BackgroundMetabolic syndrome (MetS) is a constellation of factors including abdominal obesity, hyperglycemia, dyslipidemias, and hypertension that increase morbidity and mortality from diabetes and cardiovascular diseases and affects more than a third of the population in the US. Clozapine, an atypical antipsychotic used for the treatment of schizophrenia, has been found to cause drug-induced metabolic syndrome (DIMS) and may be a useful tool for studying cellular and molecular changes associated with MetS and DIMS. Mitochondria dysfunction, oxidative stress and inflammation are mechanisms proposed for the development of clozapine-related DIMS. In this study, the effects of clozapine on mitochondrial function and inflammation in insulin responsive and obesity-associated cultured cell lines were examined.Methodology/Principal FindingsCultured mouse myoblasts (C2C12), adipocytes (3T3-L1), hepatocytes (FL-83B), and monocytes (RAW 264.7) were treated with 0, 25, 50 and 75 µM clozapine for 24 hours. The mitochondrial selective probe TMRM was used to assess membrane potential and morphology. ATP levels from cell lysates were determined by bioluminescence assay. Cytokine levels in cell supernatants were assessed using a multiplex array. Clozapine was found to alter mitochondria morphology, membrane potential, and volume, and reduce ATP levels in all cell lines. Clozapine also significantly induced the production of proinflammatory cytokines IL-6, GM-CSF and IL12-p70, and this response was particularly robust in the monocyte cell line.Conclusions/SignificanceClozapine damages mitochondria and promotes inflammation in insulin responsive cells and obesity-associated cell types. These phenomena are closely associated with changes observed in human and animal studies of MetS, obesity, insulin resistance, and diabetes. Therefore, the use of clozapine in DIMS may be an important and relevant tool for investigating cellular and molecular changes associated with the development of these diseases in the general population.

This project studied the effects of the atypical antipsychotic, clozapine, on monocyte mitochondria and the production of proinflammatory cytokines. Cultured monocytes were treated with different doses of clozapine (0, 25, 50 and 75 uM) and effects on viability, mitochondria morphology, membrane potential, ATP and cytokine secretion were assessed. With increasing doses of clozapine, mitochondria appear spotty and speckled, an indication of fission. Western blots demonstrate an increase in both profusion (OPA1) and profission (DLP1) proteins; OPA1 increased significantly with increasing clozapine relative to the control. As clozapine increased, the membrane potential increased and was significantly different (p < 0.05) from the control at all doses. An ATP luminescence assay showed ATP levels decreased significantly (p < 0.005) at all clozapine doses relative to the control. Increased clozapine caused increased cytokine production. Cytokines that were significantly altered (p < 0.05) with clozapine treatment included GMCSF, IL‐1b, IL‐10, IL‐13, and MCP1. Therefore, increasing doses of the antipsychotic clozapine negatively affects mitochondria function and causes the production of proinflammatory cytokines. Supported by SURF to DLH from the URO at St. Mary's University

Clozapine is an effective treatment for schizophrenia but causes side effects like weight gain and diabetes. Mitochondria dysfunction, oxidative stress and inflammation are mechanisms proposed for clozapine side effects as well as the development of obesity and insulin resistance. Prior investigations have focused on the brain and neurons. In this study, the effect of clozapine on insulin responsive cell types and cells associated with obesity was examined. Cultured myoblasts, adipocytes, and hepatocytes were treated with 0, 25, 50 and 75 μM clozapine. After 24 hours, a mitochondrial selective probe was used to assess morphology and membrane potential (ΔΨm). ATP lysates were also prepared and culture media collected for cytokine analyses by multiplex array. Clozapine caused mitochondria fission in all cell types. In addition, it altered mitochondria ΔΨm (p<0.05), volume (p<0.05), and ATP levels (p<0.05) relative to controls. Clozapine also significantly induced the production of the proinflammatory cytokines MCP‐1, IL‐6, and GM‐CSF. In conclusion, clozapine damages mitochondria and promotes inflammation in insulin responsive cells. Understanding how clozapine alters metabolism may provide insight on the origin of metabolic disease in the general population. Supported by Biaggini Research Fellowship, St. Mary's Univ. to VC‐S & Friends for Psychiatric Research Grant, Dept. of Psychiatry to CW‐B.