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.

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.

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.