Olanzapine's effects on hypothalamic transcriptomics and kinase activity.

Olanzapine is a second-generation antipsychotic that disrupts metabolism and is associated with an increased risk of type 2 diabetes. The hypothalamus is a key region in the control of whole-body metabolic homeostasis. The objective of the current study was to determine how acute peripheral olanzapine administration affects transcription and serine/threonine kinase activity in the hypothalamus. Hypothalamus samples from rats were collected following the pancreatic euglycemic clamp, thereby allowing us to study endpoints under steady state conditions for plasma glucose and insulin. Olanzapine stimulated pathways associated with inflammation, but diminished pathways associated with the capacity to combat endoplasmic reticulum stress and G protein-coupled receptor activity. These pathways represent potential targets to reduce the incidence of type 2 diabetes in patients taking antipsychotics.

Changes in hypothalamic mu-opioid receptor expression following acute olanzapine treatment in female rats: Implications for feeding behavior.

Advances have been made in recent years in using opioid receptor antagonists as an adjunct therapy to psychotropic medication to reduce debilitating weight gain and metabolic adverse effects associated with in particular second generation antipsychotics. However, it is unknown whether second generation antipsychotics produce a change in opioid receptor expression in the brain. The present study investigated early changes in opioid receptor expression in the female rat hypothalamus, a master controller of hunger and metabolic regulation, after acute treatment with olanzapine, a commonly used second generation antipsychotic. Using quantitative spatial in situ hybridization and receptor autoradiography, expression levels of the three opioid receptors; kappa, mu and delta, were determined at mRNA and protein level, respectively, in the five hypothalamic areas: paraventricular nucleus, arcuate nucleus, ventromedial nucleus, dorsomedial nucleus and lateral hypothalamus. After 48 h of olanzapine treatment at clinically relevant plasma concentration weight gain and food intake changes, and increased plasma glucose were observed in female rats. Olanzapine treatment also led to a significant increase in mu opioid receptor availability in the arcuate nucleus, which contains both satiety and hunger controlling neurons. No other areas showed any opioid receptor expressional changes with olanzapine treatment on neither at mRNA nor protein level. Technical difficulties made it impossible to analyze mRNA levels in the lateral hypothalamus and overall binding of delta opioid receptors. Thus, the present study provided insights in to how olanzapine at clinically relevant plasma levels already at an early stage modulated the opioid system in the hypothalamus.

Modulation of hypothalamic AMPK phosphorylation by olanzapine controls energy balance and body weight.

BACKGROUND: Second-generation antipsychotics (SGAs) are a mainstay therapy for schizophrenia. SGA-treated patients present higher risk for weight gain, dyslipidemia and hyperglycemia. Herein, we evaluated the effects of olanzapine (OLA), widely prescribed SGA, in mice focusing on changes in body weight and energy balance. We further explored OLA effects in protein tyrosine phosphatase-1B deficient (PTP1B-KO) mice, a preclinical model of leptin hypersensitivity protected against obesity. METHODS: Wild-type (WT) and PTP1B-KO mice were fed an OLA-supplemented diet (5 mg/kg/day, 7 months) or treated with OLA via intraperitoneal (i.p.) injection or by oral gavage (10 mg/kg/day, 8 weeks). Readouts of the crosstalk between hypothalamus and brown or subcutaneous white adipose tissue (BAT and iWAT, respectively) were assessed. The effects of intrahypothalamic administration of OLA with adenoviruses expressing constitutive active AMPKα1 in mice were also analyzed. RESULTS: Both WT and PTP1B-KO mice receiving OLA-supplemented diet presented hyperphagia, but weight gain was enhanced only in WT mice. Unexpectedly, all mice receiving OLA via i.p. lost weight without changes in food intake, but with increased energy expenditure (EE). In these mice, reduced hypothalamic AMPK phosphorylation concurred with elevations in UCP-1 and temperature in BAT. These effects were also found by intrahypothalamic OLA injection and were abolished by constitutive activation of AMPK in the hypothalamus. Additionally, OLA i.p. treatment was associated with enhanced Tyrosine Hydroxylase (TH)-positive innervation and less sympathetic neuron-associated macrophages in iWAT. Both central and i.p. OLA injections increased UCP-1 and TH in iWAT, an effect also prevented by hypothalamic AMPK activation. By contrast, in mice fed an OLA-supplemented diet, BAT thermogenesis was only enhanced in those lacking PTP1B. Our results shed light for the first time that a threshold of OLA levels reaching the hypothalamus is required to activate the hypothalamus BAT/iWAT axis and, therefore, avoid weight gain. CONCLUSION: Our results have unraveled an unexpected metabolic rewiring controlled by hypothalamic AMPK that avoids weight gain in male mice treated i.p. with OLA by activating BAT thermogenesis and iWAT browning and a potential benefit of PTP1B inhibition against OLA-induced weight gain upon oral treatment.

Increased growth hormone secretagogue receptor-1a (GHSR-1a) in hypothalamus during olanzapine treatment in rats.

Weight gain is the one of the most important factors which increases global burden of psychiatric disorder. Second-generation antipsychotics, olanzapine (Olz) and valproic acid (Vpa) in particular, are held responsible for weight gain. However, it is still uncertain how these drugs cause this. Thus, the rats selected for the experiment were randomly divided into 3 groups. The 1st group received only 0.5 ml saline solution intraperitoneally (n = 20, control group); the second group was given 200 mg / kg Vpa intraperitoneally (n = 20, Vpa group) and 2 mg / kg Olz was given intraperitoneally to the 3rd group (n = 20, Olz group) between 8 and 10 am for 30 days. We examined serum leptin, adiponectin, resistin, TNF-α, IL-6, ghrelin level and, the amount of ghrelin secreting cells in the stomach and growth hormone secretagogue receptor-1a (GHSR-1a, ghrelin receptor) expression in the hypothalamus. The hypothalamic GHS-1a receptor index was significantly higher in the Olz group compared with the control group and Vpa group (p = 0.036 and p = 0.016 respectively). Ghrelin immune positive cell index in stomach was statistically significantly lower in the Vpa group compared with the control and Olz groups (p = 0.028 and p = 0.013 respectively) There was no difference between the groups in terms of serum leptin, resistin, IL-6 and ghrelin levels. In the Vpa group, a statistically significant increase was found in serum adiponectin level compared with both the control group and the Olz group (p = 0009 and p = 0024 respectively) and, significant decrease was found in serum TNF-α level compared to Olz group (p = 0007). In conclusion, we found that the main cause of weight gain in Olz use was the increase in the number of hypothalamic ghrelin receptors. Investigating the mechanism by which Olz increases the number of ghrelin receptors may help to develop effective treatment strategies in preventing obesity in psychiatric patients.

Metformin ameliorates olanzapine-induced disturbances in POMC neuron number, axonal projection, and hypothalamic leptin resistance.

Antipsychotics have been widely accepted as a treatment of choice for psychiatric illnesses such as schizophrenia. While atypical antipsychotics such as aripiprazole are not associated with obesity and diabetes, olanzapine is still widely used based on the anticipation that it is more effective in treating severe schizophrenia than aripiprazole, despite its metabolic side effects. To address metabolic problems, metformin is widely prescribed. Hypothalamic proopiomelanocortin (POMC) neurons have been identified as the main regulator of metabolism and energy expenditure. Although the relation between POMC neurons and metabolic disorders is well established, little is known about the effects of olanzapine and metformin on hypothalamic POMC neurons. In the present study, we investigated the effect of olanzapine and metformin on the hypothalamic POMC neurons in female mice. Olanzapine administration for 5 days significantly decreased Pomc mRNA expression, POMC neuron numbers, POMC projections, and induced leptin resistance before the onset of obesity. It was also observed that coadministration of metformin with olanzapine not only increased POMC neuron numbers and projections but also improved the leptin response of POMC neurons in the olanzapine-treated female mice. These findings suggest that olanzapine-induced hypothalamic POMC neuron abnormality and leptin resistance, which can be ameliorated by metformin administration, are the possible causes of subsequent hyperphagia. [BMB Reports 2022; 55(6): 293-298].

The atypical antipsychotic risperidone targets hypothalamic melanocortin 4 receptors to cause weight gain.

Atypical antipsychotics such as risperidone cause drug-induced metabolic syndrome. However, the underlying mechanisms remain largely unknown. Here, we report a new mouse model that reliably reproduces risperidone-induced weight gain, adiposity, and glucose intolerance. We found that risperidone treatment acutely altered energy balance in C57BL/6 mice and that hyperphagia accounted for most of the weight gain. Transcriptomic analyses in the hypothalamus of risperidone-fed mice revealed that risperidone treatment reduced the expression of Mc4r. Furthermore, Mc4r in Sim1 neurons was necessary for risperidone-induced hyperphagia and weight gain. Moreover, we found that the same pathway underlies the obesogenic effect of olanzapine-another commonly prescribed antipsychotic drug. Remarkably, whole-cell patch-clamp recording demonstrated that risperidone acutely inhibited the activity of hypothalamic Mc4r neurons via the opening of a postsynaptic potassium conductance. Finally, we showed that treatment with setmelanotide, an MC4R-specific agonist, mitigated hyperphagia and obesity in both risperidone- and olanzapine-fed mice.

N-acetylcysteine prevents olanzapine-induced oxidative stress in mHypoA-59 hypothalamic neurons.

Olanzapine is a second-generation antipsychotic (AP) drug commonly prescribed for the treatment of schizophrenia. Recently, olanzapine has been found to cause brain tissue volume loss in rodent and primate studies; however, the underlying mechanism remains unknown. Abnormal autophagy and oxidative stress have been implicated to have a role in AP-induced neurodegeneration, while N-acetylcysteine (NAC) is a potent antioxidant, shown to be beneficial in the treatment of schizophrenia. Here, we investigate the role of olanzapine and NAC on cell viability, oxidative stress, mitochondrial mass and mitophagy in hypothalamic cells. Firstly, cell viability was assessed in mHypoA-59 and mHypoA NPY/GFP cells using an MTS assay and flow cytometric analyses. Olanzapine treated mHypoA-59 cells were then assessed for mitophagy markers and oxidative stress; including quantification of lysosomes, autophagosomes, LC3B-II, p62, superoxide anion (O2-) and mitochondrial mass. NAC (10 mM) was used to reverse the effects of olanzapine (100 µM) on O2-, mitochondrial mass and LC3B-II. We found that olanzapine significantly impacted cell viability in mHypoA-59 hypothalamic cells in a dose and time-dependent manner. Olanzapine inhibited mitophagy, instigated oxidative stress and prompted mitochondrial abnormalities. NAC was able to mitigate olanzapine-induced effects. These findings suggest that high doses of olanzapine may cause neurotoxicity of hypothalamic neurons via increased production of reactive oxygen species (ROS), mitochondrial damage and mitophagy inhibition. This could in part explain data suggesting that APs may reduce brain volume.

Role of TRPV1/TRPV3 channels in olanzapine-induced metabolic alteration: Possible involvement in hypothalamic energy-sensing, appetite regulation, inflammation and mesolimbic pathway.

Atypical antipsychotics (AAPs) have the tendency of inducing severe metabolic alterations like obesity, diabetes mellitus, insulin resistance, dyslipidemia and cardiovascular complications. These alterations have been attributed to altered hypothalamic appetite regulation, energy sensing, insulin/leptin signaling, inflammatory reactions and active reward anticipation. Line of evidence suggests that transient receptor potential vanilloid type 1 and 3 (TRPV1 and TRPV3) channels are emerging targets in treatment of obesity, diabetes mellitus and could modulate feed intake. The present study was aimed to investigate the putative role TRPV1/TRPV3 in olanzapine-induced metabolic alterations in mice. Female BALB/c mice were treated with olanzapine for six weeks to induce metabolic alterations. Non-selective TRPV1/TRPV3 antagonist (ruthenium red) and selective TRPV1 (capsazepine) and TRPV3 antagonists (2,2-diphenyltetrahydrofuran or DPTHF) were used to investigate the involvement of TRPV1/TRPV3 in chronic olanzapine-induced metabolic alterations. These metabolic alterations were differentially reversed by ruthenium red and capsazepine, while DPTHF didn't show any significant effect. Olanzapine treatment also altered the mRNA expression of hypothalamic appetite-regulating and nutrient-sensing factors, inflammatory genes and TRPV1/TRPV3, which were reversed with ruthenium red and capsazepine treatment. Furthermore, olanzapine treatment also increased expression of TRPV1/TRPV3 in nucleus accumbens (NAc), TRPV3 expression in ventral tegmental area (VTA), which were reversed by the respective antagonists. However, DPTHF treatment showed reduced feed intake in olanzapine treated mice, which might be due to TRPV3 specific antagonism and reduced hedonic feed intake. In conclusion, our results suggested the putative role TRPV1 in hypothalamic dysregulations and TRPV3 in the mesolimbic pathway; both regulate feeding in olanzapine treated mice.

Olanzapine increases AMPK-NPY orexigenic signaling by disrupting H1R-GHSR1a interaction in the hypothalamic neurons of mice.

Second generation antipsychotics, particularly olanzapine, induce severe obesity, which is associated with their antagonistic effect on the histamine H1 receptor (H1R). We have previously demonstrated that oral administration of olanzapine increases the concentration of neuropeptide Y (NPY) in the hypothalamus of rats, accompanied by hyperphagia and weight gain. However, it is unclear if the increased NPY after olanzapine administration is due to its direct effect on hypothalamic neurons and its H1R antagonistic property. In the present study, we showed that with an inverted U-shape dose-response curve, olanzapine increased NPY expression in the NPY-GFP hypothalamic neurons; however, this was not the case in the hypothalamic neurons of H1R knockout mice. Olanzapine inhibited the interaction of H1R and GHSR1a (ghrelin receptor) in the primary mouse hypothalamic neurons and NPY-GFP neurons examined by confocal fluorescence resonance energy transfer (FRET) technology. Furthermore, an H1R agonist, FMPH inhibited olanzapine activation of GHSR1a downstream signaling pAMPK and transcription factors of NPY (pFOXO1 and pCREB) in the hypothalamic NPY-GFP cell. However, an olanzapine analogue (E-Olan) with lower affinity to H1R presented negligible enhancement of pCREB within the nucleus of NPY neurons. These findings suggest that the H1R antagonist property of olanzapine inhibits the interaction of H1R and GHSR1a, activates GHSR1a downstream signaling pAMPK-FOXO1/pCREB and increases hypothalamic NPY: this could be one of the important molecular mechanisms of H1R antagonism of olanzapine-induced obesity in antipsychotic management of psychiatric disorders.

Direct effects of antipsychotic drugs on insulin, energy sensing and inflammatory pathways in hypothalamic mouse neurons.

INTRODUCTION: Second-generation antipsychotics cause serious metabolic side effects, but the mechanisms behind these effects remain largely unknown. However, emerging evidence supports that antipsychotics may act upon the hypothalamus, the primary brain region understood to regulate energy homeostasis. We have recently reported that the antipsychotics olanzapine, clozapine, and aripiprazole can directly act on hypothalamic rat neurons (rHypoE-19) to impair insulin, energy sensing, and modulate inflammatory pathways. In the current paper, we sought to replicate these findings to a mouse neuronal model. METHODS: The mouse hypothalamic neuronal cell line, mHypoE-46, was treated with olanzapine, clozapine, or aripiprazole. Western blots were used to measure the energy sensing protein AMPK, components of the insulin signalling pathway (AKT, GSK3ß), and components of the MAPK pathway (ERK1/2, JNK, p38), the latter linked to inflammation. RT-qPCR was used to measure mRNA expression of the inflammatory mediators IL-6, IL-10, and BDNF, well as putative receptors in the mHypoE-46 (current) and the rHypoE-19 (previously studied) cell lines. RESULTS: In the mHypoE-46 neurons, olanzapine and aripiprazole increased AMPK phosphorylation, while clozapine and aripiprazole inhibited insulin-induced phosphorylation of AKT. Clozapine increased JNK and aripiprazole decreased ERK1/2 phosphorylation. Olanzapine also decreased IL-6 mRNA expression, while olanzapine and clozapine increased IL-10 mRNA expression. The rHypoE-19 neurons expressed the H1, 5 H T2A, and M3 receptors, while the mHypoE-46 neurons expressed the 5 H T2A, D2, and M3 receptors. Neither cell line expressed the 5 H T2C receptor. CONCLUSION: Similar to observed effects of these agents in rat neurons, induction of AMPK by aripiprazole and olanzapine suggests impaired energy sensing, while suppression of insulin-induced pAKT by clozapine and aripiprazole suggests impaired insulin signalling, seen across both rodent derived hypothalamic cell lines. Conversely, olanzapine-induced suppression of pro-inflammatory IL-6, alongside olanzapine and clozapine-induced IL-10, demonstrate anti-inflammatory effects, which do not corroborate with our prior observations in the rat neuronal line. The different findings between cell lines could be explained by differential expression of neurotransmitters receptors and/or reflect genetic heterogeneity across the rat and mouse lines. However, overall, our findings support direct effects of antipsychotics to impact insulin, energy sensing, and inflammatory pathways in hypothalamic rodent neurons.

Olanzapine-induced endoplasmic reticulum stress and inflammation in the hypothalamus were inhibited by an ER stress inhibitor 4-phenylbutyrate.

Antipsychotics are the most important treatment for schizophrenia. However, antipsychotics, particularly olanzapine and clozapine, are associated with severe weight gain/obesity side-effects. Although numerous studies have been carried out to identify the exact mechanisms of antipsychotic-induced weight gain, it is still important to consider other pathways. Endoplasmic reticulum (ER) stress signaling and its associated inflammation pathway is one of the most important pathways involved in regulation of energy balance. In the present study, we examined the role of hypothalamic protein kinase R like endoplasmic reticulum kinase- eukaryotic initiation factor 2α (PERK-eIF2α) signaling and the inflammatory IkappaB kinase ß- nuclear factor kappa B (IKKß-NFκB) signaling pathway in olanzapine-induced weight gain in female rats. In this study, we found that olanzapine significantly activated PERK-eIF2α and IKKß-NFκB signaling in SH-SY5Y cells in a dose-dependent manner. Olanzapine treatment for 8 days in rats was associated with activated PERK-eIF2α signaling and IKKß-NFκB signaling in the hypothalamus, accompanied by increased food intake and weight gain. Co-treatment with an ER stress inhibitor, 4-phenylbutyrate (4-PBA), decreased olanzapine-induced food intake and weight gain in a dose- and time-dependent manner. Moreover, 4-PBA dose-dependently inhibited olanzapine-induced activated PERK-eIF2α and IKKß-NFκB signaling in the hypothalamus. These results suggested that hypothalamic ER stress may play an important role in antipsychotic-induced weight gain.

Antipsychotics differentially regulate insulin, energy sensing, and inflammation pathways in hypothalamic rat neurons.

INTRODUCTION: Second generation antipsychotic (AP)s remain the gold-standard treatment for schizophrenia and are widely used on- and off-label for other psychiatric illnesses. However, these agents cause serious metabolic side-effects. The hypothalamus is the primary brain region responsible for whole body energy regulation, and disruptions in energy sensing (e.g. insulin signaling) and inflammation in this brain region have been implicated in the development of insulin resistance and obesity. To elucidate mechanisms by which APs may be causing metabolic dysregulation, we explored whether these agents can directly impact energy sensing and inflammation in hypothalamic neurons. METHODS: The rat hypothalamic neuronal cell line, rHypoE-19, was treated with olanzapine (0.25-100 uM), clozapine (2.5-100 uM) or aripiprazole (5-20 uM). Western blots measured the energy sensing protein AMPK, components of the insulin signaling pathway (AKT, GSK3ß), and components of the MAPK pathway (ERK1/2, JNK, p38). Quantitative real-time PCR was performed to determine changes in the mRNA expression of interleukin (IL)-6, IL-10 and brain derived neurotrophic factor (BDNF). RESULTS: Olanzapine (100 uM) and clozapine (100, 20 uM) significantly increased pERK1/2 and pJNK protein expression, while aripiprazole (20 uM) only increased pJNK. Clozapine (100 uM) and aripiprazole (5 and 20 uM) significantly increased AMPK phosphorylation (an orexigenic energy sensor), and inhibited insulin-induced phosphorylation of AKT. Olanzapine (100 uM) treatment caused a significant increase in IL-6 while aripiprazole (20 uM) significantly decreased IL-10. Olanzapine (100 uM) and aripiprazole (20 uM) increased BDNF expression. CONCLUSIONS: We demonstrate that antipsychotics can directly regulate insulin, energy sensing, and inflammatory pathways in hypothalamic neurons. Increased MAPK activation by all antipsychotics, alongside olanzapine-associated increases in IL-6, and aripiprazole-associated decreases in IL-10, suggests induction of pro-inflammatory pathways. Clozapine and aripiprazole inhibition of insulin-stimulated pAKT and increases in AMPK phosphorylation (an orexigenic energy sensor) suggests impaired insulin action and energy sensing. Conversely, olanzapine and aripiprazole increased BDNF, which would be expected to be metabolically beneficial. Overall, our findings suggest differential effects of antipsychotics on hypothalamic neuroinflammation and energy sensing.

Effects of acute olanzapine exposure on central insulin-mediated regulation of whole body fuel selection and feeding.

The use of antipsychotics is associated with severe disruptions in whole body glucose and lipid metabolism which may in part occur through the central nervous system and impaired insulin action at the brain. Here we investigated whether olanzapine treatment might also affect the ability of central insulin treatment to regulate food intake and fuel preference in the light and dark cycle. Male Sprague-Dawley rats were treated with olanzapine (or vehicle solution; 3 mg/kg, subcutaneous) and a simultaneous acute intracerebral ventricular (ICV) infusion of insulin (or vehicle; 3 µL at 10mU; ICV) at the beginning of the 12-h light and dark cycles. Olanzapine treatment reduced RER in the dark and light phases (most consistently in the 4-hours post-treatment), while ICV insulin reduced average RER predominantly in the dark phase, but also at the end of the light cycle. The RER lowering effect of ICV-insulin during the light cycle was absent in the group co-administered olanzapine. The reduction in RER during the dark phase was mirrored by decreased food intake with ICV insulin, but not olanzapine treated rats. The reduction in food intake by ICV-insulin was abolished in rats co-administered olanzapine suggesting rapid induction of central insulin resistance. A combination of ICV-insulin and olanzapine similarly reduced RER in the dark phase, independent of changes in food intake. Olanzapine treatment, alone or in combination with ICV-insulin, significantly reduced VCO2 at regular intervals in the dark phase (specifically 3 h post-treatment), while VO2 was not significantly altered by either treatment. Finally, heat production was increased by olanzapine treatment in the light phase, though this effect was not consistent. The findings confirm that acute olanzapine treatment directly reduces RER and suggest that treatment with this drug may also override central insulin-mediated reductions in food intake at the hypothalamus (while still independently favoring fatty acid oxidation). Acute central insulin similarly reduces RER, but in contrast to olanzapine, this may represent a physiologically appropriate response to reduction in food intake.

A role of neuropeptide CART in hyperphagia and weight gain induced by olanzapine treatment in rats.

Although olanzapine is highly efficacious and most widely used second generation antipsychotic drug, the success of treatment has been hampered by its propensity to induce weight gain. While the underlying neuronal mechanisms are unclear, their elucidation may help to target alternative pathways regulating energy balance. The present study was undertaken to define the role of cocaine- and amphetamine-regulated transcript (CART), a well-known anorexic peptide, in olanzapine-induced hyperphagia and body weight gain in female rats. Olanzapine was administered daily by intraperitoneal route, alone or in combination with CART (intracerebroventricular) for a period of two weeks. Immediately after drug administrations, preweighed food was offered to the animals at the commencement of the dark phase. The food intake and body weight were measured daily just prior to next injection. Furthermore, the brains of olanzapine-treated rats were processed for the immunohistochemical analysis of CART-containing elements in the hypothalamus. Treatment with olanzapine (0.5 mg/kg) for the duration of 14 days produced a significant increase in food intake and body weight as compared to control. However, concomitant administration of CART (0.5 µg) attenuated the olanzapine-induced hyperphagia and weight gain. Olanzapine administration resulted in a significant reduction in CART immunoreactivity in the hypothalamic arcuate, paraventricular, dorsomedial and ventromedial nuclei. We suggest that decreased CART contents in the hypothalamus may be causally linked with the hyperphagia and weight gain induced by olanzapine.