Lessons from KEEPS: the Kronos Early Estrogen Prevention Study.

The Kronos Early Estrogen Prevention Study (KEEPS) was a randomized, double-blind, placebo-controlled trial designed to determine the effects of hormone treatments (menopausal hormone treatments [MHTs]) on the progression of carotid intima-medial thickness (CIMT) in recently menopausal women. Participants less than 3 years from menopause and without a history of overt cardiovascular disease (CVD), defined as no clinical CVD events and coronary artery calcium < 50 Agatston units, received either oral conjugated equine estrogens (0.45 mg/day) or transdermal 17ß-estradiol (50 µg/day), both with progesterone (200 mg/day for 12 days/month), or placebo pills and patches for 4 years. Although MHT did not decrease the age-related increase in CIMT, KEEPS provided other important insights about MHT effects. Both MHTs versus placebo reduced the severity of menopausal symptoms and maintained bone density, but differed in efficacy regarding mood/anxiety, sleep, sexual function, and deposition of ß-amyloid in the brain. Additionally, genetic variants in enzymes for metabolism and uptake of estrogen affected the efficacy of MHT for some aspects of symptom relief. KEEPS provides important information for use of MHT in clinical practice, including type, dose, and mode of delivery of MHT recently after menopause, and how genetic variants in hormone metabolism may affect MHT efficacy on specific outcomes.

Differential effects of hormone therapy on serotonin, vascular function and mood in the KEEPS.

BACKGROUND: Serotonin (5-hydroxytryptamine, 5-HT) is modulated by sex steroid hormones and affects vascular function and mood. In the Kronos Early Estrogen Prevention Cognitive and Affective Ancillary Study (KEEPS-Cog), women randomized to oral conjugated equine estrogens (oCEE) showed greater benefit on affective mood states than women randomized to transdermal 17ß-estradiol (tE2) or placebo (PL). This study examined the effect of these treatments on the platelet content of 5-HT as a surrogate measure of 5-HT synthesis and uptake in the brain. METHODS: The following were measured in a subset (n = 79) of women enrolled in KEEPS-Cog: 5-HT by ELISA, carotid intima-medial thickness (CIMT) by ultrasound, endothelial function by reactive hyperemic index (RHI), and self-reported symptoms of affective mood states by the Profile of Mood States (POMS) questionnaire. RESULTS: Mean platelet content of 5-HT increased by 107.0%, 84.5% and 39.8%, in tE2, oCEE and PL groups, respectively. Platelet 5-HT positively correlated with estrone in the oCEE group and with 17ß- estradiol in the tE2 group. Platelet 5-HT showed a positive association with RHI, but not CIMT, in the PL and oCEE groups. Reduction in mood scores for depression-dejection and anger-hostility was associated with elevations in platelet 5-HT only in the oCEE group (r = -0.5, p = 0.02). CONCLUSIONS: Effects of oCEE compared to tE2 on RHI and mood may be related to mechanisms involving platelet, and perhaps neuronal, uptake and release of 5-HT and reflect conversion of estrone to bioavailable 17ß-estradiol in platelets and the brain.

Midlife predictors of Alzheimer's disease.

Factors contributing to increased risk for Alzheimer's disease (AD) include age, sex, genes, and family history of AD. Several risk factors for AD are endogenous; however, accumulating evidence implicates modifiable risk factors in the pathogenesis of AD. Although the continued task of identifying new genes will be critical to learning more about the disease, several research findings suggest that potentially alterable environmental factors influence genetic contributions, providing targets for disease prevention and treatment. Here, we review midlife risk factors for AD, and address the potential for therapeutic intervention in midlife.

Hormone effects on fMRI and cognitive measures of encoding: importance of hormone preparation.

We compared fMRI and cognitive data from nine hormone therapy (HT)-naive women with data from women exposed to either opposed conjugated equine estrogens (CEE) (n = 10) or opposed estradiol (n = 4). Exposure to either form of HT was associated with healthier fMRI response; however, CEE-exposed women exhibited poorer memory performance than either HT-naive or estradiol-exposed subjects. These preliminary findings emphasize the need to characterize differential neural effects of various HTs.

Activation of brain regions vulnerable to Alzheimer's disease: the effect of mild cognitive impairment.

This study examined the functionality of the medial temporal lobe (MTL) and posterior cingulate (PC) in mild cognitive impairment amnestic type (MCI), a syndrome that puts patients at greater risk for developing Alzheimer disease (AD). Functional MRI (fMRI) was used to identify regions normally active during encoding of novel items and recognition of previously learned items in a reference group of 77 healthy young and middle-aged adults. The pattern of activation in this group guided further comparisons between 14 MCI subjects and 14 age-matched controls. The MCI patients exhibited less activity in the PC during recognition of previously learned items, and in the right hippocampus during encoding of novel items, despite comparable task performance to the controls. Reduced fMRI signal change in the MTL supports prior studies implicating the hippocampus for encoding new information. Reduced signal change in the PC converges with recent research on its role in recognition in normal adults as well as metabolic decline in people with genetic or cognitive risk for AD. Our results suggest that a change in function in the PC may account, in part, for memory recollection failure in AD.

Neuroprotective effects of female sex steroids in humans: current controversies and future directions.

Recent findings from the Women's Health Initiative (WHI) have raised considerable concern over prolonged use of opposed and unopposed oral conjugated equine estrogen (CEE), given the increased risk of serious adverse effects, including stroke and venous thromboembolic complications. Furthermore, results from the WHI Memory Study (WHIMS) indicated that over 5 years of therapy with Prempro impaired performance on global cognitive tests and nearly doubled the risk of dementia. These surprising findings were contradictory to cumulative evidence from basic science, epidemiological and some intervention studies suggesting hormone therapy was cardioprotective and could potentially reduce the risk of dementia. This review paper focuses on the neurobiology of estrogen, summarizing the clinical evidence for neuroprotective and cognition-enhancing efficacy of estrogen. Further, the paper briefly discusses variables that may account for the unexpected findings of WHIMS, and offers suggestions for future research.

Antecedent hyperglycemia, not hyperlipidemia, is associated with increased islet triacylglycerol content and decreased insulin gene mRNA level in Zucker diabetic fatty rats.

Type 2 diabetes is caused by a combination of beta-cell dysfunction and insulin resistance. Over time, hyperglycemia worsens, a phenomenon that has been attributed to deleterious effects of chronic hyperglycemia (glucotoxicity) or chronic hyperlipidemia (lipotoxicity) on beta-cell function and is often accompanied by increased islet triacylglycerol (TAG) content and decreased insulin gene expression. To examine these two potentially pathogenic forces, we studied Zucker rats (leptin receptor wild type, +/+; heterozygous, +/-; and mutant, -/-). First, +/+ and +/- Zucker rats were compared metabolically. At 6 weeks of age, the +/- rats had a lower level of islet insulin mRNA compared with +/+. At 12 weeks of age, differences were found in body weight and islet TAG content; however, levels of insulin mRNA were equivalent. Second, we examined whether worsening of the diabetic state in the homozygous mutant (-/-) Zucker diabetic fatty (ZDF) rat is related more to chronic hyperglycemia or to hyperlipidemia. The ZDF rats were treated for 6 weeks with either bezafibrate, a lipid-lowering drug that does not affect plasma glucose levels, or phlorizin, a drug that reduces plasma glucose without lowering lipid levels. Bezafibrate treatment lessened the rise in plasma TAG observed in nontreated rats (239 +/- 16 vs. 388 +/- 36 mg/dl, treated versus nontreated; P < 0.0001) but did not prevent the rise in fasting plasma glucose. Despite lowering plasma TAG, bezafibrate was not effective in preventing an increased islet TAG content and did not prevent the associated decrease in insulin mRNA levels. Phlorizin treatment prevented hyperglycemia (61 +/- 2 vs. 145 +/- 7 mg/dl, treated versus nontreated; P < 0.0001) and lowered islet TAG content (32.7 +/- 0.7 vs. 47.8 +/- 2.7 ng/islet, treated versus nontreated; P < 0.0001) and preserved insulin mRNA levels without preventing hypertriglyceridemia. Plasma free fatty acid level did not correlate with changes in islet TAG or insulin mRNA levels. We conclude that antecedent elevated plasma glucose levels, not plasma lipid levels, are associated with elevated islet TAG content and decreased insulin mRNA levels in ZDF animals.

Determinants of glucose toxicity and its reversibility in the pancreatic islet beta-cell line, HIT-T15.

HIT-T15 cells, a clonal beta-cell line, were cultured and passaged weekly for 6 mo in RPMI 1640 media containing various concentrations of glucose. Insulin content decreased in the intermediate- and late-passage cells as a continuous rather than a threshold glucose concentration effect. In a second series of experiments, cells were grown in media containing either 0.8 or 16.0 mM glucose from passages 76 through 105. Subcultures of passages 86, 92, and 99 that had been grown in media containing 16.0 mM glucose were switched to media containing 0.8 mM glucose and also carried forward to passage 105. Dramatic increases in insulin content and secretion and insulin gene expression were observed when the switches were made at passages 86 and 92 but not when the switch was made at passage 99. These findings suggest that glucose toxicity of insulin-secreting cells is a continuous rather than a threshold function of glucose concentration and that the shorter the period of antecedent glucose toxicity, the more likely that full recovery of cell function will occur.

In vivo prevention of hyperglycemia also prevents glucotoxic effects on PDX-1 and insulin gene expression.

Chronic exposure of pancreatic islet beta-cell lines to supraphysiologic glucose concentrations causes defects in insulin gene expression and insulin secretion. To determine whether these in vitro phenomena have pathophysiologic relevance in vivo, we studied the Zucker diabetic fatty (ZDF) rat, an animal model of type 2 diabetes. The ZDF animals had relatively higher levels of glycemia and islet insulin mRNA at 6 weeks of age than age-matched Zucker lean control (ZLC) rats. As glycemia increased in 12- and 16-week-old ZDF rats, we observed decrements in glucose-induced insulin secretion during static incubations of pancreatic islets and in insulin mRNA levels, PDX-1 mRNA levels, and PDX-1 protein binding to the insulin promoter compared with age-matched ZLC rats and 6-week-old ZDF rats. To determine whether normalization of blood glucose levels would prevent these defects, ZDF rats were treated with troglitazone beginning at 6 weeks of age. Troglitazone prevented ZDF rats from becoming hyperglycemic and preserved glucose-induced insulin responses. Furthermore, troglitazone-treated ZDF animals had greater levels of insulin and PDX-1 mRNAs compared with untreated ZDF animals of the same ages at 12 and 16 weeks. Our results demonstrate that chronic and progressive hyperglycemia resulting from type 2 diabetes in ZDF rats is associated with loss of insulin and PDX-1 mRNAs and loss of glucose-stimulated insulin secretion. Prevention of hyperglycemia prevented the associated defects in insulin and PDX-1 gene expression and improved insulin secretion. These findings provide the first in vivo evidence that prevention of progressive hyperglycemia in a model of type 2 diabetes preserves insulin and PDX-1 gene expression.

Prostaglandin E(2) mediates inhibition of insulin secretion by interleukin-1beta.

Interleukin-1beta (IL-1beta) and prostaglandin E(2) (PGE(2)), frequently co-participants in inflammatory states, are two well recognized inhibitors of glucose-induced insulin secretion. Previous reports have concluded that the inhibitory effects of these two autacoids on pancreatic beta cell function are not related because indomethacin, a potent prostaglandin synthesis inhibitor, does not prevent IL-1beta effects. However, indomethacin is not a specific cyclooxygenase inhibitor, and its other pharmacologic effects are likely to inhibit insulin secretion independently. Since we recently observed that IL-1beta induces cyclooxygenase-2 (COX-2) gene expression and PGE(2) synthesis in islet beta cells, we have reassessed the possibility that PGE(2) mediates IL-1beta effects on beta function. By using two cell lines (HIT-T15 and betaHC13) as well as Wistar rat isolated pancreatic islets, we examined the ability of two COX-2-specific antagonists, NS-398 and SC-236, to prevent IL-1beta inhibition of insulin secretion. Both drugs prevented IL-1beta from inducing PGE(2) synthesis and inhibiting insulin secretion; adding back exogenous PGE(2) re-established inhibition of insulin secretion in the presence of IL-1beta. We also found that EP3, the PGE(2) receptor subtype whose post-receptor effect is to decrease adenylyl cyclase activity and, thereby, insulin secretion, is the dominant mRNA subtype expressed. We conclude that endogenous PGE(2) mediates the inhibitory effects of exogenous IL-1beta on beta cell function.

Prevention of glucose toxicity in HIT-T15 cells and Zucker diabetic fatty rats by antioxidants.

Chronic exposure of pancreatic islets to supraphysiologic concentrations of glucose causes adverse alterations in beta cell function, a phenomenon termed glucose toxicity and one that may play a secondary pathogenic role in type 2 diabetes. However, no mechanism of action has been definitively identified for glucose toxicity in beta cells. To ascertain whether chronic oxidative stress might play a role, we chronically cultured the beta cell line, HIT-T15, in medium containing 11.1 mM glucose with and without the antioxidants, N-acetyl-L-cysteine (NAC) or aminoguanidine (AG). Addition of NAC or AG to the culture medium at least partially prevented decreases in insulin mRNA, insulin gene promoter activity, DNA binding of two important insulin promoter transcription factors (PDX-1/STF-1 and RIPE-3b1 activator), insulin content, and glucose-induced insulin secretion. These findings suggested that one mechanism of glucose toxicity in the beta cell may be chronic exposure to reactive oxygen species, i.e., chronic oxidative stress. To ascertain the effects of these drugs on diabetes, NAC or AG was given to Zucker diabetic fatty rats, a laboratory model of type 2 diabetes, from 6 through 12 weeks of age. Both drugs prevented a rise in blood oxidative stress markers (8-hydroxy-2'-deoxyguanosine and malondialdehyde + 4-hydroxy-2-nonenal), and partially prevented hyperglycemia, glucose intolerance, defective insulin secretion as well as decrements in beta cell insulin content, insulin gene expression, and PDX-1 (STF-1) binding to the insulin gene promoter. We conclude that chronic oxidative stress may play a role in glucose toxicity, which in turn may worsen the severity of type 2 diabetes.