Branched-Chain Amino Acids Are Linked with Alzheimer's Disease-Related Pathology and Cognitive Deficits.

Alzheimer's disease (AD) is an irreversible neurodegenerative disorder with a complex pathophysiology. Type 2 diabetes (T2D) is a strong risk factor for AD that shares similar abnormal features including metabolic dysregulation and brain pathology such as amyloid and/or Tau deposits. Emerging evidence suggests that circulating branched-chain amino acids (BCAAs) are associated with T2D. While excess BCAAs are shown to be harmful to neurons, its connection to AD is poorly understood. Here we show that individuals with AD have elevated circulating BCAAs and their metabolites compared to healthy individuals, and that a BCAA metabolite is correlated with the severity of dementia. APPSwe mouse model of AD also displayed higher plasma BCAAs compared to controls. In pursuit of understanding a potential causality, BCAA supplementation to HT-22 neurons was found to reduce genes critical for neuronal health while increasing phosphorylated Tau. Moreover, restricting BCAAs from diet delayed cognitive decline and lowered AD-related pathology in the cortex and hippocampus in APP/PS1 mice. BCAA restriction for two months was sufficient to correct glycemic control and increased/restored dopamine that were severely reduced in APP/PS1 controls. Treating 5xFAD mice that show early brain pathology with a BCAA-lowering compound recapitulated the beneficial effects of BCAA restriction on brain pathology and neurotransmitters including norepinephrine and serotonin. Collectively, this study reveals a positive association between circulating BCAAs and AD. Our findings suggest that BCAAs impair neuronal functions whereas BCAA-lowering alleviates AD-related pathology and cognitive decline, thus establishing a potential causal link between BCAAs and AD progression.

Oleic acid stimulates monoamine efflux through PPAR-α: Differential effects in diet-induced obesity.

Obesity continues to be a growing health concern around the world, and elevated levels of free fatty acids as a result of high-fat intake might play a role in neuroendocrine alterations leading to obesity. However, it is unclear how fatty acids affect neuroendocrine functions and energy metabolism. Since hypothalamic monoamines play a crucial role in regulating neuroendocrine functions relating to energy balance, we investigated the direct effects of oleic acid on hypothalamic monoamines and hypothesized that oleic acid would activate peroxisome proliferator-activated receptor alpha (PPAR-α), a nuclear transcription factor involved with fatty acid metabolism, to affect monoamines. We also hypothesized that this response would be subdued in diet-induced obesity (DIO). To test these hypotheses, hypothalami from Sprague Dawley and DIO rats were incubated with 0 (Control), 0.00132 mM, 0.132 mM, 1.32 mM oleic acid, 50 µM MK 886 (a selective PPAR- α antagonist), or oleic acid + MK 886 in Krebs Ringers Henseleit (KRH) solution. HPLC-EC was used to measure monoamine levels in perfusates. Oleic acid produced a significant increase in norepinephrine, dopamine, and serotonin levels in a dose-dependent manner, and incubation with MK886 blocked these effects. The effect of oleic acid on hypothalamic monoamines was attenuated in DIO rats. These findings suggest that PPARα probably plays an essential role in fatty acid sensing in the hypothalamus, by affecting monoamine efflux and DIO rats are resistant to the effects of oleic acid.

Chronic estrogen affects TIDA neurons through IL-1ß and NO: effects of aging.

Women are chronically exposed to estrogens through oral contraceptives, hormone replacement therapy or environmental estrogens. We hypothesized that chronic exposure to low levels of estradiol-17ß (E2) can induce inflammatory and degenerative changes in the tuberoinfundibular dopaminergic (TIDA) system leading to reduced dopamine synthesis and hyperprolactinemia. Young (Y; 3­4 months) and middle-aged (MA; 10­12 months) Sprague-Dawley rats that were intact or ovariectomized (OVX) were either sham-implanted or implanted with a slow-release E2 pellet (20 ng E2/day for 90 days). To get mechanistic insight, adult 3- to 4-month-old WT, inducible nitric oxide synthase (iNOS) and IL-1 receptor (IL-1R) knockout (KO) mice were subjected to a similar treatment. Hypothalamic areas corresponding to the TIDA system were analyzed. E2 treatment increased IL-1ß protein and nitrate levels in the arcuate nucleus of intact animals (Y and MA). Nitration of tyrosine hydroxylase in the median eminence increased with E2 treatment in both intact and OVX animals. There was no additional effect of age. This was accompanied by a reduction in dopamine levels and an increase in prolactin in intact animals. E2 treatment increased nitrate and reduced dopamine levels in the hypothalamus and increased serum prolactin in WT mice. In contrast, the effect of E2 on nitrate levels was blocked in IL-1R KO mice and the effect on dopamine and prolactin were blocked in iNOS KO animals. Taken together, these results show that chronic exposure to low levels of E2 decreases TIDA activity through a cytokine-nitric oxide-mediated pathway leading to hyperprolactinemia and that aging could promote these degenerative changes.

Differential effects of inhalation exposure to PM2.5 on hypothalamic monoamines and corticotrophin releasing hormone in lean and obese rats.

Acute exposure to airborne pollutants, especially particulate matter (PM2.5) is known to increase hospital admissions for cardiovascular conditions, increase cardiovascular related mortality and predispose the elderly and obese individuals to cardiovascular conditions. The mechanisms by which PM2.5 exposure affects the cardiovascular system is not clear. Since the autonomic system plays an important role in cardiovascular regulation, we hypothesized that PM2.5 exposure most likely activates the paraventricular nucleus (PVN) of the hypothalamus to cause an increase in sympathetic nervous system and/or stress axis activity. We also hypothesized that these changes may be sustained in obese rats predisposing them to higher cardiovascular risk. To test this, adult male Brown Norway (BN) rats were subjected to one day or three days of inhalation exposures to filtered air (FA) or concentrated air particulate (CAP) derived from ambient PM2.5. Corpulent JCR-LA rats were exposed to FA or CAP for four days. Animals were sacrificed 24h after the last inhalation exposure. Their brains were removed, frozen and sectioned. The PVN and median eminence (ME) were microdissected. PVN was analyzed for norepinephrine (NE), dopamine (DA) and 5-hydroxy-indole acetic acid (5-HIAA) levels using HPLC-EC. ME was analyzed for corticotrophin releasing hormone (CRH) levels by ELISA. One day exposure to CAP increased NE levels in the PVN and CRH levels in the ME of BN rats. Repeated exposures to CAP did not affect NE levels in the PVN of BN rats, but increased NE levels in JCR/LA rats. A similar pattern was observed with 5-HIAA levels. DA levels on the other hand, were unaffected in both BN and JCR/LA strains. These data suggest that repeated exposures to PM2.5 continue to stimulate the PVN in obese animals but not lean rats.

Chronic estradiol-17ß exposure suppresses hypothalamic norepinephrine release and the steroid-induced luteinizing hormone surge: role of nitration of tyrosine hydroxylase.

Chronic exposure to estrogens is known to produce a variety of deleterious effects in women including breast and ovarian cancer and anovulation. In female rats, exposure to low levels of estradiol-17ß (E2) decreases hypothalamic norepinephrine (NE) to suppress luteinizing hormone (LH) secretion and cause failure of ovulation. We hypothesized that E2 exposure most likely decreases NE release in the medial preoptic area (MPA) of the hypothalamus to produce this effect and that this may be due to E2-induced inflammatory changes in noradrenergic nuclei leading to nitration of an enzyme involved in NE synthesis. To test this, female Sprague Dawley rats were sham implanted or implanted with slow release E2 pellets (20ng/day) for 30, 60 or 90 days (E30, E60 and E90 respectively). At the end of the treatment period, the rats were implanted with a push-pull cannula in the MPA, ovariectomized and steroid primied to induce a LH surge and subjected to push-pull perfusion. Perfusates were analyzed for NE levels using HPLC-EC. Blood samples collected simultaneously were analyzed for LH levels. We measured interleukin-1ß (IL-1ß) and nitrate levels in brainstem noradrenergic nuclei that innervate the MPA. In control animals, there was a marked increase in NE levels in response to steroid priming at 1600h that was reduced in the E30 group, and completely abolished after 60 and 90 days of E2 exposure. LH profiles were similar to NE release profiles in control and E2-treated animals. We found that IL-1ß levels increased in all three (A1, A2 and A6) noradrenergic nuclei with chronic E2 exposure, while nitrate levels increased only in the A6 region. There was an increase in the nitration of the NE synthesizing enzyme in the MPA in this group as well probably contributing to reduced NE synthesis. This could be a possible mechanism by which chronic E2 exposure decreases NE levels in the MPA to suppress the LH surge.

Chronic estradiol exposure induces oxidative stress in the hypothalamus to decrease hypothalamic dopamine and cause hyperprolactinemia.

Estrogens are known to cause hyperprolactinemia, most probably by acting on the tuberoinfundibular dopaminergic (TIDA) system of the hypothalamus. Dopamine (DA) produced by TIDA neurons directly inhibits prolactin secretion and, therefore, to stimulate prolactin secretion, estrogens inhibit TIDA neurons to decrease DA production. However, the mechanism by which estrogen produces this effect is not clear. In the present study, we used a paradigm involving chronic exposure to low levels of estradiol-17ß (E(2)) to mimic prolonged exposures to environmental and endogenous estrogens. We hypothesized that chronic exposure to low levels of E(2) induces oxidative stress in the arcuate nucleus (AN) of the hypothalamus that contains TIDA neurons and causes nitration of tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of DA. This results in a significant decrease in DA and consequently, hyperprolactinemia. To investigate this, adult, intact female cycling rats were implanted with slow-release E(2) pellets (20 ng/day) for 30, 60, or 90 days and were compared with old (16-18 mo old) constant estrous (OCE) rats. Chronic E(2) exposure significantly increased the expression of glial fibrillary acidic protein and the concentrations of interleukin-1ß (IL-1ß) and nitrate in the AN that contains perikarya of TIDA neurons and increased nitration of TH in the median eminence (ME) that contains the terminals. These levels were comparable to those seen in OCE rats. We observed a significant decrease in DA concentrations in the ME and hyperprolactinemia in an exposure-dependent manner similar to that seen in OCE rats. It was concluded that chronic exposure to low levels of E(2) evokes oxidative stress in the AN to inhibit TIDA neuronal function, most probably leading to hyperprolactinemia.

Effects of central and systemic administration of leptin on neurotransmitter concentrations in specific areas of the hypothalamus.

Leptin, a hormone produced by adipocytes, has been shown to affect a number of central functions, such as regulation of the hypothalamo-pituitary-adrenal axis, feeding, and body weight regulation. Because hypothalamic monoamines are intricately involved in the regulation of these functions, we hypothesized that leptin may produce its effects by altering the activity of these neurotransmitters. To test this hypothesis, male rats received peripheral (0, 100, or 500 microg ip), or central (0 or 5 microg icv) injections of leptin. The animals were killed 5 h later, and their brains were removed, frozen, and sectioned. Serum was collected to measure leptin and corticosterone by RIA. The paraventricular nucleus (PVN), arcuate nucleus (AN), ventromedial hypothalamus (VMH), dorsomedial dorsal nucleus (DMD), median eminence (ME), and medial preoptic area (MPA) were obtained using Palkovits' microdissection technique, and monoamine concentrations in these areas were determined using HPLC-EC. Intraperitoneal administration of leptin increased serum leptin concentrations in a dose-dependent manner (P < 0.05). Both intraperitoneal and intracerebroventricular administration of leptin decreased serum corticosterone significantly (P < 0.05). Norepinephrine (NE) concentration decreased significantly in the PVN, AN, and VMH after both intraperitoneal and intracerebroventricular administration of leptin (P < 0.05). NE concentrations decreased significantly in the DMN after intracerebroventricular administration of leptin (P < 0.05). Leptin treatment (both ip and icv) decreased dopamine concentrations significantly in the PVN. Serotonin (5-HT) concentration decreased significantly in the PVN after both intraperitoneal and intracerebroventricular injections of leptin and decreased in the VMH only with intracerebroventricular treatment of leptin. Leptin did not affect any of the monoamines in the ME and MPA. These results indicate that both central and systemic administration of leptin can affect hypothalamic monoamines in a region-specific manner, which, in turn, could mediate many of leptin's central and neuroendocrine effects.

Leptin inhibits norepinephrine efflux from the hypothalamus in vitro: role of gamma aminobutyric acid.

Leptin, a hormone secreted by adipocytes, produces a number of central and neuroendocrine effects, the mechanisms behind which are not completely understood. Hypothalamic norepinephrine (NE) is involved in many of the neuroendocrine effects that are associated with leptin. Therefore, we hypothesized that leptin could affect hypothalamic NE activity to bring about its central and neuroendocrine effects. Because gamma aminobutyric acid (GABA) is known to affect the release of NE, we also tested the possibility that leptin-induced changes in NE could be mediated through GABA. The mediobasal hypothalami from adult male rats were incubated in an in vitro incubation system for four consecutive incubation periods of 60 min each at 37 degrees C in Krebs Ringers Henseleit (KRH) solution in an atmosphere of 95% O(2) and 5% CO(2.) After determining the basal release, the hypothalami were challenged with 0, 0.1, 1 or 10 nm of leptin, bicuculline (a GABA-A receptor antagonist; 10 microM) and bicuculline (10 microM) +10 nM of leptin during the second incubation period. Residual effects of leptin were measured in the third incubation where tissues were incubated with KRH alone, and the viability of tissues was determined in the fourth incubation when tissues were exposed to high K(+) KRH. NE levels in the incubation medium were measured using high-performance liquid chromatography with electrochemical detection (HPLC-EC). Leptin inhibited NE efflux from the hypothalamus in a dose-dependent manner. Moreover, incubation of hypothalami with 10 nM of leptin and bicuculline, a completely blocked the leptin-induced decrease in NE efflux. These results demonstrate for the first time that leptin could act directly on the hypothalamus to inhibit NE efflux through GABA. It was concluded that leptin could probably produce its central and neuroendocrine effects by modulating NE and GABA levels in the hypothalamus.

Central adaptation to chronic administration of interleukin-1beta (IL-1beta) in rats.

Interleukin-1beta (IL-1beta), a cytokine, has been shown to induce a number of central and neuroendocrine effects. Prolonged treatment with IL-1beta is associated with adaptive responses in feeding, body temperature and hormone profiles. The purpose of the present study was to see if these effects are accompanied by changes in hypothalamic norepinephrine (NE) and to compare it with the acute effects of IL-1beta. Adult male Sprague-Dawley rats were treated (i.p.) with 5 microg of IL-1beta once (acute) or daily for 5 consequent days (chronic). The control animals received an injection of the vehicle for IL-1beta (0.1% PBS-BSA). Body weight, food intake, and rectal temperature were monitored daily. At the end of treatment, the animals were sacrificed, and specific areas of the hypothalamus were microdissected and analyzed for NE concentrations. Corticosterone levels were measured in the serum. Both acute and chronic IL-1beta treatment produced significant changes in a number of parameters. However, there were marked differences between the two treatment regimens. While acute treatment with IL-1beta increased NE concentrations in both the paraventricular nucleus and the median eminence (ME), chronic treatment increased NE concentrations only in the ME. A corresponding increase in serum corticosterone levels was observed with acute IL treatment. Chronic treatment with IL-1beta decreased body weight, and produced an initial decrease in food intake which returned to control levels by the fourth day of treatment. Chronic IL treatment also produced an initial increase in body temperature that returned to control levels by day 4. These results indicate that the effects of IL-1beta on central and neuroendocrine functions are dependent on the duration of the treatment and that the adaptive responses observed in feeding and body temperature after chronic IL treatment are accompanied by similar responses in brain NE.

Diabetes-induced neuroendocrine changes in rats: role of brain monoamines, insulin and leptin.

Diabetes is characterized by hyperphagia, polydypsia and activation of the HPA axis. However, the mechanisms by which diabetes produces these effects are not clear. This study was conducted to examine the effects of diabetes on the neuroendocrine system and to see if treatment with insulin and/or leptin is capable of reversing these effects. Streptozotocin-induced diabetic adult male rats were subjected to the following treatments: vehicle, insulin (2 U/day, s.c.), leptin (100 microg/kg BW) or leptin+insulin every day for 2 weeks. Food intake, water intake, and body weight were monitored daily. We measured changes in monoamine concentrations in discrete nuclei of the hypothalamus at the end of treatment. Diabetes produced a marked increase in food intake and water intake and this effect was completely reversed by insulin treatment and partially reversed by leptin treatment (P<0.05). Diabetes caused an increase in norepinephrine (NE) concentrations in the paraventricular nucleus with a concurrent increase in serum corticosterone. Treatment with insulin and leptin completely reversed these effects. Induction of diabetes also increased the concentrations of NE, dopamine and serotonin in the arcuate nucleus and NE concentrations in the lateral hypothalamus, ventromedial hypothalamus (VMH) and suprachiasmatic nucleus (P<0.05). Although insulin treatment was capable of reversing all these changes, leptin treatment was unable to decrease diabetes-induced increase in NE concentrations in the VMH. These data provide evidence that hypothalamic monoamines could mediate the neuroendocrine effects of diabetes and that insulin and leptin act as important signals in this process.