Lactation and appetite-regulating hormones: increased maternal plasma peptide YY concentrations 3-6 months postpartum.

Breast-feeding is associated with maternal hormonal and metabolic changes ensuring adequate milk production. In this study, we investigate the impact of breast-feeding on the profile of changes in maternal appetite-regulating hormones 3-6 months postpartum. Study participants were age- and BMI-matched lactating mothers (n 10), non-lactating mothers (n 9) and women without any history of pregnancy or breast-feeding in the previous 12 months (control group, n 10). During study sessions, young mothers breast-fed or bottle-fed their babies, and maternal blood samples were collected at five time points during 90 min: before, during and after feeding the babies. Outcome parameters were plasma concentrations of ghrelin, peptide YY (PYY), leptin, adiponectin, prolactin, cortisol, insulin, glucose and lipid values. At baseline, circulating PYY concentrations were significantly increased in lactating mothers (100·3 (se 6·7) pg/ml) v. non-lactating mothers (73·6 (se 4·9) pg/ml, P=0·008) and v. the control group (70·2 (se 9) pg/ml, P=0·021). We found no differences in ghrelin, leptin and adiponectin values. Baseline prolactin concentrations were over 4-fold higher in lactating mothers (P<0·001). Lactating women had reduced TAG levels and LDL-cholesterol:HDL-cholesterol ratio, but increased waist circumference, when compared with non-lactating women. Breast-feeding sessions further elevated circulating prolactin (P<0·001), but induced no acute effects on appetite-regulating hormones. In summary, one single breast-feeding session did not acutely modulate circulating appetite-regulating hormones, but increased baseline PYY concentrations are associated with prolonged lactation. PYY might play a role in the coordination of energy balance during lactation, increasing fat mobilisation from maternal depots and ensuring adequate milk production for the demands of the growing infant.

Opposite effects of serum- and glucocorticoid-regulated kinase-1 and glucocorticoids on POMC transcription and ACTH release.

Serum- and glucocorticoid-regulated kinase-1 (SGK1) is a glucocorticoid early-response gene; its function, however, has been elucidated mainly in the context of mineralocorticoid signaling. Here, we investigate the expression and function of SGK1 in the pituitary gland, one of the primary glucocorticoid targets. SGK1 is expressed in the human pituitary gland and colocalizes to ACTH. The AtT-20 murine corticotroph cell line was used for functional experiments. Glucocorticoids upregulated SGK1 mRNA and protein levels, parallel to decreasing proopiomelanocortin (POMC) transcription and ACTH release. Dexamethasone-induced changes in SGK1 protein were abolished by the steroid receptor antagonist RU-486 and reduced by the inhibition of PI 3-kinase with LY-294002. SGK1 overexpression increased CREB- and activator protein-1-dependent transcription, POMC transcription, and ACTH secretion but did not influence intracellular cAMP levels. SGK1 overexpression and corticotropin-releasing hormone had additive effects on POMC promoter activity but not on ACTH secretion. SGK1 knockdown by RNA interference decreased POMC promoter activity, demonstrating the importance of SGK1 for basal POMC signaling. In summary, SGK1 is strongly stimulated by glucocorticoids in pituitary corticotrophs; however, its effects on POMC transcription are antagonistic to the classical inhibitory glucocorticoid action, suggesting a cell-regulated counterregulatory mechanism to potentially detrimental glucocorticoid effects.

Diabetic LDL triggers apoptosis in vascular endothelial cells.

This study compares the effects of LDL glycated either in vitro (LDL(iv)) or in vivo in diabetic patients (LDL(D)) on apoptosis, proliferation, and associated protein expression in cultured human umbilical vein endothelial cells. At 100 mg/l, both LDL species considerably increase apoptosis (LDL(iv) 63%, LDL(D) 40%; P < 0.05) compared with intraindividual nonglycated LDL subfractions. Considering its lower degree of glycation (LDL(D) 5-10%, LDL(iv) 42%), LDL(D)'s relative proapoptotic activity is 2.7-fold greater than that of LDL(iv). Glycated LDL-induced apoptosis is associated with increased expression of apoptosis promotors (LDL(iv): bak 88%, CPP-32 49%; LDL(D): bak 18%, CPP-32 11%; P < 0.05) and is attenuated by caspase inhibitors. Glycated LDL's antiproliferative activity (LDL(iv) -34%, LDL(D) -9%; P < 0.01) relates to reduction (P < 0.05) of cyclin D3 (LDL(iv) -27%, LDL(D) -24%) and of hypo- (LDL(iv) -22%, LDL(D) -19%) and hyperphosphorylated (LDL(iv) -53%, LDL(D) -22%) retinoblastoma protein and is paralleled by reduced expression of endothelial nitric oxide synthase (LDL(iv) -30%, LDL(D) -23%). In response to lipoprotein lipase, LDL(D) more markedly triggers endothelial apoptosis (27.1-fold) compared with LDL(iv), suggesting that LDL(D) owns a higher potential for endothelial cell damage than LDL(iv). The observed behavior of LDL(D) versus LDL(iv) could be of clinical importance and well relate to differences in structure and cellular uptake of LDL(D) compared with LDL(iv).

Thiazolidinediones inhibit apoptosis and heat shock protein 60 expression in human vascular endothelial cells.

This study evaluated direct effects of peroxisome proliferatoractivated receptor gamma(PPARgamma) agonists, including thiazolidinediones (TZDs), on vascular cell apoptosis and related protein expression to test the hypothesis that these effects are dependent on i) the respective agent's structure and ii) endothelial cells' vascular origin. Exposure (48 h) of human umbilical vein endothelial cells (HUVECs, n=6) to up to 10 microM troglitazone (TRO), rosiglitazone, pioglitazone, and to up to 50 microM RWJ241947=MCC-555 (RWJ) inhibited (p<0.05) apoptosis by 8-25%, whereas 15-deoxy-Delta(12-14)-prostaglandin J(2) (PGJ(2)) triggered (50 microM: + 400%, p<0.05) endothelial cell death versus control (=100%). Moreover, RWJ (50 microM) completely abrogated TNF-alpha(2000 U/ml) and stearic acid (200 microM) induced apoptosis in HUVECs . Similar results were obtained in human adult (saphenous) vein- and aortic endothelial cells, the latter showing no anti-apoptotic response to TRO. In HUVECs, TZDs' anti-apoptotic effects inversely correlated (r=-0.95, p<0.01) with increased (p<0.05) expression of the apoptosis-inhibitor bcl-2, whereas PGJ(2)-induced apoptosis was associated with upregulation of c-myc (+447%) and E2F-1 (+339%). Additionally, TZDs (by 25-39%) and PGJ(2) (-70%) reduced (p<0.05) expression of heat shock protein 60 (hsp60) showing no correlation with apoptosis (r=0.14, n.s.). Modulation of apoptosis by PPARgammaagonists differs in endothelial cells dependent on their vascular origin and the agonists' structure. Thiazolidinediones' ability to reduce both, endothelial apoptosis and hsp60 expression could well add to beneficial vascular effects attributed to these oral antidiabetic drugs.

Estimation of the false-negative rate in newborn screening for congenital adrenal hyperplasia.

OBJECTIVE: Newborn screening based on measurement of 17alpha-hydroxyprogesterone (17-OHP) in a dried blood spot on filter paper is an effective tool for early diagnosis of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. Its most important rationale is prevention of a life-threatening salt-wasting (SW) crisis; in moderate forms of CAH, early diagnosis and treatment may prevent permanent negative effects of androgen overproduction. Our target was to analyse if all CAH patients who had been identified clinically before puberty would have been detected by the newborn screening. METHODS: Newborn screening cards of 110 CAH patients born between 1988 and 2000 in five Middle-European countries and diagnosed prior to puberty (77 SW and 33 moderate) and cards from 920 random, healthy newborn controls were analysed. CAH screening had not yet been introduced during this time. The diagnosis was based on clinical and laboratory signs and, in most cases, on CYP21 gene mutation analysis. All 17-OHP measurements in dried blood spots were carried out using a time-resolved fluoroimmunoassay kit. RESULTS: In the newborn screening blood spots, the median of 17-OHP levels was 561 nmol/l (range 91-1404 nmol/l) in subjects with the SW form and 40 nmol/l (4-247 nmol/l) in the moderate form. All 77 SW patients would have been detected by newborn screening using the recommended cut-off limits (30 nmol/l). However, 10 of 33 patients with moderate CAH would have been missed. 17-OHP levels of all controls were below the cut-off. CONCLUSION: Newborn screening is efficient for diagnosing the SW form of CAH, but is inappropriate for identifying all patients with a moderate form of CAH. It appears that the false-negative rate is at least one-third in children with the moderate form of CAH.

Free fatty acids trigger apoptosis and inhibit cell cycle progression in human vascular endothelial cells.

Plasma free fatty acid (FFA) concentrations are increased in states of insulin resistance and impair endothelial function. Because the underlying mechanisms are largely unknown, we examined selected, purified FFAs' (100-300 micromol/l, 24-48 h) action on apoptosis, cell cycle distribution, and associated gene/protein expression in human umbilical vein endothelial cells (HUVECs). Stearic acid, but not oleic acid, time and concentration dependently increased endothelial apoptosis by fivefold (n=6, P<0.01), whereas polyunsaturated FFAs (PUFAs; linoleic, gamma-linolenic, and arachidonic acid) exerted proapoptotic activity only at 300 micromol/l (P<0.05). Proapoptotic FFA action increased with FFAs' number of double bonds and with protein expression of the apoptosis promotor bak. The G0/G1 cell cycle arrest (n=6, P<0.05) induced by stearic acid (+14%) and PUFAs (+30%) is reflected by up-regulation of p21(WAF-1/Cip1). In addition, all FFAs concentration dependently reduced (P<0.05) gene/protein expression of clusterin (-54%), NF-kappaB's inhibitor, IkappaBalpha (-50%), endothelin-1 (-44%), and endothelial NO synthase (-44%). Plasma samples obtained from individuals with elevated plasma FFAs (372+/-22 micromol/l) increased endothelial apoptosis by 4.2-fold (P<0.001, n=10) compared with intra-individually matched low plasma FFA (56+/-21 micromol/l) conditions, underlining the results obtained by defined FFA stimulation. In conclusion, FFA structure differently affects endothelial cell proliferation and apoptosis, both representing key factors in the development of micro- and macrovascular dysfunction.

The natural antioxidants, pomegranate extract and soy isoflavones, favourably modulate canine endothelial cell function.

Cardiovascular disease, preceded by vascular endothelial dysfunction, is a prominent cause of death in dogs. L-carnitine and taurine, well known for their antioxidative capacity, beneficially affect cardiovascular disease as well as certain dog cardiomyopathies. It is well established that vascular endothelial dysfunction precedes cardiovascular disease and that "vasoprotective factors" (NO and antioxidants) prevent apoptosis, whereas "risk factors" such as oxidized LDL, hyperglycemia, and free fatty acids trigger it in cultured human vascular endothelial cells. Whereas human vascular cell in vitro models are widely established and used for the characterisation of potential vasoprotective substances, such models are not available for canine endothelial cells. In the present study we therefore developed an in vitro model, which allows the testing of the effects of different substances on proliferation and apoptosis in canine aortic endothelial cells. This model was used to test L-carnitine, taurine, pomegranate extract, and Soy Isoflavones in comparison to reference substances (glutathione and pioglitazone) previously shown to modulate human endothelial cell function. L-carnitine and taurine neither exhibited antiproliferative nor antiapoptotic activities in the context of this study. However extracts from pomegranate and soy isoflavones dramatically reduced proliferation and apoptosis in a dose dependent fashion, being in line with a vasoprotective activity in dogs.

Acute effects of hydrocortisone on the metabolic response to a glucose load: increase in the first-phase insulin secretion.

BACKGROUND AND AIM: Several basic science studies support the existence of non-genomic glucocorticoid signaling in pancreas, liver, and adipocytes, but its clinical relevance has not yet been elucidated. This study aimed at investigating the rapid effects of hydrocortisone on the human metabolic response to glucose. SUBJECTS AND METHODS: In a randomized placebo-controlled crossover study, ten healthy men received an i.v. bolus of 0.6 mg/kg hydrocortisone once and placebo once 4 min before the administration of 330 mg/kg glucose. Cortisol, glucose, insulin, C-peptide, ghrelin, and peptide YY (PYY) levels were measured during the following 3 h. Minimal model analysis was performed for evaluating the metabolic response. RESULTS: Hydrocortisone attenuated the rise in plasma glucose during the initial 15 min following glucose administration (P=0.039), and it led to lower glucose levels during the first 2 h (P=0.017). This was accompanied by enhanced circulating insulin (P=0.02) and C-peptide (P=0.03) levels during the initial 15 min, and a 35% increase in the first-phase beta-cell function (P=0.003). Hydrocortisone decreased PYY concentrations during the initial 30 min (P=0.014), but it did not affect the ghrelin response to glucose. CONCLUSION: One i.v. bolus of hydrocortisone induces rapid effects on carbohydrate metabolism increasing the first-phase beta-cell function. The modulation of PYY plasma levels suggests the possible non-genomic effects of glucocorticoids on appetite-regulatory hormones.

Fatty acids induce apoptosis in human smooth muscle cells depending on chain length, saturation, and duration of exposure.

OBJECTIVE: Plasma free fatty acid (FFA) concentrations are increased in states of insulin resistance. Therefore, this study evaluated apoptosis and underlying mechanisms induced by selected nutritional FFAs, a defined FFA-mix, and human plasma containing high FFA concentrations in human smooth muscle cells (HSMCs). RESEARCH DESIGN AND METHODS: HSMCs were incubated (24-72 h) with selected FFAs (100-300 micromol/l), an FFA-mix (palmitic-/stearic-/oleic-/linoleic-/alpha-linolenic acid=2.6/1/3.6/9/1; 300-900 micromol/l), or with high FFA-plasma (600 micromol/l) versus respective control cultures. Apoptosis, caspase activation, and protein expression were determined by DNA-fragmentation assays, flow cytometry, and Western blots, respectively. RESULTS: Exposure (24h) of HSMCs to 300 micromol/l stearic-, oleic-, linoleic-, alpha-linolenic-, and arachidonic acid induced apoptosis, correlating (p<0.01) with the FFAs' chain length (r=0.602) and number of FFA double bonds (r=0.956). After 48 h, 100 micromol/l of all tested FFAs - including palmitic acid - were already sufficient to trigger HSMCs' cell death. FFA-exposure resulted in activation of caspases and apoptosis was completely abolished by co-incubation with caspase inhibitors and negatively correlated (p<0.01) with the base-excision repair protein XRCC1 (r=-0.765) and with c-myc's antagonist mad (r=-0.916), whereas positive correlations (p<0.01) were found for protein expression of the proto-oncogene c-myc (r=0.972) and the transcription factor E2F-1 (r=0.971). Exposure of HSMCs to the defined FFA-mix and to plasma samples from individuals with elevated plasma FFAs supported the results obtained by defined FFA stimulation. CONCLUSIONS: Since smooth muscle cells surround the macrophage/foam cell/lipid-laden artheromatous core of atherosclerotic lesions with a protective fibrous cap, their FFA-induced HSMC apoptosis could contribute to progression of atherosclerosis by thinning of the fibrous cap and subsequent plaque destabilization.

Different mechanisms of saturated versus polyunsaturated FFA-induced apoptosis in human endothelial cells.

Apoptosis and underlying mechanisms were evaluated in human umbilical vein endothelial cells (HUVECs), in target tissues of late diabetic vascular complications [human aortic endothelial cells (HAECs) and human retinal endothelial cells (HRECs)], and in endothelial progenitor cells (EPCs) exposed to FFAs, which are elevated in obesity and diabetes. Saturated stearic acid concentration dependently induced apoptosis that could be mediated via reduced membrane fluidity, because both apoptosis and membrane rigidity are counteracted by eicosapentaenoic acid. PUFAs triggered apoptosis at a concentration of 300 micromol/l in HUVECs, HAECs, and EPCs, but not HRECs, and, in contrast to stearic acid, involved caspase-8 activation. PUFA-induced apoptosis, but not stearic acid-induced apoptosis, strictly correlated (P < 0.01) with protein expression of E2F-1 (r = 0.878) and c-myc (r = 0.966). Lack of c-myc expression and activity owing to quiescence or transfection with dominant negative In373-Myc, respectively, renders HUVECs resistant to PUFA-induced apoptosis. Because c-myc is abundant in growing cells only, apoptosis triggered by PUFAs, but not by saturated stearic acid, obviously depends on the growth/proliferation status of the cells. Finally, this study shows that FFA-induced apoptosis depends on the vascular origin and growth/proliferation status of endothelial cells, and that saturated stearic acid-induced apoptosis and PUFA-induced apoptosis are mediated via different mechanisms.

R-(+)-alpha-lipoic acid inhibits endothelial cell apoptosis and proliferation: involvement of Akt and retinoblastoma protein/E2F-1.

Lipoic acid was recently demonstrated to improve endothelial dysfunction or retinopathy not only in rats but also in diabetic patients. We tested the hypothesis that R-(+)-alpha-lipoic acid (LA) directly affects human endothelial cell (EC) function (e.g., apoptosis, proliferation, and protein expression), independent of the cells' vascular origin. Macrovascular EC (macEC), isolated from umbilical (HUVEC) and adult saphenous veins and from aortae, as well as microvascular EC (micEC) from retinae, skin, and uterus, were exposed to LA (1 mumol/l-1 mmol/l) with/without different stimuli (high glucose, TNF-alpha, VEGF, wortmannin, LY-294002). Apoptosis, proliferation, cell cycle distribution, and protein expression were determined by DNA fragmentation assays, [(3)H]thymidine incorporation, FACS, and Western blot analyses, respectively. In macro- and microvascular EC, LA (1 mmol/l) reduced (P < 0.05) basal (macEC, -36 +/- 4%; micEC, -46 +/- 6%) and stimulus-induced (TNF-alpha: macEC, -75 +/- 11%; micEC, -68 +/- 13%) apoptosis. In HUVEC, inhibition of apoptosis by LA (500 mumol/l) was paralleled by reduction of NF-kappaB. LA's antiapoptotic activity was reduced by PI 3-kinase inhibitors (wortmannin, LY-294002), being in line with LA-induced Akt phosphorylation (Ser(437), +159 +/- 43%; Thr(308), +98 +/- 25%; P < 0.01). LA (500 mumol/l) inhibited (P < 0.001) proliferation of macEC (-29 +/- 3%) and micEC (-29 +/- 3%) by arresting the cells at the G(1)/S transition due to an increased ratio of cyclin E/p27(Kip) (4.2-fold), upregulation of p21(WAF-1/Cip1) (+104 +/- 21%), and reduction of cyclin A (-32 +/- 11%), of hyperphosphorylated retinoblastoma protein (macEC: -51 +/- 7%; micEC: -50 +/- 15%), and of E2F-1 (macEC: -48 +/- 3%; micEC: -31 +/- 10%). LA's ability to inhibit apoptosis and proliferation of ECs could beneficially affect endothelial dysfunction, which precedes manifestation of late diabetic vascular complications.

Free fatty acids normalize a rosiglitazone-induced visfatin release.

The detrimental effect of elevated free fatty acids (FFAs) on insulin sensitivity can be improved by thiazolidinediones (TZDs) in patients with type 2 diabetes mellitus. It is unknown whether this salutary action of TZD is associated with altered release of the insulin-mimetic adipocytokine visfatin. In this study, we investigated whether visfatin concentrations are altered by FFA and TZD treatment. In a randomized, double-blind, placebo-controlled, parallel-group study 16 healthy volunteers received an infusion of triglycerides/heparin to increase plasma FFA after 3 wk of treatment with rosiglitazone (8 mg/day, n = 8) or placebo (n = 8), and circulating plasma visfatin was measured. As a corollary, human adipocytes were incubated with synthetic fatty acids and rosiglitazone to assess visfatin release in vitro. The results were that rosiglitazone treatment increased systemic plasma visfatin concentrations from 0.6 +/- 0.1 to 1.7 +/- 0.2 ng/ml (P < 0.01). Lipid infusion caused a marked elevation of plasma FFA but had no effect on circulating visfatin in controls. In contrast, elevated visfatin concentrations in subjects receiving rosiglitazone were normalized by lipid infusion. In isolated adipocytes, visfatin was released into supernatant medium by acute addition and long-term treatment of rosiglitazone. This secretion was blocked by synthetic fatty acids and by inhibition of phosphatidylinositol 3-kinase or Akt. In conclusion, release of the insulin-mimetic visfatin may represent a major mechanism of metabolic TZD action. The presence of FFA antagonizes this action, which may have implications for visfatin bioactivity.