Chronic social defeat stress disrupts regulation of lipid synthesis
Several psychiatric disorders increase the risk of cardiovascular disease, including posttraumatic stress disorder and major depression. While the precise mechanism for this association has not yet been established, it has been shown that certain disorders promote an unfavorable lipid profile. To study the interaction of stress and lipid dysregulation, we utilized chronic social defeat stress (CSDS), a mouse model of chronic stress with features of posttraumatic stress disorder and major depression. Following exposure to CSDS, mice were given access to either regular chow or a Western-style diet high in fat and cholesterol (HFD). The combination of social stress and HFD resulted in significant perturbations in lipid regulation, including two key features of the metabolic syndrome: increased plasma levels of non–HDL cholesterol and intrahepatic accumulation of triglycerides. These effects were accompanied by a number of changes in the expression of hepatic genes involved in lipid regulation. Transcriptional activity of LXR, SREBP1c, and ChREBP were significantly affected by exposure to HFD and CSDS. We present CSDS as a model of social stress induced lipid dysregulation and propose that social stress alters lipid metabolism by increasing transcriptional activity of genes involved in lipid synthesis. Several psychiatric disorders increase the risk of cardiovascular disease, including posttraumatic stress disorder and major depression. While the precise mechanism for this association has not yet been established, it has been shown that certain disorders promote an unfavorable lipid profile. To study the interaction of stress and lipid dysregulation, we utilized chronic social defeat stress (CSDS), a mouse model of chronic stress with features of posttraumatic stress disorder and major depression. Following exposure to CSDS, mice were given access to either regular chow or a Western-style diet high in fat and cholesterol (HFD). The combination of social stress and HFD resulted in significant perturbations in lipid regulation, including two key features of the metabolic syndrome: increased plasma levels of non–HDL cholesterol and intrahepatic accumulation of triglycerides. These effects were accompanied by a number of changes in the expression of hepatic genes involved in lipid regulation. Transcriptional activity of LXR, SREBP1c, and ChREBP were significantly affected by exposure to HFD and CSDS. We present CSDS as a model of social stress induced lipid dysregulation and propose that social stress alters lipid metabolism by increasing transcriptional activity of genes involved in lipid synthesis. acetyl CoA carboxylase 1a carbohydrate-response element binding protein carnitine palmitoyltransferase 1a chronic social defeat stress fibroblast growth factor 21 glucose-6-phophatase glucose transporter 2 high-fat diet hepatocyte nuclear factor 4a insulin receptor substrate liver pyruvate kinase liver X receptor myosin regulatory light chain interacting protein proprotein convertase subtilisin/kexin type 9 pyruvate dehydrogenase kinase 4 phosphoenolpyruvate carboxykinase PPARγ coactivator 1a patatin-like phospholipase domain containing 2 peroxisome proliferator-activated receptor α posttraumatic stress disorder stearoyl CoA desaturase 1 sterol-regulatory element binding protein uncoupling protein 2 Patients with psychiatric disorders have a significantly elevated risk of death from cardiovascular disease (1Harris E.C. Barraclough B. Excess mortality of mental disorder.Br. J. Psychiatry. 1998; 173: 11-53Crossref PubMed Scopus (1500) Google Scholar). A Swedish study of 9,911 patients reported an increased standardized mortality ratio of 120% from cardiovascular disease in patients with anxiety neurosis (2Allgulander C. Suicide and mortality patterns in anxiety neurosis and depressive neurosis.Arch. Gen. Psychiatry. 1994; 51: 708-712Crossref PubMed Scopus (67) Google Scholar). A review of 4,328 male military veterans in the United States reported that a diagnosis of posttraumatic stress disorder (PTSD) was associated with a significant increase in the risk of death from early-age heart disease (3Boscarino J.A. A prospective study of PTSD and early-age heart disease mortality among Vietnam veterans: implications for surveillance and prevention.Psychosom. Med. 2008; 70: 668-676Crossref PubMed Scopus (323) Google Scholar). 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Recently we have validated a chronic social defeat stress (CSDS) protocol for use in mice (9Lutter M. Sakata I. Osborne-Lawrence S. Rovinsky S.A. Anderson J.G. Jung S. Birnbaum S. Yanagisawa M. Elmquist J.K. Nestler E.J. et al.The orexigenic hormone ghrelin defends against depressive symptoms of chronic stress.Nat. Neurosci. 2008; 11: 752-753Crossref PubMed Scopus (447) Google Scholar). CSDS shares features with many psychiatric disorders, including posttraumatic stress disorder, social anxiety, and major depression with comorbid anxiety (10Avgustinovich D.F. Gorbach O.V. Kudryavtseva N.N. Comparative analysis of anxiety-like behavior in partition and plus-maze tests after agonistic interactions in mice.Physiol. Behav. 1997; 61: 37-43Crossref PubMed Scopus (98) Google Scholar, 11Krishnan V. Berton O. Nestler E. The use of animal models in psychiatric research and treatment.Am. J. Psychiatry. 2008; 165: 1109Crossref PubMed Scopus (42) Google Scholar). In this model, mice subjected to repeated social aggression develop behavioral deficits similar to symptoms observed in human mental illness, including social avoidance and decreased preference for natural rewards such as sucrose. Utilizing this model we now report the effect of social stress on plasma lipid profiles and lipid regulation. We have selected the C57BL/6 mouse line for these studies because of its vulnerability to the effects chronic social stress. Additionally, we have analyzed the effect of a Western-style diet high in cholesterol and triglyceride (high-fat diet, HFD) in both groups. CSDS by itself induces insulin resistance, as demonstrated by elevated fasting glucose levels in mice exposed to CSDS. The combination of CSDS with HFD induces a synergistic elevation in the plasma level of non–HDL cholesterol and intrahepatic accumulation of triglycerides—two key features of the metabolic syndrome. We present CSDS as a model of social stress–induced metabolic dysregulation. Male 8–10-week-old C57BL6/J mice (Jackson Laboratories, Bar Harbor, ME) and CD1 retired breeder mice (Charles River, Wilmington, MA) were housed at the University of Texas Southwestern (UTSW) vivarium in a temperature-controlled environment (lights on: 0400–1600) with ad libitum access to water and standard chow (4% fat diet #7001, Harlan-Teklad, Madison, WI) or high-fat diet (protein/carbohydrate/fat: 15.2/42.7/42.0%kcal with 0.2% (w/w) cholesterol, TD.88137, Harlan-Teklad). At the completion of the 40-day study, mice were fasted overnight, and early the following morning, euthanized to acquire of blood and tissues. All animal procedures were carried out in accordance with the UT Southwestern Institutional Animal Care and Use Committee (IACUC) guidelines. Serum was collected from trunk blood after a 3-h fast. 4-(hydroxymercuri)benzoic acid Sodium Salt (#55540, Sigma Aldrich, St. Louis, MO) was added to preserve neuropeptides hormones. Insulin (#90060 Crystal Chem, Downers Grove, IL) and corticosterone (AC-15F1, Immunodiagnostic Systems, Fountain Hills, AZ) concentrations were determined from serum using ELISA kits as per the manufacturer's instructions. RIA kit (Cat. #GL-32K, Millipore, Billerica, MA) was used to measure glucagon levels. Data reported was acquired by the UTSW Mouse Metabolic Phenotyping Core (http://www.utsouthwestern.edu/utsw/home/research/metstudcore/index.html). Body composition was determined by an mq10 series Bruker Minispec (Bruker Optics, The Woodlands, TX) 28 days after defeat. CSDS was carried out using a method reported recently (12Lutter M. Krishnan V. Russo S.J. Jung S. McClung C.A. Nestler E.J. Orexin signaling mediates the antidepressant-like effect of calorie restriction.J. Neurosci. 2008; 28: 3071-3075Crossref PubMed Scopus (181) Google Scholar). Test mice were exposed to a different CD1 strain aggressor mouse each day prior to lights out (1600) for 5 min over a total of 10 days. After the 5 min of physical contact, test mice were separated from the aggressor and placed across a plastic separator with holes, where they remained in sensory contact with the CD1 aggressor for the remainder of the 24 h. Controls were handled daily in the palm of the hand for 30 s and housed in equivalent cages with members of the same strain. After the last defeat, all mice were housed individually, and a social interaction test was performed to measure the behavioral consequences of the chronic defeat stress. Briefly, mice were placed in a new arena with a small animal cage at one end, and their movements tracked for 2.5 min in the absence of another mouse, followed by 2.5 min in the presence of a caged, unfamiliar target CD1 mouse. Social interaction was quantified by comparing the amount of time the test mouse spent in the interaction zone near the small-animal cage in the presence versus the absence of the target CD1 mouse using Ethovision 3.0 software (Noldus, Leesburg, VA). Lipids were extracted from 100–200 mg of frozen tissue using the Folch method. Cholesterol and triglyceride concentrations were obtained using the Triglyceride and Cholesterol Reagent (Fischer Scientific, Pittsburgh, PA) with readings at 515 nm (triglyceride) and 500 nm (cholesterol). The epididymal white adipose, brown adipose, gastrocnemius muscle and liver of each mouse were collected, snap-frozen in liquid nitrogen, and stored at −85°C. Total RNA was isolated from these tissues using RNA STAT-60 (Tel-Test Inc., Friendswood, TX). RNA concentrations were determined by absorbance at 260 nm with a Thermo Scientific Nanodrop 100 Spectrophotometer. The total RNA was treated with RNase-free DNase (Roche, Palo Alto, CA) and reverse-transcribed into cDNA with SuperScript II reagents (Invitrogen, Carlsbad, CA) as previously described (13Kurrasch D.M. Huang J. Wilkie T.M. Repa J.J. Quantitative real-time polymerase chain reaction measurement of regulators of G-protein signaling mRNA levels in mouse tissues.Methods Enzymol. 2004; 389: PubMed Scopus Google Scholar). Quantitative real-time was performed using an and The of the used in I. The mRNA levels to the by the method real-time by the 2008; PubMed Scopus Google Scholar). mouse liver were in and were as previously described of in the of mice and Clin. 2004; PubMed Scopus Google Scholar). concentrations were using the protein kit of liver were separated on an and were to a Pittsburgh, PA) for of the with the growth factor PubMed Scopus Google Scholar) was used with as the against the of human protein was used for the of the protein J. S. O. and high for of a liver protein to the a as J. 7: PubMed Scopus Google Scholar). substrate was used to a by and of were determined by with software of Data reported as the for the number of 5 software Inc., CA) was used to all A of in the of mice and Clin. 2004; PubMed Scopus Google real-time by the 2008; PubMed Scopus Google Scholar) analysis of was performed using CSDS and diet as a interaction was observed between comparison of all was performed by and different by different significant interaction between factors and was effects of one or both factors as an for each significant was as significant was as Following a exposure to CSDS, mice were for social subjected to CSDS spent more time interacting with an than with an unfamiliar mouse social avoidance is a measure of behavior because it by not with O. McClung C.A. Krishnan V. W. Russo S.J. C.A. M. et of in the in social defeat 2006; PubMed Scopus Google Scholar). After for social the mice were into two on interaction either standard chow or a diet high in fat and cholesterol (HFD). mice given access to HFD more and fat than mice mice given either diet an increase in per both of mice a similar number of While the number of was similar between the CSDS was a significant in mice HFD significantly increased In mice on HFD demonstrated a of with significantly fat with mice on HFD we analyzed the lipid profiles of mice subjected to CSDS. The combination of CSDS and HFD significant in both total cholesterol and non–HDL cholesterol levels Serum were not significantly different in not that the of the increase in non–HDL cholesterol was of While was in mice exposed to CSDS significantly levels of than is with a report that demonstrated stress in male resulted in an in The effects of stress on serum and total cholesterol in male after PubMed Scopus Google Scholar). CSDS was associated with increased fasting glucose levels in both accompanied by elevated insulin a of insulin resistance We to this by insulin and glucose mice subjected to CSDS to the effects of resulted in in glucose levels after of not that to insulin and glucose These that the combination of CSDS and access to a HFD two key of the metabolic syndrome: insulin resistance and elevated non–HDL To the signaling underlying these metabolic we collected tissue of white brown and muscle from mice at the of the shown in the combination of social defeat stress and HFD induced a significant increase in the serum level of non–HDL real-time we the expression levels of genes involved in cholesterol synthesis. Cholesterol in the liver is by a series of genes by the factor regulatory element binding protein 2 of the of cholesterol and acid in the Clin. PubMed Scopus Google Scholar). The expression level of both of the cholesterol genes we and a similar of with a Western-style HFD in cholesterol mRNA levels of both Social defeat, increased their regular we analyzed expression levels of the is for cholesterol containing from the blood to cholesterol receptor expression is in mice on it is elevated by CSDS. as as and of the factor Transcriptional of the is and levels the same of expression with HFD and social defeat increasing mRNA levels levels of cholesterol a cholesterol levels by both cholesterol and cholesterol by The to activity after CSDS by a in intrahepatic cholesterol in mice exposed to CSDS. We this by intrahepatic levels of to HFD elevated intrahepatic cholesterol levels in both and CSDS mice that the of activity by cholesterol is after CSDS. In to transcriptional regulation, levels of receptor protein by two convertase subtilisin/kexin type 9 is an target that serum levels of by receptor protein levels M. Anderson N.N. B. 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Insulin signaling is a key of acid in the to CSDS resulted in significantly elevated expression of several genes involved in acid including the factor and its acid stearoyl CoA desaturase 1 and acetyl CoA carboxylase 1a was decreased by CSDS the that many genes by were after including is likely to the that activity is by the binding of its X receptor signaling in cardiovascular PubMed Scopus Google Scholar). To the on the expression of we the expression of genes involved in acid of the acid were decreased by CSDS, including pyruvate dehydrogenase kinase 4 uncoupling protein 2 and fibroblast growth factor 21 These genes of the factor peroxisome proliferator-activated receptor α the level of was increased by HFD The between mRNA levels and activity the that CSDS the transcriptional activity of acid The combination of fasting and observed after CSDS insulin resistance in these Insulin signaling in the to increased expression of SREBP1c, a target of insulin we analyzed of the insulin signaling involved in insulin signaling were by CSDS including insulin receptor and insulin receptor substrate 1 Insulin receptor substrate 2 expression was after the of this is given the of and The glucose transporter 2 was elevated after exposure to social defeat, it decreased by access to involved in a of While phosphoenolpyruvate carboxykinase was significantly after CSDS, effect was observed for glucose-6-phophatase Additionally, CSDS effect on the expression of factors nuclear factor 4a and PPARγ coactivator 1a that we the levels of liver pyruvate kinase a target of the carbohydrate-response element binding protein Repa J.J. The liver X receptor and hepatic The carbohydrate-response protein is a target of Biol. PubMed Scopus Google Scholar). was significantly elevated in chow after social defeat and increased in mice on HFD the of glucose into pyruvate of acid this is with a for social stress in acid We that in mice CSDS composition with significantly decreased levels of fat the levels of a of were in The combination of fat and the that tissues were as an We this by the expression of a involved in While HFD in white tissue and brown was effect of CSDS levels in muscle tissue of mice exposed to CSDS were significantly than mice Additionally, genes involved in including carnitine palmitoyltransferase 1a and patatin-like phospholipase domain containing 2 were decreased by CSDS. In expression is significantly elevated in liver of mice exposed to CSDS While mRNA levels not with protein the expression profiles and that CSDS the of several including white adipose, brown adipose, and to and plasma to the liver where elevated expression of accumulation of intrahepatic levels of transcriptional activity of SREBP1c, and ChREBP acid synthesis. The combination of increased and acid to to accumulation of intrahepatic To test this we triglyceride levels in the The interaction of CSDS and HFD a significant increase in triglyceride levels in the liver is with the increased and and decreased by the observed patterns of expression psychiatric research has on the and consequences of mental mental illness the risk of mortality from such as cardiovascular disease (1Harris E.C. Barraclough B. Excess mortality of mental disorder.Br. J. Psychiatry. 1998; 173: 11-53Crossref PubMed Scopus (1500) Google Scholar). 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