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

Highlights

• Antipsychotic (AP)s directly impact insulin and inflammatory pathways in hypothalamic neurons.

• The MAPK pathway was consistently upregulated by all APs.

• Only clozapine and aripiprazole inhibited pAKT and increased pAMPK.

• Olanzapine and aripiprazole upregulated inflammatory pathways and BDNF.

• APs have differential effects in the hypothalamus which do not align with clinical metabolic risk.

Abstract

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, GSK3β), 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.

Introduction

Antipsychotics (APs) are the gold-standard treatment for schizophrenia and are increasingly being prescribed both on- and off-label for other psychiatric conditions. However, these medications cause serious metabolic side effects. In turn, patients with schizophrenia have a two-fold increase in standardized mortality ratio from cardiovascular disease (Hennekens et al., 2005). Although the liability for metabolic side effects differs between APs (Newcomer, 2005), it has recently become clear that no single AP is devoid of this risk, particularly in younger individuals who are AP-naïve (Correll et al., 2009). Additionally, it is found that regardless of class or agent, APs increase risk of diabetes 2–3 fold, above and beyond the risk that is conferred by the illness of schizophrenia itself (Rajkumar et al., 2017).

The mechanisms of AP-induced metabolic dysregulation are unclear, but are understood to occur at least in part through the central nervous system (Kowalchuk et al., 2018). In addition, the question has arisen whether APs could be causing metabolic perturbations by directly impact brain insulin signalling (Kowalchuk et al., 2017). Insulin receptors are expressed at high levels in many brain areas, where signalling pathways (mediated through AKT, GSK3β, and the MAPKs) play a significant role in neuronal growth, memory, and energy regulation. Brain studies examining AP administration in vitro and in rodents in vivo have variably suggested upregulation of pathways downstream of the insulin receptor (i.e. AKT phosphorylation/ activation; GSK phosphorylation /deactivation), which is difficult to reconcile with the adverse metabolic effects of APs observed clinically. However, many in vitro studies have not been conducted under conditions of insulin stimulation, and thus fail to directly test effects of APs on insulin response. Insulin stimulation has been used when investigating AP effects in peripheral tissues, with variable results (Del Campo et al., 2018; Engl et al., 2005). in vivo rodent data on central insulin signalling is similarly difficult to interpret as most APs cause acute elevations in blood glucose and insulin (Boyda et al., 2010; Smith et al., 2014). Hyperglycemia and hyperinsulinemia has been shown to increase central AKT and GSK-3 phosphorylation, even in the face of insulin deficiency or disrupted insulin action (Clodfelder-Miller et al., 2005; Smith et al., 2014). Thus, it is difficult to determine if changes in activation of these proteins are due to the AP or secondary to the elevations in blood glucose. There is also the possibility that APs upregulate central insulin signalling but impair alternate energy sensing signals such as AMP-activated protein kinase (AMPK), which has been associated with AP-induced glucose dysregulation (Ikegami et al., 2013; Martins et al., 2010).

Alternatively, inflammation is also an area of interest as neuroinflammation is considered a causal factor of metabolic disease (Thaler and Schwartz, 2010). However, clinical studies evaluating AP effects on inflammation are highly inconclusive due to the many confounds which can influence inflammatory state and which are associated with the illness of schizophrenia (i.e. smoking, diet, obesity) (Tourjman et al., 2013). The heterogeneity among studies examining effects of APs on insulin and inflammatory pathways may also be attributable to a differential response of brain regions to AP exposure. Only a limited number of studies have examined APs effects on insulin signalling or energy sensing specifically in the hypothalamus (Kowalchuk et al., 2018), which is the primary brain region involved in whole body energy regulation. In addition, the hypothalamus is composed of highly heterogenous neuronal populations, and the limited in vivo papers available have examined the entire hypothalamus (Obuchowicz et al., 2006; Zhang et al., 2014). Thus, we do not know how APs may act on specific cell types. Finally, as these were whole-body experiments, we do not know if the effects seen are due to the direct impact of APs on the hypothalamus, or occur in response to feedback from other tissues signalling to this brain region.

In the present study, we set out to elucidate the direct effects of different APs (clozapine, olanzapine, aripiprazole) on insulin signalling, energy sensing and inflammation in the hypothalamus. In order to circumvent the confounding variables present in clinical studies, and to avoid changes in adiposity or acute hyperglycemia observed in rodents administered APs, we chose to employ an invitro hypothalamic cell model. We hypothesized that APs would differentially impair insulin signalling, upregulate AMPK, and induce inflammation, according to their clinical metabolic liability (clozapine = olanzapine > aripiprazole).