Amphipathic polyproline peptides stimulate cholesterol efflux by the ABCA1 transporter.

ApoA-I mimetics are short synthetic peptides that contain an amphipathic α-helix and stimulate cholesterol efflux by the ABCA1 transporter in a detergent-like extraction mechanism. We investigated the use of amphipathic peptides with a polypro helix for stimulating cholesterol efflux by ABCA1. Polypro peptides were synthesized with modified prolines, containing either a hydrophobic phenyl group (Prop) or a polar N-acetylgalactosamine (Prog) attached to the pyrrolidine ring and were designated as either PP-2, 3, 4, or 5, depending on the number of 3 amino acid repeat units (Prop-Prog-Prop). Based on molecular modeling, these peptides were predicted to be relatively rigid and to bind to a phospholipid bilayer. By CD spectroscopy, PP peptides formed a Type-II polypro helix in an aqueous solution. PP-2 was inactive in promoting cholesterol efflux, but peptides with more than 2 repeat units were active. PP-4 showed a similar Vmax as a much longer amphipathic α-helical peptide, containing 37 amino acids, but had a Km that was approximately 20-fold lower. PP peptides were specific in that they did not stimulate cholesterol efflux from cells not expressing ABCA1 and were also non-cytotoxic. Addition of PP-3, 4 and 5 to serum promoted the formation of smaller size HDL species (7 nM) and increased its capacity for ABCA1-dependent cholesterol efflux by approximately 20-35% (p < 0.05). Because of their relatively small size and increased potency, amphipathic peptides with a polypro helix may represent an alternative structural motif for the development of apoA-I mimetic peptides.

Insulin and extremity muscle mass in overweight and obese women.

BACKGROUND: Obesity disproportionately affects women, especially those of African descent, and is associated with increases in both fat and muscle masses. OBJECTIVE: Although increased extremity muscle mass may be compensatory to fat mass load, we propose that elevated insulin levels resulting from diminished insulin sensitivity may additionally contribute to extremity muscle mass in overweight or obese women. METHODS: The following measurements were performed in 197 non-diabetic women (57% black, 35% white; age 46±11 years (mean±s.d.), body mass index (BMI) range 25.0-57.7 kg m(-2)): dual-energy X-ray absorptiometry for fat and extremity muscle masses; exercise performance by duration and peak oxygen consumption (VO2 peak) during graded treadmill exercise; fasting insulin and, in 183 subjects, insulin sensitivity index (SI) calculated from the minimal model. RESULTS: SI (range 0.5-14.1 l mU(-1 )min(-1)) was negatively, and fasting insulin (range 1.9-35.6 µU ml(-1)) positively associated with extremity muscle mass (both P<0.001), independent of age and height. Sixty-seven percent of women completed 6 months of participation in a weight loss and exercise program: we found a significant association between reduction in fasting insulin and a decrease in extremity muscle mass (P=0.038), independent of reduction in fat mass or improvement in exercise performance by VO2 peak and exercise duration, and without association with change in SI or interaction by race. CONCLUSIONS: Hyperinsulinemia in overweight or obese women is associated with increased extremity muscle mass, which is partially reversible with reduction in fasting insulin concentration, consistent with the stimulatory effects of insulin on skeletal muscle.

Helix stabilization of amphipathic peptides by hydrocarbon stapling increases cholesterol efflux by the ABCA1 transporter.

Apolipoprotein mimetic peptides are short amphipathic peptides that efflux cholesterol from cells by the ABCA1 transporter and are being investigated as therapeutic agents for cardiovascular disease. We examined the role of helix stabilization of these peptides in cholesterol efflux. A 23-amino acid long peptide (Ac-VLEDSFKVSFLSALEEYTKKLNTQ-NH2) based on the last helix of apoA-I (A10) was synthesized, as well as two variants, S1A10 and S2A10, in which the third and fourth and third and fifth turn of each peptide, respectively, were covalently joined by hydrocarbon staples. By CD spectroscopy, the stapled variants at 24 °C were more helical in aqueous buffer than A10 (A10 17%, S1A10 62%, S2A10 97%). S1A10 and S2A10 unlike A10 were resistant to proteolysis by pepsin and chymotrypsin. S1A10 and S2A10 showed more than a 10-fold increase in cholesterol efflux by the ABCA1 transporter compared to A10. In summary, hydrocarbon stapling of amphipathic peptides increases their helicity, makes them resistant to proteolysis and enhances their ability to promote cholesterol efflux by the ABCA1 transporter, indicating that this peptide modification may be useful in the development of apolipoprotein mimetic peptides.

PPAR-alpha and PPAR-gamma activators induce cholesterol removal from human macrophage foam cells through stimulation of the ABCA1 pathway.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that regulate lipid and glucose metabolism and cellular differentiation. PPAR-alpha and PPAR-gamma are both expressed in human macrophages where they exert anti-inflammatory effects. The activation of PPAR-alpha may promote foam-cell formation by inducing expression of the macrophage scavenger receptor CD36. This prompted us to investigate the influence of different PPAR-activators on cholesterol metabolism and foam-cell formation of human primary and THP-1 macrophages. Here we show that PPAR-alpha and PPAR-gamma activators do not influence acetylated low density lipoprotein-induced foam-cell formation of human macrophages. In contrast, PPAR-alpha and PPAR-gamma activators induce the expression of the gene encoding ABCA1, a transporter that controls apoAI-mediated cholesterol efflux from macrophages. These effects are likely due to enhanced expression of liver-x-receptor alpha, an oxysterol-activated nuclear receptor which induces ABCA1-promoter transcription. Moreover, PPAR-alpha and PPAR-gamma activators increase apoAI-induced cholesterol efflux from normal macrophages. In contrast, PPAR-alpha or PPAR-gamma activation does not influence cholesterol efflux from macrophages isolated from patients with Tangier disease, which is due to a genetic defect in ABCA1. Here we identify a regulatory role for PPAR-alpha and PPAR-gamma in the first steps of the reverse-cholesterol-transport pathway through the activation of ABCA1-mediated cholesterol efflux in human macrophages.

The effects of a high-fruit and -vegetable, high-fiber, low-fat dietary intervention on serum concentrations of insulin, glucose, IGF-I and IGFBP-3.

OBJECTIVE: To determine the effects of dietary change on serum concentrations of insulin, glucose, IGF-I and IGFBP-3. SUBJECTS: From among participants in a randomized clinical trial of men and women without a history of diabetes who were 35 years old or older and who had at least one histologically confirmed colorectal adenoma removed during a qualifying colonoscopy within the 6 months before randomization, 750 subjects were selected for this analysis. METHODS: The authors analyzed fasting serum from 375 subjects with and 375 subjects without a recurrent polyp among participants in a randomized trial of a low-fat (20% of energy), high-fiber (18 g per 1000 kcals of energy intake) and high-fruit and -vegetable (5-8 servings per day) dietary intervention. RESULTS: After 4 years of follow-up, IGF-I concentration in the intervention group (N=248) declined by 8.86 ng/ml (initial mean of 133 ng/ml) and 7.74 ng/ml (initial mean value of 139 ng/ml) in the non-intervention group (N=502). Based on an unpaired t-test, these declines were both statistically significant, but the difference between groups for the decline in IGF-I (1.12 ng/ml ((95% confidence interval, -3.24 to 5.48)) was not. After 4 years, concentrations of IGFBP-3, insulin and glucose were not statistically different from values at baseline, and there were no differences in these serum measures between the intervention and control groups. In analysis restricted to lean (body mass index <25 kg/m(2)) subjects only, however, glucose concentrations in the intervention group decreased by 0.28 mmol/l, while they increased in the control group by 0.01 mmol/l (t-test for mean differences P=0.0003) over 4 years. CONCLUSIONS: A low-fat, high-fiber, high-fruit and -vegetable dietary intervention had minimal impact on serum concentrations of insulin, glucose, IGF-I and IGFBP-3 overall, but in lean subjects the intervention resulted in a significant reduction in serum glucose concentration.

ABCA1 overexpression leads to hyperalphalipoproteinemia and increased biliary cholesterol excretion in transgenic mice.

The discovery of the ABCA1 lipid transporter has generated interest in modulating human plasma HDL levels and atherogenic risk by enhancing ABCA1 gene expression. To determine if increased ABCA1 expression modulates HDL metabolism in vivo, we generated transgenic mice that overexpress human ABCA1 (hABCA1-Tg). Hepatic and macrophage expression of hABCA1 enhanced macrophage cholesterol efflux to apoA-I; increased plasma cholesterol, cholesteryl esters (CEs), free cholesterol, phospholipids, HDL cholesterol, and apoA-I and apoB levels; and led to the accumulation of apoE-rich HDL1. ABCA1 transgene expression delayed 125I-apoA-I catabolism in both liver and kidney, leading to increased plasma apoA-I levels, but had no effect on apoB secretion after infusion of Triton WR1339. Although the plasma clearance of HDL-CE was not significantly altered in hABCA1-Tg mice, the net hepatic delivery of exogenous 3H-CEt-HDL, which is dependent on the HDL pool size, was increased 1.5-fold. In addition, the cholesterol and phospholipid concentrations in hABCA1-Tg bile were increased 1.8-fold. These studies show that steady-state overexpression of ABCA1 in vivo (a) raises plasma apoB levels without altering apoB secretion and (b) raises plasma HDL-C and apoA-I levels, facilitating hepatic reverse cholesterol transport and biliary cholesterol excretion. Similar metabolic changes may modify atherogenic risk in humans.

A comparison of immunometric and radioimmunoassay measurement of ACTH for the differential diagnosis of Cushing's syndrome.

OBJECTIVE: Measurement of plasma ACTH levels by radioimmunoassay (RIA) is used to identify adrenal causes (AA) of Cushing's syndrome (CS) and to distinguish ectopic CS (EAS) from Cushing's disease (CD) using CRH stimulation testing and inferior petrosal sinus sampling (IPSS). We wished to determine whether diagnostic criteria developed with RIA would also be applicable for immunoradiometric (IRMA) or immunochemiluminescent (ICMA) assays. SUBJECTS AND METHODS: ACTH was measured by RIA, immunoradiometric and/or immunochemiluminescent assay on samples obtained during three types of diagnostic testing in a tertiary referral setting: a) basally (63 CD, 5 AA, 2 EAS and 37 non-CS patients); b) in 44 CD patients following CRH; c) in 6 ectopic CS and 17 CD patients during IPSS. The primary outcome was comparison of diagnostic utility. RESULTS: a) IRMA results, while lower, correlated highly with RIA (r=0.9, p<0.0001) and had similar sensitivity (100 vs 80%) and specificity (89 vs 94%) for the diagnosis of AA (p=0.3); b) the sensitivity for CD of CRH testing using IRMA was similar to that of RIA (85 vs 83%, p=1.0); c) during IPSS, IRMA had similar sensitivity (100%) and specificity (100 vs 83%) compared with ICMA or RIA (p=1.0). CONCLUSIONS: ACTH immunometric assays correlate closely to RIA and offer similar diagnostic utility.

Klotho: a humeral mediator in CSF and plasma that influences longevity and susceptibility to multiple complex disorders, including depression.

Klotho is a hormone secreted into human cerebrospinal fluid (CSF), plasma and urine that promotes longevity and influences the onset of several premature senescent phenotypes in mice and humans, including atherosclerosis, cardiovascular disease, stroke and osteoporosis. Preliminary studies also suggest that Klotho possesses tumor suppressor properties. Klotho's roles in these phenomena were first suggested by studies demonstrating that a defect in the Klotho gene in mice results in a significant decrease in lifespan. The Klotho-deficient mouse dies prematurely at 8-9 weeks of age. At 4-5 weeks of age, a syndrome resembling human ageing emerges consisting of atherosclerosis, osteoporosis, cognitive disturbances and alterations of hippocampal architecture. Several deficits in Klotho-deficient mice are likely to contribute to these phenomena. These include an inability to defend against oxidative stress in the central nervous system and periphery, decreased capacity to generate nitric oxide to sustain normal endothelial reactivity, defective Klotho-related mediation of glycosylation and ion channel regulation, increased insulin/insulin-like growth factor signaling and a disturbed calcium and phosphate homeostasis accompanied by altered vitamin D levels and ectopic calcification. Identifying the mechanisms by which Klotho influences multiple important pathways is an emerging field in human biology that will contribute significantly to understanding basic physiologic processes and targets for the treatment of complex diseases. Because many of the phenomena seen in Klotho-deficient mice occur in depressive illness, major depression and bipolar disorder represent illnesses potentially associated with Klotho dysregulation. Klotho's presence in CSF, blood and urine should facilitate its study in clinical populations.

Lipoprotein Particles in Adolescents and Young Women With PCOS Provide Insights Into Their Cardiovascular Risk.

CONTEXT: Adult women with polycystic ovarian syndrome (PCOS) have an increased risk for cardiovascular disease, but the evidence for this is controversial in adolescents and young women with PCOS. Measurement of low-density lipoprotein (LDL) particle number, measured by nuclear magnetic resonance spectroscopy is a novel technology to assess cardiovascular risk. OBJECTIVE: The objective of the study was to evaluate lipoprotein particle number and size in young women with PCOS and its relationship with insulin resistance and hyperandrogenism. DESIGN: This was a cross-sectional case control study. SETTING: The study was conducted at a clinical research center. PARTICIPANTS: Women with PCOS (n = 35) and normal controls (n = 20) participated in the study. INTERVENTIONS: Blood samples and anthropometric measures were obtained. MAIN OUTCOME MEASURES: LDL particle size and number were measured using nuclear magnetic resonance spectroscopy. A secondary outcome was to investigate the correlation of LDL particle number with high-sensitivity C-reactive protein, waist to hip ratio, hyperandrogenism, insulin resistance, and adiponectin. RESULTS: Women with PCOS had higher LDL particle number when compared with healthy controls (935 ± 412 vs 735 ± 264, P = .032); LDL particle number correlated strongly with high-sensitivity C-reactive protein (r = 0.37, P = .006) and waist-to-hip (r = 0.57, P = .0003). The higher LDL particle number was driven mainly due to differences in the small LDL particle number (sLDLp), with PCOS patients having more sLDLp (348 ± 305 vs 178 ± 195, P = .015). The sLDLp correlated with the Matsuda index (r = -0.51, P = .0001), homeostasis model assessment index of insulin resistance (r = 0.41, P = .002), and adiponectin (r = -0.46, P = .0004) but not with T. CONCLUSION: Adolescent and young women with PCOS have an atherogenic lipoprotein profile suggestive of increased cardiovascular risk that appears to be driven by the degree of visceral adiposity and insulin resistance.

The differential effect of food intake and beta-adrenergic stimulation on adipose-derived hormones and cytokines in man.

We determined whether the physiologic changes that accompany food intake or sympathetic activation by beta-adrenergic stimulation result in alterations in the secretion of leptin, tumor necrosis factor-alpha (TNF alpha), or interleukin-6 (IL-6) by serially sampling sc abdominal adipose interstitial fluid by open-flow microperfusion before and after a standardized meal and in response to isoproterenol (1 micromol/L) delivered locally. Post cibum IL-6 rose up to 5-fold, whereas leptin and TNF alpha secretion did not change; TNF alpha, but not IL-6, correlated positively with indices of lipolysis. Isoproterenol-induced lipolysis was accompanied by a transient 40% reduction in leptin and a parallel 85% elevation of TNF alpha concentration, whereas IL-6 levels did not change; again, TNF alpha correlated positively with lipolysis. These data show that secretion of some, but not all, metabolically relevant polypeptides by adipose tissue is modulated within a short time frame by food or stress stimuli, suggesting a role of these peptides in local autocrine/paracrine or distant endocrine effects on fat metabolism. TNF alpha's close correlation with lipolysis suggests that this cytokine participates in a local positive autocrine feedback loop, potentiating lipolysis and inhibiting insulin's antilipolytic actions. The regulations of adipose leptin, TNF alpha, and IL-6 secretion seem distinct from each other and different in the fed vs. fasting state.

Serum thyroxine level and cognitive decline in euthyroid older women.

BACKGROUND: Clinical and subclinical hypothyroidism is associated with cognitive impairment. OBJECTIVE: This study investigated the association between thyroxine (T(4)) and thyroid-stimulating hormone (TSH) level and change over time in cognitive performance in a sample of older women with normal thyroid gland function. METHODS: T(4) and TSH were measured at baseline in 628 women (> or = 65 years) enrolled in the Women's Health and Aging Study, a community-based study of physically impaired women. Cognitive function was assessed at baseline and after 1, 2, and 3 years, using the Mini-Mental State Examination (MMSE). Incident cognitive decline was defined as a decrease of more than one point/year in MMSE score between baseline and the end of the follow-up. The analysis included 464 subjects with normal thyroid gland function with a baseline and at least one follow-up MMSE. RESULTS: At baseline there was no association between T(4) and TSH level and cognitive function. In longitudinal analysis, adjusting for age, race, level of education, and other covariates, compared with women in the highest T(4) tertile (8.1 to 12.5 microg/dL), those in the lowest tertile (4.5 to 6.5 microg/dL) had a greater decline in MMSE score (-0.25 point/year vs -0.12 point/year; p = 0.04). A total of 95 women (20.5%) had cognitive decline during the study period (mean MMSE decline, 5.5 points). Compared with women in the highest T(4) tertile, those in the lowest tertile had a twofold risk of cognitive decline (adjusted relative risk, 1.97; 95% CI, 1.10 to 3.50). The results were not modified by baseline cognitive and physical function. There was no association between baseline TSH level and change in cognitive function. CONCLUSIONS: In older women, low T(4) levels, within the normal range, were associated with a greater risk of cognitive decline over a 3-year period. Thyroid hormone levels may contribute to cognitive impairment in physically impaired women.

Hydrolysis of phosphoproteins and inositol phosphates by cell surface phosphatase of Leishmania donovani.

Leishmania donovani promastigotes contain intense tartrate-resistant cell surface acid phosphatase (ACP1) which blocks superoxide anion production by activated human neutrophils [A.T. Remaley et al. (1984) J. Biol. Chem, 259, 11173-11175]. An extensively purified preparation of ACP1 dephosphorylates several phosphoproteins which are phosphorylated at serine residues; these include: pyruvate kinase (Km 1.6 microM; Vmax 71.4 U (mg protein)-1), phosphorylase kinase (Km 0.076 microM; Vmax 5.4 U (mg protein)-1) and histones (Km 4.86 microM; Vmax 2.2 U (mg protein)-1). However, the specific activity of the leishmanial phosphatase on these phosphoproteins is very low as compared to other phosphoprotein phosphatases. The phosphatase activity of ACP1 was also low on phosphohistone phosphorylated at tyrosine residues. Phosphatidylinositol-4,5-diphosphate (PIP2) and inositoltriphosphate (IP3) were also tested as ACP1 substrates. PIP2 was hydrolyzed rapidly by ACP1. The rate of hydrolysis of PIP2 was higher at pH 6.8 (Km 2.35 microM; Vmax 107 X 10(3) U (mg protein)-1) than at pH 5.5 (Km 4.16 microM; Vmax 71 X 10(3) U (mg protein)-1). 32P-labeled IP3 was also a substrate for ACP1; the hydrolysis products consisted of a mixture of inositoldiphosphate and inositolmonophosphate. ACP1 and ten other phosphatases were tested for their ability to dephosphorylate proteins and to inhibit O2- production by stimulated human neutrophils. There was no correlation between the protein phosphatase activity of the acid- and alkaline phosphatases and their ability to block neutrophil O2- production. The results indicate that ACP1 probably blocks the production of reduced oxygen intermediates by a mechanism that does not involve dephosphorylation of phosphoproteins; however, the possibility that the parasite's phosphatase affects phagocyte metabolism by degrading PIP2 or IP3 should be considered.

Kinetic analysis of the rapid intraoperative parathyroid hormone assay in patients during operation for hyperparathyroidism.

BACKGROUND: Rapid intraoperative parathyroid hormone (RI-PTH) assay is used to guide adequacy of resection during operation for hyperparathyroidism. We compared the RI-PTH assay (15 minutes) with a standard PTH assay, determined whether the PTH half-life varied between patients, and constructed a kinetic analysis of the RI-PTH data. METHODS: Forty-five patients with hyperparathyroidism had blood sampled at baseline and at times after parathyroid resection. Intact PTH was determined using RI-PTH and a standard assay. Values were fitted to an exponential decay curve using the baseline and the follow-up time points. PTH half-life and the new postexcision baseline value were calculated from the decay curve. RESULTS: The RI-PTH assay and the standard PTH assay correlated well. Average PTH half-life was 1.68 +/- 0.94 minutes (0.42 to 3.81 minutes). A kinetic analysis yielded a formula for the generation of a PTH decay curve. Using a 50% reduction in RI-PTH at 5 minutes as the criterion for adequate resection, 2 patients were incorrectly classified as not being cured. These patients were correctly classified using the kinetic analysis. CONCLUSIONS: PTH half-life can vary substantially. A kinetic analysis may be more accurate in assessing adequacy of resection. This method allows the surgeon to interpret RI-PTH data independent of the timing of samples.

Limitations on the quantitative determination of telomerase activity by the electrophoretic and ELISA based TRAP assays.

Telomerase is a promising new tumor marker and can be detected using the TRAP (Telomeric Repeat Amplification Protocol) method. To address factors affecting its quantitative determination, we evaluated two commercial TRAP assays, an electrophoretic and an ELISA assay formats, using cultured cells and human tumor samples. We found that both TRAP assays had a limited linearity from 250 to 5000 tumor cells, with a similar intra-assay variation. The quantification of TRAP products was affected by high cell number in sample, the presence of non-tumor cells, and interfering substances in patient specimens. Because both assays have different limitations, determination of telomerase by a combined use of the two may provide more accurate information on the telomerase activity in a specimen. Extracts of specimens should also be tested at several concentrations to insure that the result is not being falsely decreased by an inhibitor. The quantitative results for telomerase activity by the TRAP assays, however, should be interpreted cautiously.

Dual HDL/total cholesterol test: a single-tube, homogeneous assay for sequential measurement of HDL cholesterol and total cholesterol.

The quantitation of cholesterol in lipoprotein subfractions is valuable in estimating the risk for coronary artery disease, but requires multiple tests. We describe a relatively simple procedure, referred to as the dual HDL/total cholesterol (DHT) assay, which allows the sequential measurement of HDL cholesterol (HDL-C) and total cholesterol (total-C) in a single tube. HDL-C is first measured using a homogeneous assay that utilizes an anti-apolipoprotein B [apo(B)] antibody, which sterically blocks the enzymatic measurement of cholesterol on the non-HDL subfractions. Next, deoxycholate is added, which disrupts the antibody-apo(B) complex and allows the subsequent enzymatic measurement of the remaining cholesterol in the non-HDL subfractions. The DHT assay has an acceptable analytical performance and yields results similar to standard methods: for HDL-C, y(DHT) = 0.98x + 0.19, r=0.90; for total-C, y(DHT) = 1.11x - 0.09, r=0.99. In summary, the DHT assay is a homogeneous assay for both HDL-C and total-C, and provides a simple and cost-effective method for screening for hyperlipidaemia.

Measurement of low-density lipoprotein cholesterol in assessment and management of cardiovascular disease risk.

The deposition of cholesterol in the arterial wall by the infiltration of low-density lipoproteins (LDLs) is a key step in the development of atherosclerosis. In this Commentary, we discuss recent recommendations for clinical laboratory measurement of low-density lipoprotein cholesterol (LDL-C) and its utility both for assessing cardiovascular disease risk and as a tool in the management of patients receiving lipid-lowering therapy.

HDL therapy: two kinds of right?

Cardiovascular disease remains the leading cause of morbidity and mortality globally. The disease is largely controlled with interventions managing atherogenic lipids including LDL and triglycerides. However a number of studies have shown that increasing HDL levels is likely to provide better outcomes for patients suffering from this disease. There has been an extensive research effort into understanding how HDL levels are regulated in the body and which pathways can be targeted therapeutically. The HDL metabolic pathway is however overwhelmingly complex. This has provided only limited success in trialing drugs designed to raise HDL. To add to the complexity HDL itself is a heterogeneous population of particles and there is controversy surrounding which HDL particle is the most cardio-protective. In addition there is varying opinions on which of the HDL cellular receptors are more important in humans (as opposed to what has been discovered in mice) in regulating these effects. In this article we explore the evidence for and against using the currently suggested methods of raising HDL and provide some evidence for how the adverse effects of these drugs could be corrected.