HHS Public Access Author manuscript Author Manuscript
Exp Neurol. Author manuscript; available in PMC 2017 March 01. Published in final edited form as: Exp Neurol. 2016 March ; 277: 261–267. doi:10.1016/j.expneurol.2016.01.007.
The role of endoplasmic reticulum stress in hippocampal insulin resistance Catrina Sims-Robinson1,2,*, Anna Bakeman1, Rebecca Glasser1, Janet Boggs2, Crystal Pacut1, and Eva L. Feldman1 1Department
of Neurology, University of Michigan, Ann Arbor, Michigan, 48109 USA
Author Manuscript
2Department
of Neurology and Neurosurgery, Medical University of South Carolina, Charleston, South Carolina, 29425 USA
Abstract
Author Manuscript
Metabolic syndrome, which includes hypertension, hyperglycemia, obesity, insulin resistance, and dyslipidemia, has a negative impact on cognitive health. Endoplasmic reticulum (ER) stress is activated during metabolic syndrome, however it is not known which factor associated with metabolic syndrome contributes to this stress. ER stress has been reported to play a role in the development of insulin resistance in peripheral tissues. The role of ER stress in the development of insulin resistance in hippocampal neurons is not known. In the current study, we investigated ER stress in the hippocampus of 3 different mouse models of metabolic syndrome: the C57BL6 mouse on a high fat (HF) diet; apolipoprotein E, leptin, and apolipoprotein B-48 deficient (ApoE 3KO) mice; and the low density lipoprotein receptor, leptin, and apolipoprotein B-48 deficient (LDLR 3KO) mice. We demonstrate that ER stress is activated in the hippocampus of HF mice, and for the first time, in ApoE 3KO mice, but not LDLR 3KO mice. The HF and ApoE 3KO mice are hyperglycemic; however, the LDLR 3KO mice have normal glycemia. This suggests that hyperglycemia may play a role in the activation of ER stress in the hippocampus. Similarly, we also demonstrate that impaired insulin signaling is only present in the HF and ApoE 3KO mice, which suggests that ER stress may play a role in insulin resistance in the hippocampus. To confirm this we pharmacologically induced ER stress with thapsigargin in human hippocampal neurons. We demonstrate for the first time that thapsigargin leads to ER stress and impaired insulin signaling in human hippocampal neurons. Our results may provide a potential mechanism that links metabolic syndrome and cognitive health.
Author Manuscript
Keywords metabolic syndrome; brain; endoplasmic reticulum stress; insulin; hippocampus; apolipoprotein *
Address to whom all correspondence should be sent: Catrina Sims-Robinson, Ph.D., Medical University of South Carolina, Department of Neurology and Neurosurgery, 96 Jonathan Lucas Street, 309D2 Clinical Sciences Building, MSC 606, Charleston, SC 29445, (843) 792-0851 (phone), (843) 876-1220 (fax), [email protected]. Conflict of Interest The authors declare that they have no conflict of interest
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Sims-Robinson et al.
Page 2
Author Manuscript
Introduction
Author Manuscript
Metabolic syndrome is the term used for the presence of multiple conditions including hypertension, hyperglycemia, obesity, insulin resistance, and dyslipidemia. Metabolic syndrome is a risk factor for neurological disorders including stroke, dementia, and depression (Baker et al., 2010; Biessels et al., 2006; Correia et al., 2011; de la Monte, 2009; Haan, 2006; Neumann et al., 2008; Profenno et al., 2010; Zhao and Townsend, 2009). Several studies have demonstrated that factors associated with metabolic syndrome have a negative impact on cognitive health (Dahl and Hassing, 2013; Farr et al., 2008; Luchsinger et al., 2007; Novak and Hajjar, 2010; Takahashi et al., 2011). Given the prevalence of metabolic syndrome and the devastation associated with neurological disorders, it is imperative to understand the impact of metabolic syndrome on the brain and to uncover the molecular mechanisms that may drive these changes.
Author Manuscript
It is generally believed that hyperglycemia and impaired glucose tolerance are the major factors that play a role in stress activation and insulin resistance (Back and Kaufman, 2012; Boden et al., 2014; Malhotra and Kaufman, 2007). The endoplasmic reticulum (ER) plays a major role in the response to stress. ER stress is the consequence of either a physiological or pathological disturbance in protein folding, the function of intracellular calcium stores, or the synthesis of fatty acids, sterols, or phospholipids (Schroder, 2008). The increase in misfolded proteins in the ER lumen during ER stress leads to the activation of the unfolded protein response (UPR) (Lin et al., 2008; Ron and Walter, 2007; Schroder, 2008). The purpose of the UPR is to relieve the burden on the ER by increasing ER folding capacity, decreasing protein synthesis (translation), and/or degrading unfolded proteins through the ubiquitin-proteasome system or autophagy, otherwise apoptosis is activated (Eizirik et al., 2008; Lin et al., 2008; Schroder, 2008). The UPR is generated through the action of three transmembrane proteins, one of which is the inositol-requiring enzyme 1 (IRE1), the most conserved branch of the UPR (Lin et al., 2008). Activation of IRE1 signals x-box binding protein 1 (Xbp1) mRNA. Xbp1 is a transcription factor and one of the master regulators of the ER folding capacity (Sriburi et al., 2004). An intron is excised during the UPR, yielding spiced (s) Xbp1mRNA which encodes XBP1s protein (Ron and Walter, 2007). XBP1s is translocated into the nucleus to induce the expression of ER chaperones—heat shock protein 5 (also known as BiP) and heat shock protein 90 beta member 1. These heat shock proteins increase the folding capacity of the ER (Schroder, 2008).
Author Manuscript
ER stress is implicated as a mechanism underlying insulin resistance in peripheral tissues including liver, pancreas, and adipose (Hotamisligil, 2010; Ozcan et al., 2004). Insulin resistance is characterized by a decreased response to normal circulating insulin levels in a particular tissue. In the periphery, insulin resistance occurs following hyperinsulinemia and subsequent desensitization of insulin receptors, which results in decrease in the expression of the receptor (Draznin, 2006). This desensitization is due to an increase in the serine phosphorylation of the insulin receptor substrate 1 (IRS1). The serine phosphorylation of IRS1 reduces the ability of IRS proteins to attract PI 3-kinase, thereby decreasing its activation and downstream insulin signaling (Aguirre et al., 2002; Birnbaum, 2001; Patti and Kahn, 2004; Qiao et al., 1999; Qiao et al., 2002; Um et al., 2004; White, 2003). ER stress-
Exp Neurol. Author manuscript; available in PMC 2017 March 01.
Sims-Robinson et al.
Page 3
Author Manuscript
induced JNK activation leads to the phosphorylation of IRS1 at the serine 307 residue (Belgardt and Bruning, 2010; Ropelle et al., 2010). While insulin resistance is reported in both neurons and in the brain of a type 2 mouse model of diabetes (Kim et al., 2011a; Kim et al., 2011b), the mechanism underlying insulin resistance in the brain is not known. Given the fact that brain insulin plays a role in strengthening synapses, enhancing memory, and may play a role in glucose uptake in certain regions under pathologic conditions (Bingham et al., 2002; Figlewicz et al., 1993a; Figlewicz et al., 1993b; Skeberdis et al., 2001), understanding the mechanism may provide a new therapeutic target to prevent neurological complications (Sims-Robinson et al., 2010).
Author Manuscript
It is not known which aspect of the metabolic syndrome contributes to the activation of ER stress and whether or not this stress can impair insulin signaling in neurons of the hippocampus. It is generally believed that hyperglycemia and impaired glucose tolerance are the major factors that play a role in stress activation and insulin resistance (Back and Kaufman, 2012; Boden et al., 2014; Malhotra and Kaufman, 2007). Therefore, the purpose of this study was to understand which factors associated with the metabolic syndrome contribute to ER stress and brain insulin resistance. To address this we used three different animal models of the metabolic syndrome, the B6 mouse fed a HF diet, the ApoE 3KO and the LDLR 3KO mouse models. The high fat diet (HF) mouse model encompasses many of the facets of metabolic syndrome, including hyperinsulinemia, hyperglycemia, glucose intolerance, hypercholesterolemia, and hypertension (Buettner et al., 2007; Oakes et al., 1997; Tschop and Heiman, 2001; Vincent et al., 2009; Williams et al., 2003). The one major disadvantage of the model is that the major lipid carrying molecule in the B6 HF dietinduced mouse is the high-density lipoprotein (HDL), whereas the low density lipoprotein (LDL) is the major lipid carrying molecule in man (Kennedy et al., 2010). Triple knockout mice deficient in ApoB-48, leptin (ob/ob), and either Apolipoprotein E (ApoE) or LDL receptor (LDLR) have a lipoprotein profile similar to man; these models are known as the ApoE triple knockout (3KO) or the LDLR 3KO, respectively (Lloyd et al., 2008). The ApoE 3KO mice are obese, hyperinsulinemic, hyperglycemic, glucose intolerant, hypercholesterolemic, and hyperlipidemic (Lloyd et al., 2008). The LDLR 3KO mice have a similar phenotype, however, the LDLR 3KO mice are not hyperglycemic or glucose intolerant (Lloyd et al., 2008). Furthermore, to evaluate the role of ER stress in brain insulin resistance, we performed in vitro studies in human hippocampal neurons to pharmacologically induce ER stress using thapsigargin, an agent that disrupts calcium homeostasis (Samali et al., 2010).
Author Manuscript
Materials and methods Author Manuscript
Animals B6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME) at 4 weeks of age and fed either a standard 10% fat chow (Research diet #D12450B; New Brunswick, NJ) or a 54% fat chow (Research diets #D05090701) ad libitum. Triple knockout ApoE−/− ApoB100only/ob/+ and LDLR−/− ApoB100only/ob/+ mice were generated by Dr. Murielle Veniant and colleagues (Lloyd et al., 2008) and bred to obtain ApoE−/− ApoB100only/ob/+ (ApoE/ob+), ApoE−/− ApoB100only/ob/ob (ApoE 3KO), LDLR−/− ApoB100only/ob/+
Exp Neurol. Author manuscript; available in PMC 2017 March 01.
Sims-Robinson et al.
Page 4
Author Manuscript
(LDLR/ob+), and LDLR−/− ApoB100only/ob/ob (LDLR 3KO) littermates. Mice were weaned at 4 weeks of age and fed standard chow. Male mice were used in all experiments. All data were collected at 20–24 weeks of age. Mice were housed in a pathogen-free environment. All protocols and procedures were approved by the University of Michigan Committee on the Use and Care of Animals (UCUCA; approval number PRO00003694), and are in compliance with University guidelines, State and Federal regulations, and the standards of the “Guide for the Care and Use of Laboratory Animals.” The University’s Animal Welfare Assurance Number on file with NIH Office of Laboratory Animal Welfare (OLAW) is A3114-01, and the University is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC, Intl.).
Author Manuscript
Phenotypic measures Body weights were monitored monthly using a standard laboratory scale. Fasting blood glucose levels were obtained using a standard Glucometer (One Touch Ultra, Milpitas, CA) per our previously published protocol (Vincent et al., 2009). Plasma insulin levels were also measured per our previously published protocol (Hinder et al., 2013; Vincent et al., 2009). Tissue Preparation The mice were euthanized according to our published protocols (Kim et al., 2011b) with an overdose of sodium pentobarbital. The hippocampus was dissected from each hemisphere and flash frozen in liquid nitrogen and stored at −80°C until use for western immunoblotting.
Author Manuscript
Human Hippocampal Neurons
Author Manuscript
Western Immunoblotting
Primary Human Hippocampal Neurons (NEURALSTEM INC., Rockville, MD) are a human neural stem cell line of hippocampal origin that has been conditionally immortalized with cMyc-ER. Hippocampal neurons are maintained in N2b medium containing 1 μg/ml fibronectin and 10 μg/ml bFGF growth supplement, with fresh media given every other day. To exclude the effect of insulin, the culture medium is changed to treatment media (NSDM-1 without insulin) 24 hr before experimentation. ER stress is pharmacologically induced in the hippocampal neurons with the addition of 5 nM thapsigargin (Sigma Aldrich, St. Louis, MO) in DMSO for 2–48 hr. Hippocampal neurons were lysed in RIPA buffer (Pierce, Rockford, IL) containing protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN) and stored at −80°C until use for western blotting.
Western immunoblotting was performed as previously described (Kim et al., 2011a; Kim et al., 2011b). The hippocampus was homogenized in tissue protein extraction reagent (Pierce, Rockford, IL) containing a protease inhibitor cocktail (Calbiochem, San Diego, CA). The lysates were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Trisbuffered saline with Tween-20 supplemented with either 3% bovine serum albumin (BSA; Sigma Aldrich, St. Louis, MO) or 5% milk was used to block the membrane and to dilute the antibodies. Polyclonal antibodies against phospho-JNK, XBP1 (Santa Cruz Exp Neurol. Author manuscript; available in PMC 2017 March 01.
Sims-Robinson et al.
Page 5
Author Manuscript
Biotechnology, INC., Santa Cruz, CA), BiP (Abcam, Cambridge, MA), pIRS1 serine 307, IRS1 (Cell Signaling, Danvers, MA), and glyceraldehyde 3-phosphoate dehydrogenase (GAPDH; EMD Millipore, Billerica, Massachusetts) were used for western immunoblotting. The signal was visualized using LumiGLO enhanced chemiluminescence reagent (Cell Signaling Technology, Danvers, MA). Images were captured using the Chemidoc XRS system and analyzed by Quantity One software (Bio-Rad Laboratory, Hercules, CA). Statistical Analysis
Author Manuscript
Data analyses were performed using Prism, version 6 (GraphPad Software, Inc.). Assumptions about Gaussian distribution of data were made using the D’Agostino & Pearson omnibus normality test. At least 8 mice per group were used for metabolic studies. For all other measures at least 5 mice per group were used. T-test was used to compare control versus HF, ApoE/ob+ versus ApoE 3KO, or LDLR/ob+ versus LDLR 3KO. For all experiments, *p
Exp Neurol. Author manuscript; available in PMC 2017 March 01. Published in final edited form as: Exp Neurol. 2016 March ; 277: 261–267. doi:10.1016/j.expneurol.2016.01.007.
The role of endoplasmic reticulum stress in hippocampal insulin resistance Catrina Sims-Robinson1,2,*, Anna Bakeman1, Rebecca Glasser1, Janet Boggs2, Crystal Pacut1, and Eva L. Feldman1 1Department
of Neurology, University of Michigan, Ann Arbor, Michigan, 48109 USA
Author Manuscript
2Department
of Neurology and Neurosurgery, Medical University of South Carolina, Charleston, South Carolina, 29425 USA
Abstract
Author Manuscript
Metabolic syndrome, which includes hypertension, hyperglycemia, obesity, insulin resistance, and dyslipidemia, has a negative impact on cognitive health. Endoplasmic reticulum (ER) stress is activated during metabolic syndrome, however it is not known which factor associated with metabolic syndrome contributes to this stress. ER stress has been reported to play a role in the development of insulin resistance in peripheral tissues. The role of ER stress in the development of insulin resistance in hippocampal neurons is not known. In the current study, we investigated ER stress in the hippocampus of 3 different mouse models of metabolic syndrome: the C57BL6 mouse on a high fat (HF) diet; apolipoprotein E, leptin, and apolipoprotein B-48 deficient (ApoE 3KO) mice; and the low density lipoprotein receptor, leptin, and apolipoprotein B-48 deficient (LDLR 3KO) mice. We demonstrate that ER stress is activated in the hippocampus of HF mice, and for the first time, in ApoE 3KO mice, but not LDLR 3KO mice. The HF and ApoE 3KO mice are hyperglycemic; however, the LDLR 3KO mice have normal glycemia. This suggests that hyperglycemia may play a role in the activation of ER stress in the hippocampus. Similarly, we also demonstrate that impaired insulin signaling is only present in the HF and ApoE 3KO mice, which suggests that ER stress may play a role in insulin resistance in the hippocampus. To confirm this we pharmacologically induced ER stress with thapsigargin in human hippocampal neurons. We demonstrate for the first time that thapsigargin leads to ER stress and impaired insulin signaling in human hippocampal neurons. Our results may provide a potential mechanism that links metabolic syndrome and cognitive health.
Author Manuscript
Keywords metabolic syndrome; brain; endoplasmic reticulum stress; insulin; hippocampus; apolipoprotein *
Address to whom all correspondence should be sent: Catrina Sims-Robinson, Ph.D., Medical University of South Carolina, Department of Neurology and Neurosurgery, 96 Jonathan Lucas Street, 309D2 Clinical Sciences Building, MSC 606, Charleston, SC 29445, (843) 792-0851 (phone), (843) 876-1220 (fax), [email protected]. Conflict of Interest The authors declare that they have no conflict of interest
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Sims-Robinson et al.
Page 2
Author Manuscript
Introduction
Author Manuscript
Metabolic syndrome is the term used for the presence of multiple conditions including hypertension, hyperglycemia, obesity, insulin resistance, and dyslipidemia. Metabolic syndrome is a risk factor for neurological disorders including stroke, dementia, and depression (Baker et al., 2010; Biessels et al., 2006; Correia et al., 2011; de la Monte, 2009; Haan, 2006; Neumann et al., 2008; Profenno et al., 2010; Zhao and Townsend, 2009). Several studies have demonstrated that factors associated with metabolic syndrome have a negative impact on cognitive health (Dahl and Hassing, 2013; Farr et al., 2008; Luchsinger et al., 2007; Novak and Hajjar, 2010; Takahashi et al., 2011). Given the prevalence of metabolic syndrome and the devastation associated with neurological disorders, it is imperative to understand the impact of metabolic syndrome on the brain and to uncover the molecular mechanisms that may drive these changes.
Author Manuscript
It is generally believed that hyperglycemia and impaired glucose tolerance are the major factors that play a role in stress activation and insulin resistance (Back and Kaufman, 2012; Boden et al., 2014; Malhotra and Kaufman, 2007). The endoplasmic reticulum (ER) plays a major role in the response to stress. ER stress is the consequence of either a physiological or pathological disturbance in protein folding, the function of intracellular calcium stores, or the synthesis of fatty acids, sterols, or phospholipids (Schroder, 2008). The increase in misfolded proteins in the ER lumen during ER stress leads to the activation of the unfolded protein response (UPR) (Lin et al., 2008; Ron and Walter, 2007; Schroder, 2008). The purpose of the UPR is to relieve the burden on the ER by increasing ER folding capacity, decreasing protein synthesis (translation), and/or degrading unfolded proteins through the ubiquitin-proteasome system or autophagy, otherwise apoptosis is activated (Eizirik et al., 2008; Lin et al., 2008; Schroder, 2008). The UPR is generated through the action of three transmembrane proteins, one of which is the inositol-requiring enzyme 1 (IRE1), the most conserved branch of the UPR (Lin et al., 2008). Activation of IRE1 signals x-box binding protein 1 (Xbp1) mRNA. Xbp1 is a transcription factor and one of the master regulators of the ER folding capacity (Sriburi et al., 2004). An intron is excised during the UPR, yielding spiced (s) Xbp1mRNA which encodes XBP1s protein (Ron and Walter, 2007). XBP1s is translocated into the nucleus to induce the expression of ER chaperones—heat shock protein 5 (also known as BiP) and heat shock protein 90 beta member 1. These heat shock proteins increase the folding capacity of the ER (Schroder, 2008).
Author Manuscript
ER stress is implicated as a mechanism underlying insulin resistance in peripheral tissues including liver, pancreas, and adipose (Hotamisligil, 2010; Ozcan et al., 2004). Insulin resistance is characterized by a decreased response to normal circulating insulin levels in a particular tissue. In the periphery, insulin resistance occurs following hyperinsulinemia and subsequent desensitization of insulin receptors, which results in decrease in the expression of the receptor (Draznin, 2006). This desensitization is due to an increase in the serine phosphorylation of the insulin receptor substrate 1 (IRS1). The serine phosphorylation of IRS1 reduces the ability of IRS proteins to attract PI 3-kinase, thereby decreasing its activation and downstream insulin signaling (Aguirre et al., 2002; Birnbaum, 2001; Patti and Kahn, 2004; Qiao et al., 1999; Qiao et al., 2002; Um et al., 2004; White, 2003). ER stress-
Exp Neurol. Author manuscript; available in PMC 2017 March 01.
Sims-Robinson et al.
Page 3
Author Manuscript
induced JNK activation leads to the phosphorylation of IRS1 at the serine 307 residue (Belgardt and Bruning, 2010; Ropelle et al., 2010). While insulin resistance is reported in both neurons and in the brain of a type 2 mouse model of diabetes (Kim et al., 2011a; Kim et al., 2011b), the mechanism underlying insulin resistance in the brain is not known. Given the fact that brain insulin plays a role in strengthening synapses, enhancing memory, and may play a role in glucose uptake in certain regions under pathologic conditions (Bingham et al., 2002; Figlewicz et al., 1993a; Figlewicz et al., 1993b; Skeberdis et al., 2001), understanding the mechanism may provide a new therapeutic target to prevent neurological complications (Sims-Robinson et al., 2010).
Author Manuscript
It is not known which aspect of the metabolic syndrome contributes to the activation of ER stress and whether or not this stress can impair insulin signaling in neurons of the hippocampus. It is generally believed that hyperglycemia and impaired glucose tolerance are the major factors that play a role in stress activation and insulin resistance (Back and Kaufman, 2012; Boden et al., 2014; Malhotra and Kaufman, 2007). Therefore, the purpose of this study was to understand which factors associated with the metabolic syndrome contribute to ER stress and brain insulin resistance. To address this we used three different animal models of the metabolic syndrome, the B6 mouse fed a HF diet, the ApoE 3KO and the LDLR 3KO mouse models. The high fat diet (HF) mouse model encompasses many of the facets of metabolic syndrome, including hyperinsulinemia, hyperglycemia, glucose intolerance, hypercholesterolemia, and hypertension (Buettner et al., 2007; Oakes et al., 1997; Tschop and Heiman, 2001; Vincent et al., 2009; Williams et al., 2003). The one major disadvantage of the model is that the major lipid carrying molecule in the B6 HF dietinduced mouse is the high-density lipoprotein (HDL), whereas the low density lipoprotein (LDL) is the major lipid carrying molecule in man (Kennedy et al., 2010). Triple knockout mice deficient in ApoB-48, leptin (ob/ob), and either Apolipoprotein E (ApoE) or LDL receptor (LDLR) have a lipoprotein profile similar to man; these models are known as the ApoE triple knockout (3KO) or the LDLR 3KO, respectively (Lloyd et al., 2008). The ApoE 3KO mice are obese, hyperinsulinemic, hyperglycemic, glucose intolerant, hypercholesterolemic, and hyperlipidemic (Lloyd et al., 2008). The LDLR 3KO mice have a similar phenotype, however, the LDLR 3KO mice are not hyperglycemic or glucose intolerant (Lloyd et al., 2008). Furthermore, to evaluate the role of ER stress in brain insulin resistance, we performed in vitro studies in human hippocampal neurons to pharmacologically induce ER stress using thapsigargin, an agent that disrupts calcium homeostasis (Samali et al., 2010).
Author Manuscript
Materials and methods Author Manuscript
Animals B6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME) at 4 weeks of age and fed either a standard 10% fat chow (Research diet #D12450B; New Brunswick, NJ) or a 54% fat chow (Research diets #D05090701) ad libitum. Triple knockout ApoE−/− ApoB100only/ob/+ and LDLR−/− ApoB100only/ob/+ mice were generated by Dr. Murielle Veniant and colleagues (Lloyd et al., 2008) and bred to obtain ApoE−/− ApoB100only/ob/+ (ApoE/ob+), ApoE−/− ApoB100only/ob/ob (ApoE 3KO), LDLR−/− ApoB100only/ob/+
Exp Neurol. Author manuscript; available in PMC 2017 March 01.
Sims-Robinson et al.
Page 4
Author Manuscript
(LDLR/ob+), and LDLR−/− ApoB100only/ob/ob (LDLR 3KO) littermates. Mice were weaned at 4 weeks of age and fed standard chow. Male mice were used in all experiments. All data were collected at 20–24 weeks of age. Mice were housed in a pathogen-free environment. All protocols and procedures were approved by the University of Michigan Committee on the Use and Care of Animals (UCUCA; approval number PRO00003694), and are in compliance with University guidelines, State and Federal regulations, and the standards of the “Guide for the Care and Use of Laboratory Animals.” The University’s Animal Welfare Assurance Number on file with NIH Office of Laboratory Animal Welfare (OLAW) is A3114-01, and the University is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC, Intl.).
Author Manuscript
Phenotypic measures Body weights were monitored monthly using a standard laboratory scale. Fasting blood glucose levels were obtained using a standard Glucometer (One Touch Ultra, Milpitas, CA) per our previously published protocol (Vincent et al., 2009). Plasma insulin levels were also measured per our previously published protocol (Hinder et al., 2013; Vincent et al., 2009). Tissue Preparation The mice were euthanized according to our published protocols (Kim et al., 2011b) with an overdose of sodium pentobarbital. The hippocampus was dissected from each hemisphere and flash frozen in liquid nitrogen and stored at −80°C until use for western immunoblotting.
Author Manuscript
Human Hippocampal Neurons
Author Manuscript
Western Immunoblotting
Primary Human Hippocampal Neurons (NEURALSTEM INC., Rockville, MD) are a human neural stem cell line of hippocampal origin that has been conditionally immortalized with cMyc-ER. Hippocampal neurons are maintained in N2b medium containing 1 μg/ml fibronectin and 10 μg/ml bFGF growth supplement, with fresh media given every other day. To exclude the effect of insulin, the culture medium is changed to treatment media (NSDM-1 without insulin) 24 hr before experimentation. ER stress is pharmacologically induced in the hippocampal neurons with the addition of 5 nM thapsigargin (Sigma Aldrich, St. Louis, MO) in DMSO for 2–48 hr. Hippocampal neurons were lysed in RIPA buffer (Pierce, Rockford, IL) containing protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN) and stored at −80°C until use for western blotting.
Western immunoblotting was performed as previously described (Kim et al., 2011a; Kim et al., 2011b). The hippocampus was homogenized in tissue protein extraction reagent (Pierce, Rockford, IL) containing a protease inhibitor cocktail (Calbiochem, San Diego, CA). The lysates were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Trisbuffered saline with Tween-20 supplemented with either 3% bovine serum albumin (BSA; Sigma Aldrich, St. Louis, MO) or 5% milk was used to block the membrane and to dilute the antibodies. Polyclonal antibodies against phospho-JNK, XBP1 (Santa Cruz Exp Neurol. Author manuscript; available in PMC 2017 March 01.
Sims-Robinson et al.
Page 5
Author Manuscript
Biotechnology, INC., Santa Cruz, CA), BiP (Abcam, Cambridge, MA), pIRS1 serine 307, IRS1 (Cell Signaling, Danvers, MA), and glyceraldehyde 3-phosphoate dehydrogenase (GAPDH; EMD Millipore, Billerica, Massachusetts) were used for western immunoblotting. The signal was visualized using LumiGLO enhanced chemiluminescence reagent (Cell Signaling Technology, Danvers, MA). Images were captured using the Chemidoc XRS system and analyzed by Quantity One software (Bio-Rad Laboratory, Hercules, CA). Statistical Analysis
Author Manuscript
Data analyses were performed using Prism, version 6 (GraphPad Software, Inc.). Assumptions about Gaussian distribution of data were made using the D’Agostino & Pearson omnibus normality test. At least 8 mice per group were used for metabolic studies. For all other measures at least 5 mice per group were used. T-test was used to compare control versus HF, ApoE/ob+ versus ApoE 3KO, or LDLR/ob+ versus LDLR 3KO. For all experiments, *p
