Life Extension Magazine®

Issue: Feb 2009


High omega-3 fat intake improves insulin sensitivity and reduces CRP and IL6, but does not affect other endocrine axes in healthy older adults.

Aging diminishes hormone secretion and target cell responsiveness, possibly due to loss of cell membrane fluidity or alteration of membrane phospholipids affecting signal transduction. We investigated whether a high omega-3 polyunsaturated fatty acid diet would improve endocrine function in 6 men and 6 women aged over 60 years. Subjects first ate an isocaloric control diet for 6 weeks, followed by an 8-week experimental diet, which included 720 g of fatty fish weekly plus 15 ml of sardine oil daily. In the last week, we measured RBC membrane fatty acids on each diet, performed pituitary, adrenal, hepatic, and Leydig cell endocrine provocative testing, and assayed selected cytokines. We also assessed insulin sensitivity utilizing octreotide insulin suppression testing and assessed free fatty acid (FFA) responses to isoproteronol. Insulin sensitivity increased significantly after 8 weeks on the omega-3 diet and FFA responses trended lower. Serum C-reactive protein was significantly reduced and a trend towards lower IL-6 was noted. No differences were found in other metabolic parameters, adiponectin levels, or hormone responses. We conclude that, in older people, high omega-3 consumption increases insulin sensitivity, may reduce FFA mobilization by catecholamines, and reduces inflammatory markers, but did not alter endocrine responsiveness after 8 weeks.

Horm Metab Res. 2008 Mar;40(3):199-205

Chemokines in cardiovascular risk prediction.

In consideration of the important role of inflammation in plaque progression and stability, recent work has focused on whether plasma markers of inflammation can non-invasively diagnose and predict coronary artery disease (CAD) and other forms of atherosclerotic disorders. Although several studies support an important pathogenic role of chemokines in atherogenesis and plaque destabilization, potentially representing attractive therapeutic targets in atherosclerotic disorders, this does not necessarily mean that chemokines are suitable parameters for risk prediction. In fact, the ability to reflect up-stream inflammatory activity, stable levels in individuals and high stability of the actual protein (e.g. long half-life and negligible circadian variation), are additional important criteria for an ideal biomarker in cardiovascular disease. Although plasma/serum levels of certain chemokines (e.g. interleukin 8 and monocyte chemoattractant protein-1) have been shown to be predictive for future cardiac events in some studies, independent of traditional cardiovascular risk factors and C-reactive protein, and although certain gene polymorphisms of chemokines/chemokine receptors (e.g. fractalkine receptor) have been shown to be predictive of future atherosclerotic disease, further prospective studies, including a larger number patients, are needed to make any firm conclusion. While the demonstrations of an association between chemokines and CAD are a necessary first step, such studies do not establish the full clinical utility of a biomarker, which is a more demanding process that requires validation in multiple cohorts, and clear demonstration of incremental prognostic value over traditional risk models. If successful, such new biomarker will be a useful indicator for better risk assessment, diagnosis, and prognosis, as well as monitoring pharmacological treatments for atherosclerosis.

Thromb Haemost. 2007 May;97(5):748-54

Characteristics of catechin- and theaflavin-mediated cardioprotection.

Catechins and theaflavins-the main polyphenolic substances of green and black tea, respectively-exert a plethora of beneficial effects on the cardiovascular system. In a model of H(2)O(2)-mediated oxidative stress, we investigated the effects of epigallocatechin-3-gallate (EGCG) and theaflavin-3,3’-digallate (TF3) on neonatal rat cardiomyocytes. Pretreatment with EGCG or TF3 1 hr prior to induction of oxidative stress by H(2)O(2) effectively protected cardiac myocytes as determined by measuring release of lactate dehydrogenase after 24 hrs. Longer pre-incubation times resulted in significant loss of protection. To enable further mechanistic insight, we investigated expression of antioxidative enzymes and activation of prosurvival signaling cascades. Whereas mRNA levels of glutathione peroxidase 3, superoxide dismutase 1, and catalase were not influenced by both polyphenols, heme oxygenase (HO-1) was selectively upregulated by EGCG-but not by TF3. However, inhibition of HO-1 did not diminish polyphenol-mediated cardioprotection. While EGCG and TF3 activated Akt, extracellular signal-regulated kinase 1/2, and p38 mitogen-activated protein kinase, inhibition of these kinases did not attenuate polyphenol-mediated protection. Loading of cardiomyocytes with dichlorofluorescein revealed that intracellular levels of reactive oxygen species were significantly reduced after treatment with EGCG or TF3 as early as 30 mins after induction of oxidative stress. In conclusion, activation of prosurvival signaling kinases and upregulation of antioxidative enzymes do not play a major role in tea polyphenol-mediated cardioprotection.

Exp Biol Med (Maywood). 2008 Apr;233(4):427-33

The efficacy of black tea in ameliorating endothelial function is equivalent to that of green tea.

Consumption of tea has been shown to improve endothelial function. It is assumed that catechins are the tea components responsible for these beneficial effects. In black tea, catechin concentrations are significantly lower than in green tea. The present study was designed to compare green and black tea with regard to amelioration of endothelial function. Endothelial function in response to both teas was assessed in bovine aortic endothelial cells (BAEC) and rat aortic rings. To elucidate whether these findings are also applicable to humans, flow-mediated dilation (FMD) and nitro-mediated dilation (NMD) were assessed by ultrasound in twenty-one healthy women before and 2 h after consumption of green and black tea (2 h of FMD and NMD), in comparison with water (control). In BAEC, green and black tea significantly increased endothelial NO synthase activity to the same extent. Similarly, both teas induced comparable endothelial-dependent vasodilation in rat aortic rings. In human subjects, ingestion of green and black tea led to significant increases in FMD: from 5.4 (sd 2.3) to 10.2 (sd 3) % (baseline-adjusted difference (BAD) for 2 h of FMD, green tea v. water: 5.0 (95 % CI 3.0, 7.0) %; P < 0.001) and from 5 (sd 2.6) to 9.1 (sd 3.6) % (BAD for 2 h of FMD, black tea v. water: 4.4 (95 % CI 2.3, 6.5) %; P < 0.001), respectively. The increase in FMD was not significantly different between the two tea preparations (BAD for 2 h of FMD, green tea v. black tea: 0.66 (95 % CI - 0.76, 2.09) %; P = 0.36). NMD did not vary between any of the groups. In conclusion, green and black tea are equally effective in improving endothelial function.

Br J Nutr. 2008 Apr;99(4):863-8

Rosuvastatin to prevent vascular events in men and women with elevated C-reactive protein.

BACKGROUND: Increased levels of the inflammatory biomarker high-sensitivity C-reactive protein predict cardiovascular events. Since statins lower levels of high-sensitivity C-reactive protein as well as cholesterol, we hypothesized that people with elevated high-sensitivity C-reactive protein levels but without hyperlipidemia might benefit from statin treatment. METHODS: We randomly assigned 17,802 apparently healthy men and women with low-density lipoprotein (LDL) cholesterol levels of less than 130 mg per deciliter (3.4 mmol per liter) and high-sensitivity C-reactive protein levels of 2.0 mg per liter or higher to rosuvastatin, 20 mg daily, or placebo and followed them for the occurrence of the combined primary end point of myocardial infarction, stroke, arterial revascularization, hospitalization for unstable angina, or death from cardiovascular causes. RESULTS: The trial was stopped after a median follow-up of 1.9 years (maximum, 5.0). Rosuvastatin reduced LDL cholesterol levels by 50% and high-sensitivity C-reactive protein levels by 37%. The rates of the primary end point were 0.77 and 1.36 per 100 person-years of follow-up in the rosuvastatin and placebo groups, respectively (hazard ratio for rosuvastatin, 0.56; 95% confidence interval [CI], 0.46 to 0.69; P<0.00001), with corresponding rates of 0.17 and 0.37 for myocardial infarction (hazard ratio, 0.46; 95% CI, 0.30 to 0.70; P=0.0002), 0.18 and 0.34 for stroke (hazard ratio, 0.52; 95% CI, 0.34 to 0.79; P=0.002), 0.41 and 0.77 for revascularization or unstable angina (hazard ratio, 0.53; 95% CI, 0.40 to 0.70; P<0.00001), 0.45 and 0.85 for the combined end point of myocardial infarction, stroke, or death from cardiovascular causes (hazard ratio, 0.53; 95% CI, 0.40 to 0.69; P<0.00001), and 1.00 and 1.25 for death from any cause (hazard ratio, 0.80; 95% CI, 0.67 to 0.97; P=0.02). Consistent effects were observed in all subgroups evaluated. The rosuvastatin group did not have a significant increase in myopathy or cancer but did have a higher incidence of physician-reported diabetes. CONCLUSIONS: In this trial of apparently healthy persons without hyperlipidemia but with elevated high-sensitivity C-reactive protein levels, rosuvastatin significantly reduced the incidence of major cardiovascular events.

N Engl J Med. 2008 Nov 20;359(21):2195-207

Inflammation is a crucial feature of atherosclerosis and a potential target to reduce cardiovascular events.

Contrary to popular opinion, atherosclerosis is not a disease unique to modern civilization. In fact, atherosclerotic lesions have been found in the arteries of mummies dating back to 1,500 B.C., and yet our understanding of this complex process is still evolving. A fusion of basic science advances and clinical research findings has radically altered our traditional concepts about the pathogenesis and treatment of the clinical complications of atherosclerosis. Most physicians previously regarded the artery as a being merely a blood conduit that became encrusted with lipid detritus as part of the aging process. Modern-day treatment of atherosclerosis has arisen primarily from an understanding of the epidemiology of the disease rather than its pathophysiology, in that risk factors have traditionally been targeted. Our concepts of atherogenesis have evolved from vague ideas of inevitable degeneration to a much better defined scenario of molecular and cellular events. As we enhance our understanding of its fundamental mechanism, we can begin to approach atherogenesis as a modifiable rather than ineluctable process. Indeed, as we recognize now that inflammation plays a pivotal role in the process of atherosclerosis, it is noteworthy to evaluate the effect of modern therapies on this facet of the disease.

Handb Exp Pharmacol. 2005;(170):697-722

Inflammaging as a major characteristic of old people: can it be prevented or cured?

Widespread aging at the population level is a recent phenomenon that emerged in affluent societies. Inflammation is necessary to cope with damaging agents and is crucial for survival, particularly to cope with acute inflammation during our reproductive years. But chronic exposure to a variety of antigens, especially to some viruses such as cytomegalovirus, for a period much longer than that predicted by evolution, induces a chronic low-grade inflammatory status that contributes to age-associated morbidity and mortality. This condition carries the proposed name “inflammaging.” Centenarians are unique in that, despite high levels of pro-inflammatory markers, they also exhibit anti-inflammatory markers that may delay disease onset. The key to successful aging and longevity is to decrease chronic inflammation without compromising an acute response when exposed to pathogens.

Nutr Rev. 2007 Dec;65(12 Pt 2):S173-6

Achieving and maintaining cognitive vitality with aging.

Cognitive vitality is essential to quality of life and survival in old age. With normal aging, cognitive changes such as slowed speed of processing are common, but there is substantial interindividual variability, and cognitive decline is clearly not inevitable. In this review, we focus on recent research investigating the association of various lifestyle factors and medical comorbidities with cognitive aging. Most of these factors are potentially modifiable or manageable, and some are protective. For example, animal and human studies suggest that lifelong learning, mental and physical exercise, continuing social engagement, stress reduction, and proper nutrition may be important factors in promoting cognitive vitality in aging. Manageable medical comorbidities, such as diabetes, hypertension, and hyperlipidemia, also contribute to cognitive decline in older persons. Other comorbidities such as smoking and excess alcohol intake may contribute to cognitive decline, and avoiding these activities may promote cognitive vitality in aging. Various therapeutics, including cognitive enhancers and protective agents such as antioxidants and anti-inflammatories, may eventually prove useful as adjuncts for the prevention and treatment of cognitive decline with aging. The data presented in this review should interest physicians who provide preventive care management to middle-aged and older individuals who seek to maintain cognitive vitality with aging.

Mayo Clin Proc. 2002 Jul;77(7):681-96

Immunoproteasomes and immunosenescence.

Aging is a complex process which is accompanied with the decline and the reshaping of different functions of the body. In particular the immune system is characterized, during ageing (immunosenescence) by a remodeling of innate immunity (well preserved, up-regulated) and clonotypical immunity (severely altered) and by the occurrence of a chronic inflammatory process (inflammaging) which are, at least in part, genetically controlled. In this scenario, it can be anticipated that a crucial role is played by age-related structural and functional alterations and modifications of proteasomes and immunoproteasomes, the last being a key component of antigen processing and MHC class I antigen presentation. A variety of experimental data are available, suggesting that proteasomes are affected by age, and that in centenarians they are relatively preserved. On the contrary, few data are available on immunoproteasomes, likely as a consequence of the poverty of suitable cellular models. Lymphoblastoid cell lines from EBV immortalized B cells from old donors is envisaged as a possible model for the study of immunoproteasomes in humans and their changes with age. Thus, basic questions such as those related to possible consequences, for immune responses in infectious diseases and cancer, of age-related alterations of antigen processing and presenting, change with age of self-antigen repertoire, and the genetic basis of immunoproteasome activity and its change with age, remain largely unanswered.

Ageing Res Rev. 2003 Oct;2(4):419-32

Is inflammaging an auto[innate]immunity subclinical syndrome?

The low-grade, chronic, systemic inflammatory state that characterizes the aging process (inflammaging) results from late evolutive-based expression of the innate immune system. Inflammaging is characterized by the complex set of five conditions which can be described as 1. low-grade, 2. controlled, 3. asymptomatic, 4. chronic, 5. systemic, inflammatory state, and fits with the antagonistic pleiotropy theory on the evolution of aging postulating that senescence is the late deleterious effect of genes (pro-inflammatory versus anti-inflammatory) that are beneficial in early life. Evolutionary programming of the innate immune system may act via selection on these genetic traits. Here I propose that the already acquired knowledge in this field may pave the way to a new chapter in the pathophysiology of autoimmunity: the auto-innate-immunity syndromes. Indeed, differently from the well known chapter of conventional autoimmune diseases and syndromes where the main actor is the adaptive immunity, inflammaging may constitute the subclinical paradigm of a new chapter of autoimmunity, namely that arising from an autoimmune inflammatory response of the innate-immune-system, an old actor of immunity and yet a new actor of autoimmunity, also acting as a major determinant of elderly frailty and age-associated diseases.

Immun Ageing. 2006 Dec 16;3:12

Role of environmental and genetic factor interaction in age-related disease development: the gastric cancer paradigm.

The association of Helicobacter pylori (Hp) infection with gastric cancer is well known and might be considered a paradigmatic example of the role that interaction among environmental factors and individual background might play in inducing age-associated disease. To evaluate the role of interaction of Hp infection with genetic background, gastric cancer and chronic gastritis patients as well as random selected controls were typed for five inflammation-related polymorphisms of IL-1 and IL-10 cytokine genes. No association among IL-10 or IL-1 variants with an increased risk of gastric cancer was found, whereas an Hp-independent association of IL-1beta -511T positive genotypes to an increased risk of chronic gastritis was found (Hp-/511T+ OR 1.89, 95% CI: 1.01-3.54; Hp+/-511T+ OR 1.83, 95% CI: 1.05-3.19). Stratification of gastric cancer group according to Hp infection does not allow finding a statistically significant association of Hp+ to the higher histological grading (G3) of gastric cancer (OR 1.54, 95% CI: 0.46-5.11). Our findings seem to confirm that cytokine genetic variants might contribute to determining the background for inflammaging in which H. pylori infection might facilitate cancer development.

Rejuvenation Res. 2008 Apr;11(2):509-12

The effect of age and telomere length on immune function in the horse.

Telomeres, specialized structures present at the ends of linear eukaryotic chromosomes, function to maintain chromosome stability and integrity. Telomeres shorten with each cell division eventually leading to replicative senescence, a process thought to be associated with age-related decline in immune function. We hypothesized that shortened PBMC telomere length is a factor contributing to immunosenescence of the aged horse. Telomere length was assessed in 19 horses ranging in age from 1 to 25 years. Mitogen-induced 3H-thymidine incorporation, total serum IgG, and pro-inflammatory cytokine expression was also determined for each horse. Relative telomere length (RTL) was highly correlated with overall age. RTL was positively correlated with 3H-thymidine incorporation and total IgG. Expression of pro-inflammatory cytokines was negatively correlated with RTL. These measures were also correlated with age, as expected. However, RTL was not correlated with immunosenescence and inflammaging in the oldest horse.

Dev Comp Immunol. 2008;32(12):1409-15

Relative safety profiles of high dose statin regimens.

Recent clinical trials recommend achieving a low-density lipoprotein cholesterol level of <100 mg/dl in high-risk and <70 mg/dl in very high risk patients. To attain these goals, however, many patients will need statins at high doses. The most frequent side effects related to the use of statins, myopathy, rhabdomyolysis, and increased levels of transaminases, are unusual. Although low and moderate doses show a favourable profile, there is concern about the tolerability of higher doses. During recent years, numerous trials to analyze the efficacy and tolerability of high doses of statins have been published. This paper updates the published data on the safety of statins at high doses.

Vasc Health Risk Manag. 2008;4(3):525-33

Effect of Chyawanprash and vitamin C on glucose tolerance and lipoprotein profile.

Chyawanprash is an ancient Indian dietary supplement containing vitamin C (34 mg/100 g) derived from amla (Emblica officinalis). In addition, Chyawanprash also contains several other herbal products. The present study was designed to compare the effects of vitamin C with those of Chyawanprash. Ten normal healthy adult male volunteers (age 20-32 years) participated in the 16-week study. They were placed randomly in either the Chyawanprash group (n = 5) or vitamin C group (n = 5). Those in the former received 15 g/d of Chyawanprash while those in the latter received 500 mg/d vitamin C during the first 8 weeks of the study. For the next 8 weeks, no supplement was given. For each individual, an oral glucose tolerance test was performed, and lipoprotein profile in peripheral serum samples was determined at 0 weeks, 4 weeks, 8 weeks, 12 weeks and 16 weeks. In the Chyawanprash group, the 8 weeks Vs 0 weeks value (mean +/- S.D.) respectively for various indices which were significantly different were fasting plasma glucose (100.2 +/- 5.58 mg/dl vs 116.2 +/- 11.6 mg/dl), area under 2-h plasma glucose curve (245.9 +/- 15.13 mg.dl-1.h vs 280.8 +/- 37.09 mg.dl-1.h), HDL cholesterol (53.2 +/- 4.56 mg/dl vs 42.7 +/- 7.17 mg/dl), LDL cholesterol (82.4 +/- 8.80 mg/dl vs 98.26 +/- 12.07 mg/dl), LDL/HDL ratio (1.56 +/- 0.28 vs 2.38 +/- 0.63). In the Vitamin C group, only the LDL/HDL ratio was significantly lower at 8 weeks than at 0 weeks (1.99 +/- 0.44 vs 2.29 +/- 0.43). All the variables that changed significantly were no longer significantly different from the 0 weeks value at 16 weeks. Chyawanprash reduces postprandial glycemia in the oral glucose tolerance test and reduces blood cholesterol level to a significantly greater extent than vitamin C.

Indian J Physiol Pharmacol. 2001 Jan;45(1):71-9

Mechanisms of cancer prevention by green and black tea polyphenols.

Drinking green tea is associated with decreased frequency of cancer development. This review outlines the wide range of mechanisms by which epigallocatechin gallate (ECGC) and other green and black tea polyphenols inhibit cancer cell survival. EGCG suppressed androgen receptor expression and signalling via several growth factor receptors. Cell cycle arrest or apoptosis involved caspase activation and altered Bcl-2 family member expression. EGCG inhibited telomerase activity and led to telomere fragmentation. While at high concentrations polyphenols had pro-oxidative activities, at much lower levels, anti-oxidative effects occurred. Nitric oxide production was reduced by EGCG and black tea theaflavins by suppressing inducible nitric oxide synthase via blocking nuclear translocation of the transcription factor nuclear factor-kappaB as a result of decreased IkappaB kinase activity. Polyphenols up- or down-regulated activity of a number of key enzymes, including mitogen-activated protein kinases and protein kinase C, and increased or decreased protein/mRNA levels, including that of cyclins, oncogenes, and tumor suppressor genes. Metastasis was inhibited via effects on urokinase and matrix metalloproteinases. Polyphenols reduced angiogenesis, in part by decreasing vascular endothelial growth factor production and receptor phosphorylation. Recent work demonstrated that EGCG reduced dihydrofolate reductase activity, which would affect nucleic acid and protein synthesis. It also acted as an aryl hydrocarbon receptor an-tagonist by directly binding the receptor’s molecular chaperone, heat shock protein 90. In conclusion, green and black tea polyphenols act at numerous points regulating cancer cell growth, survival, and metastasis, including effects at the DNA, RNA, and protein levels.

Anticancer Agents Med Chem. 2006 Sep;6(5):389-406

Pharmacokinetics and systemic endocrine effects of the phyto-oestrogen 8-prenylnaringenin after single oral doses to postmenopausal women.

AIMS: Pre-clinical data suggest that the racemic phyto-oestrogen 8-prenylnaringenin (8-PN) may have beneficial effects in postmenopausal women and may become an alternative to classical hormone replacement therapy (HRT) treatment regimes. The aim of this study was to investigate the pharmacokinetics, endocrine effects and tolerability of chemically synthesized 8-PN in postmenopausal women. METHODS: The study was performed using a randomized, double-blind, placebo-controlled, dose-escalation design with three groups of eight healthy postmenopausal women. In each group six subjects received 8-PN and two subjects placebo. 8-PN was given orally in doses of 50, 250 or 750 mg. Drug concentrations in serum, urine and faeces were measured up to 48 h and follicle-stimulating hormone/luteinizing hormone (LH) concentrations up to 24 h. RESULTS: All treatments were well tolerated and associated with a low incidence of (drug unrelated) adverse events. Serum concentrations of free 8-PN showed rapid drug absorption and secondary peaks suggestive of marked enterohepatic recirculation. Independent of the treatment group, approximately 30% of the dose was recovered in excreta as free compound or conjugates over the 48-h observation period. The first C(max) and AUC(0-48 h) showed dose linearity with ratios of 1 : 4.5 : 13.6 (C(max)) and 1 : 5.2 : 17.1 (AUC). The750- mg dose decreased LH concentrations by 16.7% (95% confidence interval 0.5, 30.2). CONCLUSION: Single oral doses of up to 750 mg 8-PN were well tolerated by postmenopausal women. The pharmacokinetic profile of 8-PN was characterized by rapid and probably complete enteral absorption, high metabolic stability, pronounced enterohepatic recirculation and tight dose linearity. The decrease in LH serum concentrations found after the highest dose demonstrates the ability of 8-PN to exert systemic endocrine effects in postmenopausal women.

Br J Clin Pharmacol. 2006 Sep;62(3):288-96

Estrogenic activity of 7-hydroxymatairesinol potassium acetate (HMR/lignan) from Norway spruce (Picea abies) knots and of its active metabolite enterolactone in MCF-7 cells.

Lignans are plant polyphenols which may possess anticancer, antioxidant, antimicrobial, anti-inflammatory and immunomodulatory activities. In particular, the lignan 7-hydroxymatairesinol (HMR/lignan, HMR) is a novel precursor of the mammalian lignan enterolactone (EL). In the present study, we investigated the estrogenicity of HMR and of EL in comparison to estradiol (E2), by measuring their effects on growth and apoptotic markers in the human estrogen-sensitive cell line MCF-7. HMR, EL and E2 concentration-dependently increased the percentage of MCF-7 cells in the S phase of the cell cycle, with the following relative potencies: E2 congruent with EL>>HMR, and efficacies: E2>HMR>>EL. Treatment of MCF-7 cells with either HMR, EL or E2 also increased the Bcl-2/Bax mRNA ratio. The effects of HMR and EL were reduced in the presence of the estrogen receptor (ER) antagonist tamoxifene. We conclude that both HMR and its metabolite EL are endowed with estrogenic activity, which is likely to be exerted through the contribution of ER-dependent pathways and to target the same intracellular mechanisms acted upon by E2. The estrogenicity of HMR and EL is however milder than that of E2, as indicated by the lower potencies and efficacies of both lignans. The present results support the notion that dietary supplementation with HMR may result in a mild estrogenic activity, both directly and by providing a suitable source for endogenous EL.

Pharmacol Res. 2007 Aug;56(2):140-7

Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results From theWomen’s Health Initiative randomized controlled trial.

CONTEXT: Despite decades of accumulated observational evidence, the balance of risks and benefits for hormone use in healthy postmenopausal women remains uncertain. OBJECTIVE: To assess the major health benefits and risks of the most commonly used combined hormone preparation in the United States. DESIGN: Estrogen plus progestin component of the Women’s Health Initiative, a randomized controlled primary prevention trial (planned duration, 8.5 years) in which 16608 postmenopausal women aged 50-79 years with an intact uterus at baseline were recruited by 40 US clinical centers in 1993-1998. INTERVENTIONS: Participants received conjugated equine estrogens, 0.625 mg/d, plus medroxyprogesterone acetate, 2.5 mg/d, in 1 tablet (n = 8506) or placebo (n = 8102). MAIN OUTCOMES MEASURES: The primary outcome was coronary heart disease (CHD) (nonfatal myocardial infarction and CHD death), with invasive breast cancer as the primary adverse outcome. A global index summarizing the balance of risks and benefits included the 2 primary outcomes plus stroke, pulmonary embolism (PE), endometrial cancer, colorectal cancer, hip fracture, and death due to other causes. RESULTS: On May 31, 2002, after a mean of 5.2 years of follow-up, the data and safety monitoring board recommended stopping the trial of estrogen plus progestin vs placebo because the test statistic for invasive breast cancer exceeded the stopping boundary for this adverse effect and the global index statistic supported risks exceeding benefits. This report includes data on the major clinical outcomes through April 30, 2002. Estimated hazard ratios (HRs) (nominal 95% confidence intervals [CIs]) were as follows: CHD, 1.29 (1.02-1.63) with 286 cases; breast cancer, 1.26 (1.00-1.59) with 290 cases; stroke, 1.41 (1.07-1.85) with 212 cases; PE, 2.13 (1.39-3.25) with 101 cases; colorectal cancer, 0.63 (0.43-0.92) with 112 cases; endometrial cancer, 0.83 (0.47-1.47) with 47 cases; hip fracture, 0.66 (0.45-0.98) with 106 cases; and death due to other causes, 0.92 (0.74-1.14) with 331 cases. Corresponding HRs (nominal 95% CIs) for composite outcomes were 1.22 (1.09-1.36) for total cardiovascular disease (arterial and venous disease), 1.03 (0.90-1.17) for total cancer, 0.76 (0.69-0.85) for combined fractures, 0.98 (0.82-1.18) for total mortality, and 1.15 (1.03-1.28) for the global index. Absolute excess risks per 10 000 person-years attributable to estrogen plus progestin were 7 more CHD events, 8 more strokes, 8 more PEs, and 8 more invasive breast cancers, while absolute risk reductions per 10 000 person-years were 6 fewer colorectal cancers and 5 fewer hip fractures. The absolute excess risk of events included in the global index was 19 per 10,000 person-years. CONCLUSIONS: Overall health risks exceeded benefits from use of combined estrogen plus progestin for an average 5.2-year follow-up among healthy postmenopausal US women. All-cause mortality was not affected during the trial. The risk-benefit profile found in this trial is not consistent with the requirements for a viable intervention for primary prevention of chronic diseases, and the results indicate that this regimen should not be initiated or continued for primary prevention of CHD.

JAMA. 2002 Jul 17;288(3):321-33

Prenylflavonoids: a new class of non-steroidal phytoestrogen (Part 1). Isolation of 8-isopentenylnaringenin and an initial study on its structure-activity relationship.

Bioassay-guided fractionation of a methanolic extract of a Thai crude drug, derived from heartwood of Anaxagorea luzonensis A. Gray (Annonaceae), resulted in the isolation of 8-isopentenylnaringenin (1) as an estrogen agonist with a activity of about an order of magnitude greater than genistein. Various flavonoids possessing isopentenyl side chains in the A-ring have been prepared and evaluated for their ability to bind estrogen receptor. In addition, enantiomers of 1 were separated and the respective enantiomers were assayed. These studies have demonstrated that the presence of an 8-isopentenyl group is an important factor for binding. Flavones, flavanones and flavonols having an isopentenyl substituent at C-8 exhibited an appreciable affinity for estrogen receptor. Conversely, isoflavones possessing an 8-isopentenyl substituent at C-8 did not show this activity. Movement of the isopentenyl group from position 8 to 6 resulted in loss of the activity. No significant difference was observed between 2(S)- and 2(R)-enantiomers of 1 in their binding affinity. Prenylflavonoids are reported to possess a wide range of biological activities; however, estrogenic activity has not been described.

Planta Med. 1998 Aug;64(6):511-5

Leptin signaling in the hypothalamus: emphasis on energy homeostasis and leptin resistance.

Leptin, the long-sought satiety factor of adipocytes origin, has emerged as one of the major signals that relay the status of fat stores to the hypothalamus and plays a significant role in energy homeostasis. Understanding the mechanisms of leptin signaling in the hypothalamus during normal and pathological conditions, such as obesity, has been the subject of intensive research during the last decade. It is now established that leptin action in the hypothalamus in regulation of food intake and body weight is mediated by a neural circuitry comprising of orexigenic and anorectic signals, including NPY, MCH, galanin, orexin, GALP, alpha-MSH, NT, and CRH. In addition to the conventional JAK2-STAT3 pathway, it has become evident that PI3K-PDE3B-cAMP pathway plays a critical role in leptin signaling in the hypothalamus. It is now established that central leptin resistance contributes to the development of diet-induced obesity and ageing associated obesity. Central leptin resistance also occurs due to hyperleptinimia produced by exogenous leptin infusion. A defective nutritional regulation of leptin receptor gene expression and reduced STAT3 signaling may be involved in the development of leptin resistance in DIO. However, leptin resistance in the hypothalamic neurons may occur despite an intact JAK2-STAT3 pathway of leptin signaling. Thus, in addition to defective JAK2-STAT3 pathway, defects in other leptin signaling pathways may be involved in leptin resistance. We hypothesize that defective regulation of PI3K-PDE3B-cAMP pathway may be one of the mechanisms behind the development of central leptin resistance seen in obesity.

Front Neuroendocrinol. 2003 Dec;24(4):225-53

Fat storage in adipocytes requires inactivation of leptin’s paracrine activity: implications for treatment of human obesity.

Hyperleptinemia rapidly depletes adipocyte fat in lean rats, whereas comparable hyperleptinemia produced by adipocytes in diet-induced obesity does not, implying a leptinergic blockade in adipocytes during overnutrition. Indeed, activated STAT-3 in white adipose tissue (WAT) of normal rats was less on a 60% high fat diet (HFD) than on 4% fat, despite a 10-fold higher plasma leptin. In 6 days of a HFD, mRNA of the postreceptor leptin inhibitor, suppressor of cytokine signaling-3, increased 22-fold in WAT, while leptin receptor (Lepr-b) mRNA gradually disappeared, implying leptinergic blockade at both postreceptor and receptor levels. Adipocyte-specific Lepr-b overexpression of a Lepr-b transgene completely prevented the adipocyte hypertrophy and hyperplasia and the increase in body fat induced in wild-type mice by HFD. Activated STAT-3 and AMP-activated protein kinase (AMPK), and the mRNA of lipooxidative enzymes, peroxisome proliferator-activated receptor-gamma-coactivator-1alpha, and uncoupling protein-1 and -2 were increased in WAT. Body temperature was elevated in the transgenic mice, suggesting uncoupled fatty acid oxidation of surplus fatty acids. In conclusion, storage of surplus calories in WAT and the development of diet-induced obesity require the blockade of a latent leptin-stimulated caloric sump in white adipocytes.

Proc Natl Acad Sci U S A. 2005 Dec 13;102(50):18011-6.

Weight-reducing effects of the plasma protein encoded by the obese gene.

The gene product of the ob locus is important in the regulation of body weight. The ob product was shown to be present as a 16-kilodalton protein in mouse and human plasma but was undetectable in plasma from C57BL/6J ob/ob mice. Plasma levels of this protein were increased in diabetic (db) mice, a mutant thought to be resistant to the effects of ob. Daily intraperitoneal injections of either mouse or human recombinant OB protein reduced the body weight of ob/ob mice by 30% after 2 weeks of treatment with no apparent toxicity but had no effect on db/db mice. The protein reduced food intake and increased energy expenditure in ob/ob mice. Injections of wild-type mice twice daily with the mouse protein resulted in a sustained 12% weight loss, decreased food intake, and a reduction of body fat from 12.2 to 0.7%. These data suggest that the OB protein serves an endocrine function to regulate body fat stores.

Science. 1995 Jul 28;269(5223):543-6

Serum immunoreactive-leptin concentrations in normal-weight and obese humans.

BACKGROUND. Leptin, the product of the ob gene, is a hormone secreted by adipocytes. Animals with mutations in the ob gene are obese and lose weight when given leptin, but little is known about the physiologic actions of leptin in humans. METHODS. Using a newly developed radioimmunoassay, wer measured serum concentrations of leptin in 136 normal-weight subjects and 139 obese subjects (body-mass index, > or = 27.3 for men and > or = 27.8 for women; the body-mass index was defined as the weight in kilograms divided by the square of the height in meters). The measurements were repeated in seven obese subjects after weight loss and during maintenance of the lower weight. The ob messenger RNA (mRNA) content of adipocytes was determined in 27 normal-weight and 27 obese subjects. RESULTS. The mean (+/- SD) serum leptin concentrations were 31.3 +/- 24.1 ng per milliliter in the obese subjects and 7.5 +/- 9.3 ng per milliliter in the normal-weight subjects (P < 0.001). There was a strong positive correlation between serum leptin concentrations and the percentage of body fat (r = 0.85, P < 0.001). The ob mRNA content of adipocytes was about twice as high in the obese subjects as in the normal-weight subjects (P < 0.001) and was correlated with the percentage of body fat (r = 0.68, P < 0.001) in the 54 subjects in whom it was measured. In the seven obese subjects studied after weight loss, both serum leptin concentrations and ob mRNA content of adipocytes declined, but these measures increased again during the maintenance of the lower weight. CONCLUSIONS. Serum leptin concentrations are correlated with the percentage of body fat, suggesting that most obese persons are insensitive to endogenous leptin production.

N Engl J Med. 1996 Feb 1;334(5):292-5

Metabolic syndrome and stroke.

The metabolic syndrome is highly prevalent worldwide, and its cardiovascular toll is expected to rise with the growing obesity epidemic. Mounting evidence points to an association between metabolic syndrome and first or recurrent stroke. This article discusses the emerging data supporting a link between stroke and the metabolic syndrome and underscores the need to better understand the syndrome’s pathophysiology, with a goal to appropriately and intensively limit the burden of this multiple risk factor entity.

Curr Diab Rep. 2008 Feb;8(1):37-41

Central leptin gene therapy fails to overcome leptin resistance associated with diet-induced obesity.

The objective of this study was to determine if central overexpression of leptin could overcome the leptin resistance caused by 100 days of high-fat feeding. Three-month old-F344XBN male rats were fed either control low fat chow (Chow), which provides 15% of energy as fat, or a high-fat/high-sucrose diet (HF), which provides 59% of energy as fat. Over several weeks, the HF-fed animals spontaneously split into two groups of animals: those that became obese on the HF diet (DIO) and those that did not gain extra weight on the HF diet [diet resistant (DR)]. After 100 days of HF feeding, animals were given a single intracerebroventricular injection containing 5.75E10 particles of rAAV encoding leptin (rAAV-leptin) or control virus (rAAV-con). Chow animals responded robustly to rAAV-leptin, including significant anorexia, weight loss, and lipopenia. In contrast, DIO were completely unresponsive to rAAV-leptin. DR rats responded to rAAV-leptin, but in a more variable fashion than Chow. Unlike what was observed in Chow, the anorectic response to rAAV-leptin rapidly attenuated and was no longer significant by day 14 postvector delivery. Both DIO and DR animals were found to have reduced long-form leptin receptor expression and enhanced basal P-STAT-3 in the hypothalamus with respect to Chow. rAAV-leptin caused an increase in STAT3 phosphorylation and proopiomelanocortin expression in the hypothalamus and an increase in uncoupling protein-1 in brown adipose tissue in both Chow and DR animals, but failed to do so in DIO. This suggests that central overexpression of leptin is not a viable strategy to reverse diet-induced obesity.

Am J Physiol Regul Integr Comp Physiol. 2003 Nov;285(5):R1011-20.

Plasma leptin and insulin relationships in obese and nonobese humans.

Hyperinsulinemia. is associated with an overexpression of mRNA for the ob protein leptin in rodent models of genetic obesity, and insulin has been reported to directly stimulate leptin mRNA in rat adipocytes. Human obesity is also associated with increased leptin mRNA as well as plasma levels, but there have been no reports of the effect of insulin on leptin secretion. We, therefore, tested the hypothesis that insulin stimulates leptin secretion in humans. Using a newly developed leptin assay, immunoreactive leptin was measured in fasting and postprandial plasma samples from 27 healthy adults and in samples before and during euglycemic-hyperinsulinemic then stepped hypoglycemic (hourly steps at 85, 75, 65, 55, and 45 mg/dl) clamps from 10 healthy subjects and 11 patients with IDDM. Plasma leptin was correlated (r = 0.84, P = 0.0005) with BMI in obese but not nonobese subjects and with fasting (r = 0.75, P = 0.008) but not postprandial plasma insulin levels. (Leptin levels did not change postprandially.) Euglycemic hyperinsulinemia did not alter leptin levels, nor did hyperinsulinemic hypoglycemia. Thus, because circulating leptin levels are not increased during postprandial hyperinsulinemia or during euglycemic (or hypoglycemic) hyperinsulinemia, we conclude that, at least in the short term, insulin does not increase leptin secretion in humans and that hyperleptinemia in obese individuals is not likely the result of hyperinsulinemia.

Diabetes. 1996 May;45(5):695-8

Rapid transformation of white adipocytes into fat-oxidizing machines.

Adenovirus-induced hyperleptinemia rapidly depletes body fat in normal rats without increasing free fatty acids and ketogenesis, implying that fat-storing adipocytes are oxidizing the fat. To analyze the ultrastructural changes of adipocytes accompanying this functional transformation, we examined the fat tissue by electron microscopy. After 14 days of hyperleptinemia, adipocytes had become shrunken, fatless, and encased in a thick basement-membrane-like matrix. They were crowded with mitochondria that were much smaller than those of brown adipocytes. Their gene expression profile revealed striking up-regulation of peroxisome proliferator-activated receptor gamma coactivator 1alpha (an up-regulator of mitochondrial biogenesis not normally expressed in white fat), increased uncoupling proteins-1 and -2, and down-regulation of lipogenic enzymes. Phosphorylation of both acetyl CoA carboxylase and AMP-activated protein kinase was increased, thus explaining the increase in fatty acid oxidation. The ability to transform adipocytes into unique fat-burning cells may suggest novel therapeutic strategies for obesity.

Proc Natl Acad Sci USA. 2004 Feb 17;101(7):2058-63

Combined leptin actions on adipose tissue and hypothalamus are required to deplete adipocyte fat in lean rats: implications for obesity treatment.

Intense hyperleptinemia completely depletes adipocyte fat of normal rats within 14 days. To determine the mechanism, epididymal fat pads from normal wild-type (+/+) and obese (fa/fa) Zucker Diabetic Fatty (ZDF) donor rats were transplanted into normal +/+ and fa/fa ZDF recipients. Hyperleptinemia induced by adenovirus-leptin administration depleted all fat from native fat pads and from fat transplants from +/+ donors but not from transplants from ZDF(fa/fa) donors with defective leptin receptors. In both native and transplanted +/+ fat pads, large numbers of mitochondria were apparent, and genes involved in fatty acid oxidation were up-regulated. However, +/+ fat pads transplanted into fa/fa recipients did not respond to hyperleptinemia, suggesting lack of an essential leptin-stimulated cohormone(s). In +/+ but not in fa/fa rats, plasma catecholamine levels rose, and both P-STAT3 and P-CREB increased in adipose tissue, suggesting that both direct and indirect (hypothalamic) leptin receptor-mediated actions of hyperleptinemia are involved in depletion of adipocyte fat.

J Biol Chem. 2006 Dec 29;281(52):40283-91

Low-dose leptin reverses skeletal muscle, autonomic, and neuroendocrine adaptations to maintenance of reduced weight.

Maintenance of a reduced body weight is accompanied by decreased energy expenditure that is due largely to increased skeletal muscle work efficiency. In addition, decreased sympathetic nervous system tone and circulating concentrations of leptin, thyroxine, and triiodothyronine act coordinately to favor weight regain. These “weight-reduced” phenotypes are similar to those of leptin-deficient humans and rodents. We examined metabolic, autonomic, and neuroendocrine phenotypes in 10 inpatient subjects (5 males, 5 females [3 never-obese, 7 obese]) under 3 sets of experimental conditions: (a) maintaining usual weight by ingesting a liquid formula diet; (b) maintaining a 10% reduced weight by ingesting a liquid formula diet; and (c) receiving twice-daily subcutaneous doses of leptin sufficient to restore 8 am circulating leptin concentrations to pre-weight-loss levels and remaining on the same liquid formula diet required to maintain a 10% reduced weight. During leptin administration, energy expenditure, skeletal muscle work efficiency, sympathetic nervous system tone, and circulating concentrations of thyroxine and triiodothyronine returned to pre-weight-loss levels. These responses suggest that the weight-reduced state may be regarded as a condition of relative leptin insufficiency. Prevention of weight regain might be achievable by strategies relevant to reversing this leptin-insufficient state.

J Clin Invest. 2005 Dec;115(12):3579-86

Leptin—from regulation of fat metabolism to stimulation of breast cancer growth.

Leptin restricts intake of calories as a satiety hormone. It probably stimulates neoplastic proliferation in breast cancer, too. Growth of malignant cells could be regulated by various leptin-induced second messengers like STAT3 (signal transducers and activators of transcription 3), AP-1 (transcription activator protein 1), MAPK (mitogen-activated protein kinase) and ERKs (extracellular signal-regulated kinases). They seem to be involved in aromatase expression, generation of estrogens and activation of estrogen receptor alpha (ERalpha) in malignant breast epithelium. Leptin may maintain resistance to antiestrogen therapy. Namely, it increased activation of estrogen receptors, therefore, it was suspected to reduce or even overcome the inhibitory effect of tamoxifen on breast cell proliferation. Although several valuable reviews have been focused on the role of leptin in breast cancer, the status of knowledge in this field changes quickly and our insight should be continuously revised. In this summary, we provide refreshed interpretation of intensively reported scientific queries of the topic.

Pathol Oncol Res. 2006;12(2):69-72

High leptin levels are associated with stroke.

BACKGROUND AND PURPOSE: Leptin, an important hormone for body weight regulation, may be involved in the pathogenesis of cardiovascular manifestations of obesity. We tested whether leptin may be an independent risk marker for stroke in a case-referent study. METHODS: Definitive acute stroke events, defined by MONICA criteria, were identified from October 1, 1995 to April 30, 1999. Referents without known cardiovascular disease were randomly selected from a population census. Patient characteristics were taken from hospital files and leptin was analyzed in stored samples. Logistic regression analysis was used to determine possible differences in leptin levels between groups. RESULTS: One hundred and thirty-seven cases with ischemic stroke and 69 cases with hemorrhagic stroke were identified. In comparison with referents, male patients with stroke had significantly higher leptin levels. Both male and female stroke patients had increased blood pressure compared with the referents. In multivariate analyses, high leptin levels were associated with both ischemic (OR = 4.89; 95% CI: 1.89-12.62) and hemorrhagic (OR = 3.86; 95% CI: 1.13-13.16) stroke in men, and with ischemic stroke in women (OR = 4.10; 95% CI: 1.45-11.62). The combination of high leptin levels and increased blood pressure (systolic or diastolic) was associated with a strong positive interaction in males with hemorrhagic stroke. CONCLUSION: Leptin may be an important link for the development of cerebrovascular disease in the insulin resistance syndrome in men.

Cerebrovasc Dis. 2003;15(1-2):63-9

The use of a Cissus quadrangularis/Irvingia gabonensis combination in the management of weight loss: a double-blind placebo-controlled study.

AIM: To evaluate the effects of two formulations, Cissus quadrangularis-only and a Cissus quadrangularis/Irvingia gabonensis combination, on weight loss in overweight and obese human subjects. METHODS: The study was a 10 week randomized, double-blind, placebo-controlled design involving 72 obese or overweight participants (45.8% male; 54.2% female; ages 21-44; mean age = 29.3). The participants were randomly divided into three equal (n = 24) groups: placebo, Cissus quadrangularis-only, and Cissus quadrangularis/Irvingia gabonensis combination. Capsules containing the placebo or active formulations were administered twice daily before meals; no major dietary changes nor exercises were suggested during the study. A total of six anthropomorphic and serological measurements (body weight, body fat, waist size; total plasma cholesterol, LDL cholesterol, fasting blood glucose level) were taken at baseline and at 4, 8, and 10 weeks. RESULTS: Compared to the placebo group, the two active groups showed a statistically significant difference on all six variables by week 10. The magnitude of the differences was noticeable by week 4 and continued to increase over the trial period. CONCLUSION: Although the Cissus quadrangularis-only group showed significant reductions on all variables compared to the placebo group, the Cissus quadrangularis/Irvingia gabonensis combination resulted in even larger reductions. This apparently synergistic formulation should prove helpful in the management of obesity and its related complications.

Lipids Health Dis. 2008 Mar 31;7:12

The effect of Irvingia gabonensis seeds on body weight and blood lipids of obese subjects in Cameroon.

Dietary fibres are frequently used for the treatment of obesity. The aim of this study was to evaluate the efficacy of Irvingia gabonensis seeds in the management of obesity. This was carried out as a double blind randomised study involving 40 subjects (mean age 42.4 years). Twenty-eight subjects received Irvingia gabonensis (IG) (1.05 g three time a day for one month) while 12 were on placebo (P) and the same schedule. During the one-month study period all subjects were on a normocaloric diet evaluated every week by a dietetic record book. At the end, the mean body weight of the IG group was decreased by 5.26 +/- 2.37% (p < 0.0001) and that of the placebo group by 1.32 +/- 0.41% (p < 0.02). The difference observed between the IG and the placebo groups was significant (p < 0.01). The obese patients under Irvingia gabonensis treatment also had a significant decrease of total cholesterol, LDL-cholesterol, triglycerides, and an increase of HDL-cholesterol. On the other hand, the placebo group did not manifest any changes in blood lipid components. Irvingia gabonensis seed may find application in weight lose.

Lipids Health Dis. 2005 May 25;4:12

Blueberry-induced changes in spatial working memory correlate with changes in hippocampal CREB phosphorylation and brain-derived neurotrophic factor (BDNF) levels.

Phytochemical-rich foods have been shown to be effective at reversing age-related deficits in memory in both animals and humans. We show that a supplementation with a blueberry diet (2% w/w) for 12 weeks improves the performance of aged animals in spatial working memory tasks. This improvement emerged within 3 weeks and persisted for the remainder of the testing period. Memory performance correlated well with the activation of cAMP-response element-binding protein (CREB) and increases in both pro- and mature levels of brain-derived neurotrophic factor (BDNF) in the hippocampus. Changes in CREB and BDNF in aged and blueberry-supplemented animals were accompanied by increases in the phosphorylation state of extracellular signal-related kinase (ERK1/2), rather than that of calcium calmodulin kinase (CaMKII and CaMKIV) or protein kinase A. Furthermore, age and blueberry supplementation were linked to changes in the activation state of Akt, mTOR, and the levels of Arc/Arg3.1 in the hippocampus, suggesting that pathways involved in de novo protein synthesis may be involved. Although causal relationships cannot be made among supplementation, behavior, and biochemical parameters, the measurement of anthocyanins and flavanols in the brain following blueberry supplementation may indicate that changes in spatial working memory in aged animals are linked to the effects of flavonoids on the ERK-CREB-BDNF pathway.

Free Radic Biol Med. 2008 Aug 1;45(3):295-305

A blueberry-enriched diet provides cellular protection against oxidative stress and reduces a kainate-induced learning impairment in rats.

Young male Fischer-344 rats were fed a diet containing 2% blueberry (BB) extract or control diet for at least 8 weeks and then received bilateral hippocampal injections of kainic acid (KA 200 ng/0.5 microl) or phosphate buffered saline (PBS). One week later rats were trained in one-way active footshock avoidance in a straight runway followed the next day by training in a footshock motivated 14-unit T-maze with documented sensitivity to hippocampal glutamatergic manipulations. Based on analyses of several performance variables, KA-treated rats exhibited clearly impaired learning performance; however, the BB diet significantly reduced this impairment. Supporting the behavioral findings, stereological assessment of CA1 pyramidal neurons documented greater neuronal loss in KA-treated controls compared to KA-treated rats on the BB diet. In an in vitro experiment, FaO cells grown in medium supplemented with serum from BB-fed rats had enhanced viability after exposure to hydrogen peroxide. These findings suggest that BB supplementation may protect against neurodegeneration and cognitive impairment mediated by excitotoxicity and oxidative stress.

Neurobiol Aging. 2008 Nov;29(11):1680-9

Blueberry polyphenols attenuate kainic acid-induced decrements in cognition and alter inflammatory gene expression in rat hippocampus.

Cognitive impairment in age-related neurodegenerative diseases such as Alzheimer’s disease may be partly due to long-term exposure and increased susceptibility to inflammatory insults. In the current study, we investigated whether polyphenols in blueberries can reduce the deleterious effects of inflammation induced by central administration of kainic acid by altering the expression of genes associated with inflammation. To this end, 4-month-old male Fischer-344 (F344) rats were fed a control, 0.015% piroxicam (an NSAID) or 2% blueberry diet for 8 weeks before either Ringer’s buffer or kainic acid was bilaterally micro-infused into the hippocampus. Two weeks later, following behavioral evaluation, the rats were killed and total RNA from the hippocampus was extracted and used in real-time quantitative RT-PCR (qRT-PCR) to analyze the expression of inflammation-related genes. Kainic acid had deleterious effects on cognitive behavior as kainic acid-injected rats on the control diet exhibited increased latencies to find a hidden platform in the Morris water maze compared to Ringer’s buffer-injected rats and utilized non-spatial strategies during probe trials. The blueberry diet, and to a lesser degree the piroxicam diet, was able to improve cognitive performance. Immunohistochemical analyses of OX-6 expression revealed that kainic acid produced an inflammatory response by increasing the OX-6 positive areas in the hippocampus of kainic acid-injected rats. Kainic acid up-regulated the expression of the inflammatory cytokines IL-1beta and TNF-alpha, the neurotrophic factor IGF-1, and the transcription factor NF-kappaB. Blueberry and piroxicam supplementations were found to attenuate the kainic acid-induced increase in the expression of IL-1beta, TNF-alpha, and NF-kappaB, while only blueberry was able to augment the increased IGF-1 expression. These results indicate that blueberry polyphenols attenuate learning impairments following neurotoxic insult and exert anti-inflammatory actions, perhaps via alteration of gene expression.

Nutr Neurosci. 2008 Aug;11(4):172-82

Dopamine and Abeta-induced stress signaling and decrements in Ca2+ buffering in primary neonatal hippocampal cells are antagonized by blueberry extract.

We have shown previously that dietary blueberry (BB) extract supplementation (S) reversed several parameters of neuronal and behavioral (e.g., cognition) aging in rodents. Additionally, findings indicate that COS-7 cells transfected with muscarinic receptor subtypes (e.g., M1) showed decrements in Ca;{2+} clearance following depolarization (Ca;{2+} Recovery time, Ca;{2+}RT) that were antagonized by BB. Since it has been postulated that at least part of the loss of cognitive function in aging may be dependent upon a dysregulation in calcium homeostasis (i.e., Ca;{2+}RT), we assessed whether: a) Ca;{2+}RT would be altered in dopamine (DA)- or amyloid beta (Abeta)-exposed cultured primary hippocampal neuronal cells (HNC), and b) BB pre-treatment of the cells would prevent these deficits. Thus, control or BB (0.5 mg/ml)-treated HNC were exposed to DA (0.1 mM, 2 hrs), Abeta(40) (25 microM, 24 hrs), Abeta(42) (25 microM, 24 hrs), and Abeta(25-35) (25 microM, 24 hrs), and Ca;{2+}RT following KCl-induced depolarization assessed. Ca;{2+}RT was assessed as the % of HNC showing recovery to 50%-70% of control at 5, 10, or 15 min after depolarization. Results indicated that DA significantly lowered Ca;{2+}RT in the HNC at all time points examined after depolarization. However, BB treatment selectively prevented these declines in Ca;{2+}RT. In the case of Abeta, the greatest effects on Ca;{2+}RT were seen when the hippocampal cells were Abeta(42)-treated. These effects were antagonized by BB treatment. Abeta(40) produced fewer deficits on Ca;{2+}RT than those seen when the HNC were pre-treated with either A;{2+}(42) or A;{2+}(25-35), but BB was relatively ineffective in antagonizing the deficits in Ca;{2+}RT produced by A;{2+}(40) or A;{2+}(25-35). Additional analyses indicated that BBs may be exerting their protective effects in the hippocampal cells by altering levels of phosphorylated MAPK, PKCgamma, and phosphorylated CREB. Therefore it appears that at least part of the protective effect of BBs may involve alterations in stress signaling.

J Alzheimers Dis. 2007 Jul;11(4):433-46

Modulation of hippocampal plasticity and cognitive behavior by short-term blueberry supplementation in aged rats.

During aging, reductions in hippocampal neurogenesis are associated with memory decline indicating a causal relationship. Indeed, insulin-like growth factor-1 (IGF-1), a major activator of the extracellular receptor kinase pathway that is central in learning and memory processes, is also a key modulator of hippocampal neurogenesis. Previously, we showed that age-related declines in spatial memory tasks can be improved by antioxidant-rich diets containing blueberries. In this study, to begin to understand the mechanisms responsible for the beneficial effects of blueberries, we assessed changes in hippocampal plasticity parameters such as hippocampal neurogenesis, extracellular receptor kinase activation, and IGF-1 and IGF-1R levels in blueberry-supplemented aged animals. Our results show that all these parameters of hippocampal neuronal plasticity are increased in supplemented animals and aspects such as proliferation, extracellular receptor kinase activation and IGF-1 and IGF-1R levels correlate with improvements in spatial memory. Therefore, cognitive improvements afforded by polyphenolic-rich fruits such as blueberries appear, in part, to be mediated by their effects on hippocampal plasticity.

Nutr Neurosci. 2004 Oct-Dec;7(5-6):309-16

Dietary supplementation with blueberry extract improves survival of transplanted dopamine neurons.

The exact mechanisms contributing to poor neuronal survival in cell transplantation paradigms for Parkinson’s disease (PD) are unknown. However, transplantation-induced host immune response, inflammation, and subsequent oxidative stress are likely contributors to cell death since dopamine (DA) neurons are exquisitely sensitive to oxidative damage. Multiple studies have attempted to improve cell survival by treating transplant material with antioxidant and antiinflammatory compounds, whereas far fewer studies have attempted to modify the host environment to reduce these threats. Flavonoids, phytochemicals found in fruits and vegetables, have antioxidant, antiinflammatory, and immunomodulatory properties. For example, supplementation with dietary blueberry extract (BBE) prevents oxidative stress-associated impairment of striatal motor function during aging and restores lost motor function in aged rats. We hypothesized that dietary supplementation of rodent diets with BBE would improve the survival of embryonic DA neurons transplanted into the unilaterally DA-depleted striatum. Inclusion of 2% BBE in a custom chow diet significantly increased the survival of implanted DA neurons and ameliorated rotational behavior asymmetries as compared to transplanted animals consuming a standard diet. These findings provide support for the potential of dietary phytochemicals as an easily administered and well-tolerated therapy that can be used to improve the effectiveness of DA neuron replacement.

Nutr Neurosci. 2006 Oct-Dec;9(5-6):251-8

Fruit polyphenols and their effects on neuronal signaling and behavior in senescence.

The onset of age-related neurodegenerative diseases superimposed on a declining nervous system could exacerbate the motor and cognitive behavioral deficits that normally occur in senescence. It is likely that, in cases of severe deficits in memory or motor function, hospitalization and/or custodial care would be a likely outcome. This means that unless some way is found to reduce these age-related decrements in neuronal function, healthcare costs will continue to rise exponentially. Thus, it is extremely important to explore methods to retard or reverse the age-related neuronal deficits as well as their subsequent, behavioral manifestations. Applying molecular biological approaches to slow aging in the human condition may be years away. So it is important to determine what methods can be used today to increase healthy aging, forestall the onset of these diseases, and create conditions favorable to obtaining a “longevity dividend” in both financial and human terms. In this regard, epidemiological studies indicate that consumption of diets rich in antioxidants and anti-inflammatory compounds, such as those found in fruits and vegetables, may lower the risk of developing age-related neurodegenerative diseases, such as Alzheimer’s or Parkinson’s diseases (AD and PD). Research suggests that the polyphenolic compounds found in fruits, such as blueberries, may exert their beneficial effects by altering stress signaling and neuronal communication, suggesting that interventions may exert protection against age-related deficits in cognitive and motor function. The purpose of this article is to discuss the benefits of these interventions in rodent models and to describe the putative molecular mechanisms involved in their benefits.

Ann N Y Acad Sci. 2007 Apr;1100:470-85

Effect of blueberry feeding on plasma lipids in pigs.

Two feeding trials were conducted with pigs to determine the effects of blueberry supplementation on plasma lipid levels and other indices of cardiovascular benefit. In the first trial, where basal diets contained a high level of plant-based components (70% soya, oats and barley), supplementation with 1, 2 and 4% blueberries resulted in a decrease in total, LDL- and HDL-cholesterol. The greatest reduction was observed in the 2% blueberry-fed pigs, where total, LDL- and HDL-cholesterol were reduced 11.7, 15.1 and 8.3%, respectively. In the second trial where basal diets contained only 20% (w/w) of soya, oats and barley, the lipid-modulating effect of blueberries was attenuated, so that supplementation with 1.5% blueberries reduced total cholesterol by 8%, which occurred only in pigs whose diets had been supplemented with cholesterol (0.08%), NaCl (0.11%) and fructose (9%). In the first feeding trial, blueberry supplementation had no effect on blood platelet activity. Blueberry supplementation also had no effect on the susceptibility of leucocyte DNA to oxidation in the first trial and no effect on the susceptibility of LDL to oxidation in the second trial. Results of these two feeding trials are discussed in relation to the effects of basal diet composition on lipid-modulating effects of blueberries.

Br J Nutr. 2008 Jul;100(1):70-8

Effect of Blueberin on fasting glucose, C-reactive protein and plasma aminotransferases, in female volunteers with diabetes type 2: double-blind, placebo controlled clinical study.

In a 4-week randomized placebo-controlled clinical trial we investigated the effect of 300 mg Blueberin, a phytomedicine containing 250 mg Blueberry leaves (Vaccinium arctostaphylos L, Ericaceae) extract providing minimum 50 mg 3,4-caffeoylquinic (chlorogenic) acid, and 50 mg myricetin, on fasting plasma glucose, alanine aminotransferases (ALT), aspartate aminotransferases (AST), glutamyltransferase (GGT) enzymes levels, and serum inflammatory C-Reactive proteins (CRP) in forty-two volunteer subjects (46+/-15 year of age, BMI 25+/-3 kgs/(m2)) diagnosed with Type 2 diabetes. During the 4-week trial, the Blueberin supplement was administered three times per day, 15-30 minutes prior to a meal along with 100 ml of water. Results of this trial revealed that the supplementation of Blueberin reduced fasting plasma glucose from 143+/-5,2mg/L to 104+/-5,7 mg/L (p<0,001), whereas there was no statistically significant changes in the Placebo group from 138+/-4,8 mg/L to 126+/-5,1mg/L (p>0,05). The reduction of fasting glucose was correlated with the reduction of serum CRP and in the Blueberin group from 5,18+/-1,4 mg/l to 2,14+/-1,8 mg/L (p<0,05), whereas in the Placebo group CRP levels were not significantly reduced from 5,11+/-1,7 mg/l to 4,94+/-1,1mg/L (p>0,05). Furthermore, the Blueberin also significantly reduced the levels of plasma enzymes ALT, AST and GGT, indicating that, in addition to anti-diabetes effects, the Blueberin also possess pharmacologically relevant anti-inflammatory properties.

Georgian Med News. 2006 Dec;(141):66-72

Availability of blueberry phenolics for microbial metabolism in the colon and the potential inflammatory implications.

Blueberries are a rich source of phenylpropanoid-derived phytochemicals, widely studied for their potential health benefits. Of particular interest for colonic health are the lower molecular weight phenolic acids and their derivatives, as these are the predominant phenolic compounds detected in the colon. Blueberries contained a wide variety of phenolic acids, the majority of which (3371.14 +/- 422.30 mg/kg compared to 205.06 +/- 45.34 mg/kg for the free phenolic acids) were attached to other plant cell-wall components and therefore, likely to become available in the colon. Cytokine-induced stimulation of the inflammatory pathways in colon cells was four-fold up-regulated in the presence of the free phenolic acid fraction. Incubation of the bound phenolic acids with human faecal slurries resulted in qualitative and quantitative differences in the phenolic compounds recovered. The metabolites obtained by incubation with faecal slurries from one volunteer significantly decreased (1.67 +/- 0.69 ng/cm(3)) prostanoid production, whereas an increase (10.78 +/- 5.54 ng/cm(3)) was obtained with faecal slurries from another volunteer. These results suggest that any potential protective effect of blueberry phenolics as anti-inflammatory agents in the colon is a likely result of microbial metabolism. Studies addressing a wide-range of well-characterised human volunteers will be required before such health claims can be fully established.

Mol Nutr Food Res. 2007 Jun;51(6):726-31

Probiotics and blueberry attenuate the severity of dextran sulfate sodium (DSS)-induced colitis.

We studied the anti-inflammatory properties of probiotic strains and blueberry in a colitis model. The disease activity index (DAI) was significantly lower on days 9 and 10 in all groups compared to the colitis control. Myeloperoxidase (MPO) and bacterial translocation to the liver and to the mesenteric lymph nodes (MLN) decreased significantly in all groups compared to colitis control. Cecal Enterobacteriaceae count decreased significantly in blueberry with and without probiotics compared to the other groups. Lactobacillus plantarum reisolated from the cecal content in the presence of blueberry, contrary to Lactobacillus fermentum. Colonic MDA decreased significantly in all groups, except the L. fermentum group, compared to the colitis control. The cecal concentration of acetic, propionic, and butyricbutyric acid was significantly higher in the L. plantarum group, while the L. fermentum group yielded the highest concentration of lactic acid compared with all other groups. Lactobacillus plantarum DSM 15313, Lactobacillus fermentum 35D, and blueberry alone and in combination improve the DAI, reduce bacterial translocation, and reduce inflammation.

Dig Dis Sci. 2008 Sep;53(9):2464-73

Blueberry prevents bone loss in ovariectomized rat model of postmenopausal osteoporosis.

The objective of the present study was to explore the bone protective role of blueberry in an ovariectomized rat model. Thirty 6-month-old female Sprague-Dawley rats were either sham-operated (Sham) or ovariectomized (Ovx) and divided into three groups: Sham, Ovx (control), Ovx+blueberry (5% blueberry w/w). After 100 days of treatment, rats were euthanized, and blood and tissues were collected. Bone mineral density (BMD) and content of whole body, right tibia, right femur and fourth lumbar vertebra were assessed via dual-energy X-ray absorptiometry. As expected, Ovx resulted in loss of whole-body, tibial, femoral, and 4th lumbar BMD by approximately 6%. Blueberry treatment was able to prevent the loss of whole-body BMD and had an intermediary effect on prevention of tibial and femoral BMD when compared to either Sham or Ovx controls. The bone-protective effects of blueberry may be due to suppression of Ovx-induced increase in bone turnover, as evident by lowered femoral mRNA levels of alkaline phosphatase, collagen type I and tartrate-resistant acid phosphatase to the Sham levels. Similarly, serum osteocalcein levels were also lower in the blueberry group when compared to the Ovx control group, albeit not significantly. In summary, our findings indicate that blueberry can prevent bone loss as seen by the increases in BMD and favorable changes in biomarkers of bone metabolism.

J Nutr Biochem. 2008 Oct;19(10):694-9

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