome years, compared with an incidence of 6 per 100,000 in the general population, and a cumulated rate of incidence of 14, 73, 78, and 50 per 100,000 among 0-19, 20-29, 30-39, and 40+ year olds, respectively. CONCLUSION: Aortic dissection is extremely common in the setting of Turner's syndrome, and occurs early in life. Patients with Turner's syndrome should be offered a protocol for clinical follow-up similar to that provided for patients with Marfan syndrome, and each clinic should embrace a programme for follow-up.

Glycosaminoglycans increase levels of free and bioactive IGF-I in vitro.

BACKGROUND: It is unclear how IGFs become separated from their IGF-binding proteins (IGFBPs) in vivo. However, the IGFBPs possess binding sites for glycosaminoglycans (GAGs) and interaction with GAGs alters IGFBP ligand affinity. Accordingly, GAGs may control IGF bioavailability. To test this hypothesis, we investigated the effect of GAGs on serum levels of free and bioactive IGF-I, total IGF-I, and IGFBPs in vitro. METHODS: Serum was incubated with increasing concentrations of six different GAGs (heparin, tinzaparin (Innohep), dermatan sulfate, heparan sulfate, non-anticoagulant (nac) heparin, and nac low-molecular weight heparin). To investigate for reversibility, heparin was co-incubated with protamine sulfate (PS). Finally, the effect of heparin was studied in serum from pregnant and post partum women, normal subjects and patients with type 1 diabetes. RESULTS: All GAGs increased free IGF-I in a dose-dependent manner (P<0.0001), whereas total IGF-I and IGFBP levels remained unchanged. However, the potency of the GAGs differed significantly (P<0.0001) and did not relate to their anti-coagulating activity. The effect of heparin on free IGF-I was fully reversed by PS. Heparin increased free and bioactive IGF-I in all tested sera (P<0.0001), but the increase was most pronounced in samples from pregnant women (P<0.0001). CONCLUSION: All tested GAGs stimulated the release of free and bioactive IGF-I in several types of serum, most likely by reversible interaction with the IGFBPs. The effect was most pronounced in pregnancy sera, which are characterized by extensive IGFBP-3 proteolysis. Our findings support the view that GAGs localized in the vessel wall and attached to the extracellular matrix control IGF-I tissue accessibility and bioactivity.

Endotoxemia-induced lymphocyte apoptosis is augmented by a hyperinsulinemic-euglycemic clamp.

BACKGROUND: Sepsis and endotoxemia are associated with lymphocyte apoptosis. This has been regarded as harmful, contributing to further immune suppression in already immune-compromised patients. Because normalization of blood glucose improves outcome in critically ill patients, the authors hypothesized that one of the effects of insulin and normoglycemia would be inhibition of lymphocyte apoptosis. Therefore, in this experimental study in pigs, the authors examined the separate and combined effects of acute endotoxemia and a hyperinsulinemic-euglycemic clamp (HEC) on lymphocyte apoptosis. METHODS: After 60 min of stabilization, 38 anesthetized and mechanically ventilated pigs (weight, 35-40 kg) were divided (by randomization performed before the experiment) into four groups and were then studied for 570 min. Group 1 received no intervention. Group 2 received a HEC (5 mm p-glucose, insulin infusion rate of 0.6 mU . kg (-1). min(-1)) for 570 min. Group 3 received a lipopolysaccharide infusion for 180 min. Group 4 was given a combination of a HEC and a lipopolysaccharide infusion. After the 570-min study period, the pigs were killed, and tissue was sampled from the spleen and frozen. In four sections of each sample, the apoptosis of B and T lymphocytes were analyzed using stereologic methods: The number of apoptotic B and T cells was estimated by fluorescence immunohistochemistry with anti-active caspase-3 and either anti-CD21 (B lymphocytes) or anti-CD3epsilon (T lymphocytes). The number of apoptotic B and T lymphocytes was then compared using two-way analysis of variance, and the interaction between endotoxemia and the clamp (hyperinsulinemia and euglycemia) was investigated. RESULTS: Endotoxemia induced apoptosis of B (P < 0.001) and T lymphocytes (P = 0.016) in the spleen, and this effect was independent of the clamp. The ratios of apoptotic cells in the spleen tissue of pigs with and without endotoxemia were 2.4 (confidence interval, 1.7-3.4) and 1.6 (confidence interval, 1.1-2.2) for B and T lymphocytes, respectively. Independent of endotoxin infusion, HEC increased the number of apoptotic lymphocytes (P = 0.029 and P = 0.038 for B and T lymphocytes, respectively). The ratios of the number of apoptotic spleen cells in pigs treated and not treated with HEC were 1.5 (confidence interval, 1.0-2.1) and 1.5 (confidence interval, 1.0-2.1) for B and T lymphocytes, respectively. CONCLUSION: In this porcine model, both endotoxemia and a HEC increased the number of apoptotic B and T lymphocytes in the spleen. Contrary to our hypothesis, lymphocyte apoptosis during acute endotoxemia was augmented by a HEC.

Expression levels and functional aspects of the hyaluronan receptor CD44. Effects of insulin, glucose, IGF-I, or growth hormone on human arterial smooth muscle cells.

An increased amount of hyaluronan (HA) in the arterial wall is a feature of the diabetic macroangiopathy. The functional consequences of accumulated HA are mediated through binding to CD44. The regulation of this receptor by diabetic metabolic and hormonal factors is, however unknown. The objective of this study was to examine the influence of glucose, insulin, insulin-like growth factor I (IGF-I), and human growth hormone (hGH) on the formation and function of the HA receptor CD44 in cultures of human aortic smooth muscle cells (SMCs). Migration of nonproliferating SMCs were determined by estimating the area covered by cells 6 days after removal of a barrier. Cellular content of standard CD44 and its isoforms, CD44v3 and CD44v6, and HA-binding capacity were measured using a modified enzyme-linked immunosorbent assay procedure. The analysis is made either with antibodies against CD44 or with HA as a ligand. The migration assay showed that glucose, insulin, and IGF-I were able to stimulate SMC migration (2 P < .01). Anti-CD44 antibody inhibited the stimulated migration at most concentrations. Insulin increased HA binding at 100 to 1000 micro U/mL insulin (2 P < .03). CD44 expression was only elevated at 1000 micro U/mL insulin (2 P < .03), whereas CD44 content decreased at 2 ng/mL hGH and increased at 16 ng/mL hGH (2 P < .01). Glucose and IGF-I reduced the amount of the variant isoform CD44v3 (2 P < .01) but did not change the amount of total CD44. CD44v6 was not present on human arterial SMCs. In conclusion, the present data obtained with human arterial SMCs in vitro support a role of CD44 and its isoform, CD44v3, in the SMC response to the metabolic and hormonal disorders of diabetes.

Overexpression of hyaluronan in the tunica media promotes the development of atherosclerosis.

The arterial content of hyaluronan (HA) undergoes diffuse changes as part of the diabetic macroangiopathy. Because HA influences the phenotype of vascular cells in vitro such as proliferation, migration, and secretion, it is tempting to speculate that diabetes-induced hastened cardiovascular disease may be linked to the increased amount of HA. To explore the pathophysiological role of altered HA content in the arterial wall in vivo, we created transgenic (Tg) mice with HA overexpression in smooth muscle cells (SMCs) in large and small vessels, targeted by the alpha smooth-muscle-cell-actin (alphaSMA) promoter fused to the human hyaluronan synthase 2 (hHAS2) cDNA. RT-PCR demonstrated hHAS2 mRNA expression in the tunica media of large and small vessels. In situ hybridization confirmed that hHAS2 mRNA was targeted to the SMCs. The aortic HA content was elevated in the Tg mice, and by immunohistochemistry, it was seen that HA accumulated in the tunica media. The secretory profile of high- and low-molecular HA was similar in wild-type and Tg animals. Overproduction of HA in the aorta resulted in thinning of the elastic lamellae in Tg mice. Our data suggest that this may lead to increased mechanical stiffness and strength, as determined by controlled stretching until failure. Finally, overproduction of HA on the genetic background of the ApoE-deficient mouse strain promoted atherosclerosis development in the aorta. These results indicate that a single component of the diabetic macroangiopathy, diffuse accumulation of HA, accelerates the progression of atherosclerosis.

Homocysteine and the production of collagens, proliferation and apoptosis in human arterial smooth muscle cells.

Homocysteine (H(e)) is an important and independent risk factor for atherosclerosis. We showed that human aortic smooth muscles in cultures proliferated significantly at a concentration of 25 micromol/L H(e) without the presence of serum. There was no effect of H(e) on apoptosis as determined by TUNEL-assay and gene expression of proapoptotic protein bax, caspases and TNFalpha families. However, collagen types I, III and IV increased significantly in a dose-dependent manner at elevated concentrations of H(e) and the amount of type VI collagen was significantly reduced in a dose-dependent manner. H(e) induced increased cell replication with an unaffected apoptosis rate. The present observations suggest that H(e) may contribute to accelerated progression of atherosclerotic lesions with collagen alterations which transform the injury into fibrotic plaques.

Growth hormone receptor expression and function in pituitary adenomas.

OBJECTIVE AND DESIGN: Hypopituitarism, in particular GH deficiency, is prevalent in patients with clinically nonfunctioning pituitary adenomas (NFPAs) both before and after surgery. The factors regulating the growth of pituitary adenomas in general and residual tumour tissue in particular are not fully characterized, and the effect of GH and IGF-I on human pituitary cell proliferation has not previously been reported. In NFPA tissue from 14 patients we evaluated GH receptor (GHR) expression and signal transduction, and the effect of GH and IGF-I exposure on cell proliferation and hormone secretion in vitro. MEASUREMENTS: Tissue samples from 14 NFPAs were investigated. Expression of GHR in tissue samples was assessed by reverse transcription polymerase chain reaction (RT-PCR). Six tumours were immunostained with a GHR antibody. In the cell cultures, STAT5 (signal transducer and activator of transcription 5) phosphorylation was measured by Western blot analysis as an index of GHR signalling; cell proliferation was evaluated by [H3]-thymidine incorporation and glycoprotein hormone production analysed by radioimmunoassay (RIA). RESULTS: All adenomas investigated expressed the GHR, but there was no detection of STAT5 phosphorylation. Overall, GH and IGF-I administration did not significantly stimulate cell proliferation in vitro, although some individual adenomas exhibited a proliferative response to various extents. GH also did not significantly influence glycoprotein hormone secretion in vitro. CONCLUSION: GH receptors are expressed in human pituitary adenoma cells but their functional role is uncertain. GH and IGF-I do not consistently influence the proliferation of cultured pituitary adenoma cells.

Growth hormone increases vascular cell adhesion molecule 1 expression: in vivo and in vitro evidence.

We investigated the impact of GH administration on endothelial adhesion molecules, vascular cell adhesion molecule-1 (VCAM-1) and E-selectin, in vivo and in vitro. Soluble VCAM-1, E-selectin, and C-reactive protein concentrations were measured before and after treatment in 25 healthy subjects and 25 adult GH-deficient (GHD) patients randomized to GH treatment or placebo. Furthermore, we studied the direct effect of GH and IGF-I and serum from GH-treated subjects on basal and TNF alpha-stimulated expression of VCAM-1 and E-selectin on cultured human umbilical vein endothelial cells. Baseline levels of VCAM-1, but not E-selectin, were significantly lower in GHD patients than in healthy subjects (362 +/- 15 microg/liter vs. 516 +/- 21 microg/liter, P < 0.001) and increased in GHD patients during GH treatment, compared with placebo [net difference between groups 151.8 microg/liter (95% confidence interval: 95.0-208.7 microg/liter); P < 0.0001]. In human umbilical vein endothelial cells, there was no direct stimulatory effect of either GH or IGF-I on the expression of VCAM-1 and E-selectin, but serum from GH-treated healthy subjects significantly increased the expression of VCAM-1 (P < 0.01). Our findings are compatible with the notion that GH may stimulate the expression of VCAM-1 indirectly through modulation of circulating factors. VCAM-1-mediated leukocyte extravasation is implicated in several illnesses including atherosclerosis and multiple-organ failure in sepsis, and we hypothesize that enhanced expression of VCAM-1 may contribute to the detrimental effects of GH in critically ill patients.

Localisation and phenotypical characterisation of collagen-producing cells in TGF-beta 1-induced renal interstitial fibrosis.

Transforming growth factor beta 1 (TGF-beta 1) contributes to the accumulation of extracellular matrix (ECM) in the tubulointerstitial space in chronic renal diseases. Identification of target cells and the contribution of epithelial-mesenchymal transformation (EMT) in TGF-beta 1-induced fibrosis in vivo are currently under investigation. We have developed a transgenic model of slowly developing TGF-beta 1-driven tubulointerstitial fibrosis (TIF). By using this model our aim was to localise the ECM-producing cells, to investigate the temporal and spatial distribution of the cellular markers alpha-smooth muscle cell actin (alpha SM-actin), Fsp1 and Hsp47 and to explore the possible involvement of EMT in TGF-beta1-induced TIF in vivo. We utilised a combination of in situ hybridisation, immunohistochemistry and western blotting techniques and found that alpha SM-actin-positive interstitial cells are the main source of collagen types I and III and fibronectin, whereas collagen type IV(alpha 1/alpha 2) originates mainly from the tubular epithelial cells. Furthermore, macrophages are not important combatants during the early course of TGF-beta 1-induced TIF. Finally, EMT is not necessary for the initiation of TGF-beta 1-induced TIF. We conclude, that intervention directed against the recruitment of activated interstitial cells may avoid the development of end-stage renal disease.

A highly sensitive and specific assay for determination of IGF-I bioactivity in human serum.

At present, the circulating bioactivity of insulin-like growth factor I (IGF-I) is estimated by immunological measurements of IGF-I levels. However, immunoassays ignore the modifying effects of the IGF-binding proteins (IGFBPs) on the interaction between IGF-I and the IGF-I receptor (IGF-IR). Therefore, we developed an IGF-I kinase receptor activation assay (KIRA) based on cells transfected with the human IGF-IR gene. The bioassay was sensitive (detection limit 0.08 microg/l), specific (cross-reactivity of insulin, insulin analogs, and proinsulin was <1%; IGF-II cross-reactivity was 12%), and accurate (within- and between-assay coefficients of variation <7 and <15%). The operational range of the assay (0.25-10.0 microg/l) allowed for determination of IGF-I bioactivity in serum from patients with, for example, growth hormone deficiency, type 1 diabetes, and acromegaly. Addition of IGFBPs dose dependently reduced the KIRA signal, whereas addition of IGF-II to preformed complexes (1:1 molar ratio) of IGF-I and IGFBP dose dependently increased IGF-I bioactivity by displacement of bound IGF-I. In conclusion, the KIRA will enable us to compare IGF-I bioactivity with existing immunological measurements of IGF-I in serum and, hopefully, to elucidate the factors that determine IGF-I bioactivity in vivo.

Regulation of insulin-like growth factor binding protein secretion by human granulosa luteal cells in a polycystic ovary-like environment.

OBJECTIVE: To determine the effect of androstenedione (A), insulin, and LH on secretion of insulin-like growth factor binding proteins (IGFBPs) from human granulosa luteal cells. DESIGN: Human granulosa cells were cultured for a total of 4 days in serum-free medium containing A (10(-6) mol/L), with or without insulin (100 microU/mL-800 microU/mL), LH (1 microU/mL-10 microg/L), and A (10(-5) mol/L). SETTING: Granulosa cells were obtained from IVF procedures. PATIENT(S): Women undergoing IVF for tubal disease. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Immunoassay and autoradiographs of Western ligand blotting detected IGFBP accumulations in the medium. RESULT(S): Cultured granulosa cells secreted IGFBP-1 through IGFBP-4. Insulin (100 microU/mL-800 microU/mL), LH (1 microg/L-10 microg/L), and A (10(-5) mol/L) caused a significant decrease in IGFBP-1 accumulation in the medium both alone and when added in combination. The release of IGFBP-2 and IGFBP-4 was significantly stimulated by insulin, whereas LH had no effect. Elevated levels of androgen (10(-5) mol/L) significantly stimulated the secretion of IGFBP-2, whereas the release of IGFBP-4 was reduced. CONCLUSION(S): These results demonstrate that androgen and insulin are important regulators of IGFBP release and that elevated levels of the two hormones may contribute to the altered IGFBP profile found in PCOS follicles, compared with the case of estrogen-dominant follicles.

Long-term AICAR administration reduces metabolic disturbances and lowers blood pressure in rats displaying features of the insulin resistance syndrome.

The insulin resistance syndrome is characterized by several risk factors for cardiovascular disease. Chronic chemical activation of AMP-activated protein kinase by the adenosine analog 5-aminoimidazole-4-carboxamide-1-beta -D-ribofuranoside (AICAR) has been shown to augment insulin action, upregulate mitochondrial enzymes in skeletal muscles, and decrease the content of intra-abdominal fat. Furthermore, acute AICAR exposure has been found to reduce sterol and fatty acid synthesis in rat hepatocytes incubated in vitro as well as suppress endogenous glucose production in rats under euglycemic clamp conditions. To investigate whether chronic AICAR administration, in addition to the beneficial effects on insulin sensitivity, is capable of improving other phenotypes associated with the insulin resistance syndrome, obese Zucker (fa/fa) rats (n = 6) exhibiting insulin resistance, hyperlipidemia, and hypertension were subcutaneously injected with AICAR (0.5 mg/g body wt) daily for 7 weeks. Obese control rats were either pair-fed (PF) (n = 6) or ad libitum-fed (AL) (n = 6). Lean Zucker rats (fa/-) (n = 8) served as a reference group. AICAR administration significantly reduced plasma triglyceride levels (P < 0.01 for AICAR vs. AL, and P = 0.05 for AICAR vs. PF) and free fatty acids (P < 0.01 for AICAR vs. AL, and P < 0.05 for AICAR vs. PF) and increased HDL cholesterol levels (P < 0.01 for AICAR vs. AL and PF). AICAR treatment also lowered systolic blood pressure by 14.6 +/- 4.3 mmHg (P < 0.05), and AICAR-treated animals exhibited a tendency toward decreased intra-abdominal fat content. Furthermore, AICAR administration normalized the oral glucose tolerance test and decreased fasting concentrations of glucose and insulin close to the level of the lean animals. Finally, in line with previous findings, AICAR treatment was also found to enhance GLUT4 protein expression and to increase maximally insulin-stimulated glucose transport in primarily white fast-twitch muscles. Our data provide strong evidence that long-term administration of AICAR improves glucose tolerance, improves the lipid profile, and reduces systolic blood pressure in an insulin-resistant animal model. The present study gives additional support to the hypothesis that AMPK activation might be a potential future pharmacological strategy for treating the insulin resistance syndrome.