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Heat shock protein expression in response to stress and diabetes Hoekstra, Kenneth Andrew 1997

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HEAT SHOCK PROTEIN EXPRESSION IN RESPONSE TO STRESS AND DIABETES by KENNETH ANDREW HOEKSTRA B.Sc, Trinity Western University, 1993 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE FACULTY OF GRADUATE STUDIES (Department of Animal Science) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA MAY 1997 © KENNETH ANDREW HOEKSTRA, 1997 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of /9y? rV&vaJ, Sc2.) The University of British Columbia Vancouver, Canada Date ^ P r S ? _ , 7 J T 1 DE-6 (2/88) 11 A B S T R A C T The exposure of cells to a wide range of stressors results in a highly synchronized, genetically determined response, initiated by detection of the stressor, which in turn leads to a regulatory response that involves the elevated synthesis of a specific set of proteins, termed heat shock proteins (HSPs), which serve to counteract the initial damage,and re-establish cellular homeostasis. The expression of HSP30, 60, 70, and 90 was measured in the heart, liver, kidney, lung, and gonads of Japanese quail exposed to seven different stressors (mild restraint, loud noise, inescapable irritation, cold temperature, isolation in darkness and two stressful social situations), and in the heart, liver, kidney and lung of non-diabetic and diabetic rats exposed to seven individual stressors. Tonic Immobility (Tl) tests were also conducted on Japanese quail to assess whether or not the stressors increased fear response. Increased expression of HSP70 was found in the heart tissue of birds exposed to loud noise, inescapable irritation, cold temperature and isolation in darkness. The expression of other HSPs was not apparent in the heart or any of the other tissues examined. Longer Tl was observed only in birds exposed to the noise stress. Increased expression of HSP30 and HSP70 was found in the heart tissue of non-diabetic, stressed rats. Increased expression of HSP30 and HSP70 was also found in acute (4 weeks) and chronic (12 weeks) streptozotocin rats. In the liver, increased HSP70 expression was found for acute Ill and chronic diabetic treatments. In the kidney, increased HSP70 expression was found only in chronic diabetes. No change in HSP 60 or 90 was detected in any tissues examined and the lung did not show any HSP increase. These results support the possibility of a tissue- and class-specific HSP response when exposed to a variety of stressors. TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iv LIST OF FIGURES vi LIST OF TABLES viii LIST OF ABBREVIATIONS ix ACKNOWLEDGMENTS xi CHAPTER ONE: 1.1 General Introduction 1 1.2 The Heat Shock Proteins 2 1.2.1 HSP30 4 1.2.2 HSP60 5 1.2.3 HSP70 7 1.2.4 HSP90 10 CHAPTER TWO: Whole-animal stress and the in vivo expression of heat shock protein-70 in birds 13 2.1 Introduction 13 2.2 Materials and Methods 14 2.2.1 Experimental Treatments 14 2.2.2 Tl Testing 16 2.2.3 Tissue Sampling 17 2.2.4 Western Blotting 18 2.2.5 Densitometric quantification of HSP levels 20 2.2.6 Statistical Analysis 20 2.3 Results 21 2.3.1 HSP expression 2.3.1.1 HSP70 22 2.3.1.2 HSP30, 60, and 90 22 2.3.2 Tl duration 32 2.4 Discussion 33 CHAPTER THREE The effects of restraint stress on the expression of heat shock proteins in the diabetic rat 36 3.1 Introduction 36 3.2 Methods 38 3.2.1 Treatment protocol 38 3.2.2 Tissue Preparation 41 3.2.3 Statistical Analysis 42 3.3 Results 42 3.3.1 Heart Tissue 43 3.3.2 Liver Tissue 52 3.3.3 Kidney Tissue 52 3.3.4 Lung Tissue 54 3.4 Discussion 59 CHAPTER FOUR General summary and conclusions 67 REFERENCES 70 vi L I S T O F F I G U R E S Page Figure 2.1 HSP70 expression and mean temperature decline after cold temperature stress 23 Figure 2.2 Expression of HSP70 in the heart following isolation in darkness stress 24 Figure 2.3 Expression of HSP70 in the heart following noise stress 25 Figure 2.4 Expression of HSP70 in the heart following cold temperature stress 26 Figure 2.5 Expression of HSP70 in the heart following inescapable irritation 27 Figure 2.6 Summary of results from isolation in darkness stress on HSP70 expression in heart tissue 28 Figure 2.7 Summary of results from noise stress on HSP70 expression in heart tissue 29 Figure 2.8 Summary of results from cold temperature stress on HSP70 expression in heart tissue 30 Figure 2.9 Summary of results from inescapable irritation on HSP70 expression in heart tissue 31 Figure 3.1 Expression of HSP70 in the heart/vascular tissue of rats following restraint and/or four weeks diabetic 44 Figure 3.2 Summary of HSP70 expression in the heart/vascular tissue of restraint and/or four weeks diabetic 45 Figure 3.3 Expression of HSP70 in the heart/vascular tissue of rats following restraint and/or 12 weeks diabetic 46 Figure 3.4 Summary of HSP70 expression in the heart/vascular tissue of restraint and/or 12 weeks diabetic 47 Figure 3.5 Expression of HSP30 in the heart/vascular tissue of following restraint and/or 2 wks diabetes 48 Figure 3.6 Summary of results of HSP30 expression in heart/vascular tissue 49 Figure 3.7 Expression of HSP30 in the heart/vascular tissue of following restraint and/or 10 wks diabetes 50 Figure 3.8 Summary of results of HSP30 expression in heart/vascular tissue 51 Figure 3.9 Expression of HSP70 in liver tissue of rats after 2 weeks diabetes and/or restraint stress 53 Figure 3.10 Summary of HSP70 expression in the liver tissue of rats after 2 weeks diabetes and/or restraint stress 54 Figure 3.11 Expression of HSP70 in liver tissue of rats after 10 weeks diabetes and/or restraint stress 55 Figure 3.12 Summary of HSP70 expression in the liver tissue of rats after 10 weeks diabetes and/or restraint stress 56 Figure 3.13 Expression of HSP70 in the kidney tissue of rats after induction of diabetes 57 Figure 3.14 Summary of results of HSP70 expression in the kidney 58 viii LIST OF TABLES P A G E Table 1 Summary of heat shock protein expression 42 ix LIST OF ABBREVIATIONS ATP -adenosine 5'-triphosphate BSA -bovine serum albumin cDNA -complementary deoxyribonucleic acid cpn -chaperonins DNA -deoxyribonucleic acid EDTA -ethelenediaminetetetraacetic acid HSC -constitutive heat shock protein HSP30 -heat shock protein 30 HSP60 -heat shock protein 60 HSP70 -heat shock protein 70 HSP90 -heat shock protein 90 IDDM -insulin-dependent diabetes mellitus NIDDM -non-insulin dependent diabetes mellitus IL-1 -interleukin -1 min -minute ml_ -milliliters P D G F -platelet derived growth factor P M S F -phenylmethylsulfonylfluroide rubisco -ribulose-bisphosphate carboxylase SDS -sodium dodecyl sulfate sec -second S D S - P A G E -sodium dodecyl sulfate-polyacrylamide gel electrophoresis STZ -streptozotocin TCP-1 -tailless complex polypeptide Tl -tonic immobility Tris -Tris [hydroxymethyl] aminomethane ACKNOWLEDGMENTS I wish to thank Dr. Kimberly Cheng for his encouragement, support, and generosity throughout this study. I thank Drs. G. K. Iwama, D. V. Godin, P. Candido for support and comments on this manuscript. I am grateful to Carl Mazur and Robert Forsyth for enjoyable conversations and technical advice. I thank Sulemon Bhatti for his friendship and many enjoyable conversations both inside and outside the lab. I thank my family for their ongoing support of my ideas and love for research. I especially wish to thank Kamla Jagroop whose everlasting love, support, friendship, and patience never faulted and to whom I will always be grateful 1 C H A P T E R O N E 1.1 GENERAL INTRODUCTION Successful adaptation to stress is a prerequisite for the survival of all organisms living in an environment in which noxious stimuli are constantly present. It is not surprising that organisms have developed an array of integrated stress-responsive systems that work in concert to return the host to a sustainable homeostatic plane. Higher organisms, including human beings, have developed complex mechanisms to tolerate the myriad of insults that occur to cellular constituents and organ systems as a result of trauma and disease. The short-term effects result in rapid actions such as cardiovascular and metabolic responses that benefit the host in a "fight or flight" reaction. The long-term effects generally occur through alterations in gene transcription that prepare the host for or adapt the host to repetitive or chronic stress. Both short and long-term responses lead to the production of stress-responsive proteins. These stress-responsive proteins, termed heat shock proteins (HSPs), have an enormous functional capacity: they alter enzymatic pathways, modulate hormone levels, and regulate the expression of subsequent HSPs (Ellis, 1996). The importance of the HSPs in the host response to short and long-term stress and their intimate association with the activation of other physiological pathways have only recently begun to be appreciated (Schoel and Kaufmann, 1996). It was the purpose of these investigations to study the effects of different stresses on HSP expression in normal and diabetic animals. A brief introduction to the various heat shock protein families follows and will precede a description of two related but independent investigations, the methods used, experimental results, and their individual discussion. The last chapter will consist of a general summary and conclusions. 1.2 THE HEAT SHOCK PROTEINS Traditionally, the HSPs are defined as those whose synthesis is dramatically induced at relatively high temperatures. Although much investigation has focused on their synthesis following heat shock, it is apparent that they have many functions in the unstressed cell as well. Heat shock proteins are involved in multiple stages of protein biogenesis beginning with synthesis and involvement in the subsequent events of folding, translocation, and degradation. Thus, It is important to realize that HSPs are critical to maintaining cell homeostasis and integrity during normal physiological growth and differentiation, as well as in response to the stressful event of imminent loss of normal physiology (Ellis, 1996). Heat shock proteins are defined as those proteins essential for the survival of cells of all species under various aspects of cellular 3 homeostasis through protective or adaptive functions (Hartl et al., 1994; Hendrick and Hartl, 1993; Gething and Sambrook, 1992). As some key features of each HSP class are discussed, it will become apparent that, in many cases, HSPs from different families cooperate with one another in modulating a number of cellular processes (Minowada and Welch, 1996). One such process for example is molecular chaperoning. The term "molecular chaperone" was originally used to describe nucleoplasms and then the chloroplast ribulose-bisphosphate carboxylase (rubisco)-binding protein, which were observed to promote the oligomeric assembly of nucleosomes and rubisco (Ellis, 1987). In addition it has been proposed that molecular chaperones are involved in disassembly of oligomeric structures (Pelham, 1986). The term molecular chaperones has subsequently been extended to include proteins which bind to and stablize the non-native conformations of other proteins and facilitate their correct folding by releasing them in a controlled manner. This "molecular chaperone" concept can accommodate at least some aspects of the stress response by supposing that the need for these chaperones increases when proteins are damaged by stress; if the cell is to survive the stress, such damaged proteins need to be refolded correctly or removed by proteolysis whereas undamaged proteins need to be protected against subsequent stresses that might create incorrect assemblies (Ellis, 1987). Thus, the stress response can be viewed as an extension of a basic chaperone function that all cells 4 require under normal conditions rather than a unique function required only under stress conditions (Ellis, 1996). The predominant role of molecular chaperones appears to be preventing the incorrect intermolecular association of unfolded polypeptide chains which results in their aggregation. The formation of a stable tertiary structure requires the presence of a complete protein domain (-100 amino acids). However, proteins emerge from the ribosomes as unfolded chains with their amino terminus first and are therefore unable to fold stably until a domain has been synthesized. A second situation also occurs with polypeptide chains that are transported in an extended state across organellar membranes within the cell (Gething and Sambrook, 1992). The fully extended or partically folded polypeptide chains expose hydrophobic surfaces to the aqueous solvent, which become buried within the interior of the newly formed polypeptide. In the unfolded state, polypeptides could potentially interact with each other through these hydrophobic surfaces, forming aggregates even at relatively low concentrations. So, given the high concentration in cellular compartments of both total protein (20-30%) and nascent polypeptides (30-50itM in E. coli) unproductive reactions would be strongly preferred in vivo over the correct folding pathway. 1.2.1 HSP30 5 Very little is known about the smaller stress proteins. The group of low-molecular weight HSPs is very heterogeneous; they have few similar amino acid sequences in different organisms (Arrigo and Welch, 1987). These small HSPs do show sequence homology to the carboxyterminal region of the major eye lens protein, alpha-B-crystallin, which protects proteins against aggregation in vitro. (Klemenz et al, 1991; Horwitz, 1992). It has been suggested that this homology raises the possibility that the evolution of an eye lens protein from such a chaperone reflects not only its suitability for optical purposes, but that its chaperone activity is important in maintaining the integrity of lens proteins against aggregation that could impair their optical properties (Jakob et al., 1993). Within the resting cell, HSP30 is associated with the Golgi complex (Jakob et al., 1993). Following heat shock, much of the smaller HSPs are relocated in the nucleus, but following recovery they return to the vicinity of the Golgi complex (Arrigo et al., 1988). In summary, the HSP30 is a diverse group of stress proteins and various organisms contain very different numbers of them. While they are the least understood in terms of specific functions, they do appear to have a chaperone function (Arrigo et al., 1988). 1.2.2 HSP60 The heat shock protein-60 (HSP60) family was originally identified as host-encoded proteins, essential for the growth of Escherichia coli at all temperatures (Hendrick and Hartl, 1993). An operon called groE was identified encoding two 6 protein subunits, one around 60-kDa called GroEL and the other around 10-kDa called GroES (Friedman et al., 1984). The precise function of GroEL was unknown until a subunit of a plastid protein required in the assembly of the chloroplast enzyme rubisco, was found to be 46% identical in amino acid sequence to GroEL (Hemmingsen et al., 1988). Further investigation with the protozoan Tetrahymena thermophila revealed that HSP60 in the mitochondrial matrix showed a high sequence similarity to both GroEL and chloroplast protein (McMullin and Hallberg, 1988). Because the chloroplast protein was regarded as a molecular chaperone, and the action of GroEL in phage morphogenesis was similar, HSP60 was dubbed "chaperonin", to define a subclass of sequence-related molecular chaperones including stress-inducible and non-inducible members found in the bacterial cytosol, and in the inner space of mitochondria and chloroplasts. The HSP60s in bacteria, mitochondria, and chloroplasts functionally cooperate with a smaller co-chaperone protein of 10 kDa subunit size (termed "chaperonin 10"). Thus, the two subunits have become known as cpn 60 (i.e. HSP60) and cpn 10 (i.e. GroES) for bacteria and mitochondria, respectively (Musgrove and Ellis, 1986). Subsequent work revealed sequence similarity between cpn 60 and TCP-1 (tailless complex polypeptide), a protein in the eukaryotic cytosol. These similarities led to a proposal that the chaperonins be defined as a family of related chaperones containing two distinct subfamilies: the groE subfamily, found in bacteria, plastids, and mitochondria, and the TCP-1 subfamily found in the eukaryotic cytosol (Ellis, 1992). 7 Evidence has shown that both subfamilies assist in the correct folding of newly synthesized polypeptides in the cytosol of prokaryotes and eukaryotes (Hendrick and Hartl, 1993; Craig et al., 1993; Georgopoulos and Welch, 1993). For example, GroEL mutants of E.coli fail to assemble head structures of phage /V and tail assemblies of phage T5 (Georgopoulos et al., 1973). Secondly, a complex found in the chloroplast stromal compartment between cytoplasmically synthesized large subunit of chloroplast rubisco and a nuclear-encoded 60-kDa binding protein (i.e. HSP60) was shown to be an intermediate in the assembly of this enzyme from its eight large and eight small subunits (Cannon et al., 1986). The partial formation of polypeptides in association with the chaperonins supports their functional role in effectively preventing unproductive interactions between folding polypeptides. Chaperonins appear to be absent from four intracellular compartments where proteins are known to fold: the periplasmic space of some bacteria, the endoplasmic reticulum, the intramitochondrial membrane space, and the chloroplast thylakoid lumen (Horwich et al., 1993). All secreted proteins pass through the endoplasmic reticulum and many of them are of medical interest. The endoplasmic reticulum creates the disulfide bonds common in secreted proteins and HSP 70 and 90 families (Ellis, 1996). How proteins fold within these compartments in the absence of the cpns remains unknown (Ellis, 1996). 1.2.3 HSP70 8 The first members of the 70-kDa heat shock protein family were identified and elucidated in the 1970's. In 1974, Tissieres and colleagues observed that the synthesis of certain proteins in Drosophila salivary glands was greatly enhanced by heat shock (Tissieres et al, 1974). Additionally, studies on bacteriophage X yielded an E.coli mutant which was temperature-sensitive for growth at 42 °C (Georgopoulos, 1977). The product of the mutant gene was isolated and identified as the DnaK protein; the mutant became known as dnaK756 (Georgopoulos, 1979). Since HSP70 is the most widely studied of all the HSPs, the following paragraphs explain the structure of this exciting HSP. Sequencing of the E.coli DnaK protein revealed similarity to the previously sequenced Drosophila 70-kDa heat shock protein. This high degree of sequence conservation of 70-kDa heat shock protein genes within eukaryotes allowed the Drosophila gene to be used as a hybridization probe in cloning the cDNAs of other HSP70's (Gupta and Singh, 1992). The purification of HSP70 from numerous species soon followed. It became apparent that HSP70 homologues occur in all known groups of organisms: archebacteria, the eubacteria, and the eukaryotes (Gupta and Singh, 1992).Thus, HSPs are highly conserved proteins having an amino acid sequence homology of 50% from bacteria to man (Subjeck and Shyy, 1986). To date, no full-length HSP70 has proven amenable to crystallization and structure determination. The aminoterminal two thirds of the HSP70 contains an ATPase site and is more highly conserved that the carboxyterminal portion that 9 is thought to contain a peptide binding site (Chappell et al., 1987). The crystal structure of the 44-kDa segment of mammalian HSP70 (the clathrin-uncoating ATPase) reveals four structural domains that form two lobes enclosing a cleft in which ATP binds a structure very similar to the ATP-binding domain of G-actin (Flaherty et al., 1990; Flaherty et al., 1991). The high level df sequence conservation in the heat shock 70 family implies that the ATPase fragments of other members are also likely to have a similar tertiary structure. Recently, the crystal structure of a peptide complex with the substrate-binding unit of bacterial HSP70, termed DnaK, was determined (Zhu et al., 1996). The analysis identified the structure as a two-domain unit, a p-sandwich subdomain followed by a-helical segments. The peptide is bound to DnaK in an extended conformation through a channel defined by loops from the (3-sandwich. The oc-helical domain stabilizes the complex, but does not contact the peptide directly. This domain is rotated in the molecules of a second crystal lattice, suggesting a model of conformation-dependent substrate binding featuring a "latch mechanism" for maintaining the binding of the peptide (Zhu et al., 1996). Although the expression of HSP70 is increased under conditions of heat shock, it also became apparent that many members of this family of proteins were present under normal conditions in the cell, their presence not being strictly stress-related (Beckmann et al., 1990). The mammalian heat shock 70 protein consists of two members: a constitutive 73 kDa protein that is present in all cells and is only modestly increased after heat stress and a highly stress-inducible 72 10 kDa protein that is also present in unstressed cells but can reach a very high level after cellular stress (Beckmann et al., 1990). Although the HSP70 is not completely understood, all these observations are consistent with a model in which the normal role of constitutive HSP70 (HSC70) appears to bind transiently to hydrophobic regions of extended polypeptides as these emerge in several processes [eg. during synthesis in the ribosome, during transport from a lipid bilayer, or on the surface of folded proteins (Brown et al., 1993)]. Thus, under normal growth conditions, this binding serves a chaperone function, thereby reducing incorrect interactions between transiently exposed interacting surfaces (Brown et al., 1993). Under stress conditions, this binding is required to a greater extent, because the exposure of potential sites of interaction increases as a result of stress-induced denaturation (Brown et al., 1993). 1.2.4 HSP90 Heat shock 90 protein is the most abundant constitutively expressed stress protein in the eukaryotic cytosol (Georgopoulos and Welch, 1993). This group of stress proteins is highly conserved in bacteria, yeast, and mammals. Heat shock 90 protein is present in unstressed cells, but its synthesis increases approximately five-fold following heat stress (Georgopoulos and Welch, 1993). Postulated functions of HSP90 include interaction with a variety of cellular 11 proteins and steroid receptors to perform such functions as the regulation of protein synthesis and translocation of receptors within the cell (Ellis, 1996). It is not clear how many cellular proteins are potentially regulated by HSP90, but the studies to date point to a vital role for HSP90 binding to partly folded proteins, thereby preventing their aggregation and forming complexes with other proteins, thereby preserving their function (Ellis, 1996). The vertebrate cytosolic HSP90 is bound to a variety of proteins such as viral tyrosine kinases and steroid hormone receptors (Pratt et al., 1992). Heat shock protein 90 and HSP70 are found in murine cells as a complex with the glucocorticoid receptor. The formation of this complex prevents receptor binding to DNA and is a precursor for the receptor to develop the ability to bind steroid hormone (Picard et al., 1990). Hormone binding triggers the release of HSP90 and allows the receptor to bind to its DNA (Brugge, 1986). Thus, HSP90 complexes are inhibitory in maintaining th receptors in an unactivated state, but they have an additional purpose in managing the conformation of the receptor so that it can bind and respond to the hormone. The mechanistic details of HSP90 functions have yet to be defined but like the HSP60 and HSP70 proteins, HSP90 proteins possess ATPase activity which may facilitate polypeptide substrate binding and release (Hendrick and Hartl, 1993). It has been found that HSP90 binds transcriptional factors that regulate the expression of HSP genes (Nadeau et al., 1993). The operation of a 12 regulatory feedback system in which HSP90 concentrations would be matched with its target proteins has been suggested (Nadeau et al., 1993). Heat shock protein 90 is an extremely abundant constitutively expressed HSP which transiently interacts with many target proteins. However, HSP90 may bind to or mask domains of target proteins which are critical for their biological activation, thus inhibiting their functioning (Minowada and Welch, 1996). CHAPTER TWO 13 WHOLE-ANIMAL STRESS AND THE IN VIVO EXPRESSION OF HEAT SHOCK PROTEIN-70 IN THE JAPANESE QUAIL 2.1 INTRODUCTION Cells respond to a variety of adverse conditions, such as extremes of heat and cold (Chretian and Landry, 1988; Flanagen et al., 1995; Holland et al., 1993), hypoxia (Iwaki et al., 1993), ischemia (Cairo et al., 1985), viral infections (Garry et al., 1983) or surgical stress (Udelsman et al., 1991) by increasing the synthesis of a family of HSPs (Lee et al., 1990; Morimoto et al., 1994). Heat shock proteins are highly conserved in organisms studied so far, from bacteria to humans (Kelly and Schlesinger, 1982; Subjeck and Shyy, 1986) and they may protect cells by preventing damage to other cellular proteins (Welch and Suhan, 1986). The majority of studies in HSPs have been performed in vitro on cultured cells and much less is known about HSP expression in vivo. To my knowledge, studies of HSP expression in whole animals have only been conducted on a few mammals (Gower et al., 1989, Udelsman et al., 1993) and fish (Dyer et al., 1993) with very few stressors other than disease and extreme temperatures. In mammals, HSPs have recently been shown to be induced by exposure of whole animals to stressors such as restraint, surgery, and heat shock (Blake et al., 1991; Udelsman et al., 1991; Brown, 1990). We therefore exposed an 14 atherosclerosis-susceptible strain of Japanese quail to stressors hypothesizing that whole body exposure to stressors would also induce cellular HSP synthesis. This model was used with hopes to eventually study the interplay of stress and hypercholesterolemia. HSP expression was examined in organ tissues which have been shown to be susceptible to cellular damage during a physiological stress (Blake et al., 1990; Cairo et al., 1985; Welch et al., 1986) and explored a number of stressors that would not be expected to cause direct damage to cells or tissues. Previous studies have shown a correlation between the increases in the level of plasma corticosterone in response to restraint and the duration of tonic immobility (Tl) in Japanese quail (Satterlee and Jones, 1995). I therefore also used the Tl test to assess whether or not the stressors elicited stress and fear response in these individuals (Jones, 1986; Jones and Faure, 1981). I hoped to be able to determine whether or not the levels of synthesis or accumulation of HSPs may be useful in determining to what extent a particular environmental factor is perceived by the bird as stressful. 2.2 MATERIALS AND METHODS 2.2.1 EXPERIMENTAL TREATMENTS Forty 12 week old male Japanese quail [Coturnix japonica) were acquired from the UBC Quail Genetic Resource Center. They were housed in individual cages 15 (but not visually or auditorily isolated) under a 14L/10D light - dark cycle and fed a commercial quail diet (26% protein) and water ad libitum. Groups of five birds were randomly assigned to one of 8 treatments: Treatment 1 -Control; the birds were not handled, except for the weighing and the Tl tests (see below). For treatments 2 to 8, each bird was taken out of its home cage, put in a white plastic carrying case and taken to another room for the stress treatment. Body weights of individual birds were taken before the start of the first stress session and after the last stress session. Two birds from each treatment group were sacrificed at the end of 5 days while the remaining 3 birds from each treatment group were sacrificed at the end of the 10 days. This procedure was repeated each day at the same time of day, between 9am and 11am. Treatment 2 --The birds were placed in individual cardboard boxes (44 x 44 x 33 cm) with the lid closed and were left in the dark for 60 min before being returned to their respective home cages via the carrying case. Treatment 3 - Each bird was restrained by taping both wings to the body and returned to their home cage. The birds were released after 60 min. Treatment 4 — Each bird was restrained with by taping both wings to the body and left in the carrying case while exposed to loud Rock and Roll music (74db, range = 67.8 to 76.5 db) (Micro-5 Noise Dosimeter; Quest Electronics, Wise.) for 60 min before being released and returned to their home cages. 16 Treatment 5 -- The body temperature was measured via the cloaca. Each bird was restrained by taping both wings to the body, left in the carrying case and put in a refrigerator (4°C) for 30 min. Body temperature was taken again and the birds were released and returned to their home cages. Treatment 6 - A piece of velcro tape was taped loosely around the neck of each bird. The birds were left in the carrying case for 60 min, after which time the tape was removed and the birds returned to their home cages. Treatment 7 - Each bird was taken to a room where there were large floor pens holding about 150 birds each. The treatment birds were individually let loose in each pen for 60 min, by the end of which time they were caught and returned to their home cages. Treatment 8 -Each bird was taken to a room where there were cages holding 3 males each. Each treatment bird was placed in one of these cages with the 3 strange males (and their territory) for 60 min before being returned to their home cages. The treatment birds were put in with a different group of males each day. 2.2.2 T l TESTING Tl tests were conducted on individual birds prior to the study and about an hour after the last stress session. Each bird was taken out of its home cage, put in a 17 white plastic carrying case and taken to a room where a platform with a stop-watch was set up (Cheng et al., 1990). The door of the room was closed to buffer noise from the outside. All the lights in the room were turned off except for a shaded 150w red lamp about 90 cm above the platform. Tl was induced by placing the bird on its back on the platform and held down for 15 sec, at the end of which time the hand was removed from the bird. The experimenter started timing and also retreated quietly to a chair 1.8 m away from the platform (and in the dark). If the bird remained on its back for another 15 sec, timing continued until the bird righted itself. Timing stopped as soon as both feet of the bird touched the surface of the platform. A maximum of 10 min was allowed and if the bird remained in Tl at the end of 10 min, the test was terminated. If the bird righted itself before the end of 15 sec after the removal of the experimenter's hand, the induction was deemed not successful and the procedure would be repeated. A maximum of 5 inductions was allowed for each test. 2.2.3 TISSUE SAMPLING After the end of the last stress session (on either the 5th or the 10th day ), the bird was allowed to recover for 20 h before being sacrificed by decapitation. Five tissues (heart, liver, gonads, kidneys and lungs) were dissected, blotted dry and stored in sealed packets at -70°C until processed further for protein extraction (Lee and Dunbar, 1994). 18 Each specimen was diced into small fragments and 0.5 g of tissue was transferred to a 15 mL test-tube containing 57 mM phenylmethylsulfonylfluroide (PMSF), 100 L I M pepstatin, 50 \xM leupeptin, 3 L I M aprotinin, 15 mM ethelenediaminetetetraacetic acid (EDTA) and 0.1% , sodium dodecyl sulfate (SDS) in a final volume of 2 mL (pH= 7.5). After homogenization (Omni International, Waterbury CT) at 4°C for 30 sec, the suspension was centrifuged (17,000g, 90 min, 4°C). One portion (650 ul) of the supernatant collected was transferred to a 1.5 mL eppendorf tube to which an equal volume of electro-phoresis sample buffer (p-2-mercaptoethanol, glycerol, SDS, Tris [hydroxymethyl] aminomethane-HCI (Tris-HCI), pH 6.8, bromophenol blue) was added and the mixture was boiled for 5 min prior to storage at -70°C for later sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SOS-PAGE). Another aliquot (400 L I L ) of supernatant was transferred to a 0.5 mL Eppendorf tube and stored at -70°C for later determination of total protein using bovine serum albumin (BSA) as a standard (Smith et al., 1985). During one of the extractions there was a power outage and some samples were lost. 2.2.4 WESTERN BLOTTING Equal amounts (25 u.g) of individual total protein were resolved on 12% polyacrylamide gels under reducing conditions (Laemmli, 1970) using a minigel 19 setup (Bio-Rad Laboratories, Richmond, CA). Prestained protein molecular weight standards were run with every gel (Life Technologies, Gaithersburg, MD). Proteins were electrophoretically blotted from the gel onto a nitro-cellulose membrane in transfer buffer (48mM Tris, 39mM glycine, and 20% methanol, pH 9.2) at 200 mA for 30 min using a semi-dry transfer apparatus (Bio-Rad). Prestained protein standards also served to monitor protein transfer. After blocking all available binding sites on the blots with 2% nonfat dry milk for 1 h at room temperature, the blots were washed twice in TTBS (Tris-buffered Saline, pH 7.5 , 0.05% Tween-20) for 5 min intervals. Bound proteins were probed with HSP30 (rabbit polyclonal antibody against fish HSP30), diluted 1:600 in TTBS. Other probes used were a mouse monoclonal HSP60 (1:600), a mouse monoclonal HSP72/73 antibody (1:500), or a mouse monoclonal HSP90 antibody (1:600). AN antibodies were purchased from StressGen (Victoria, B.C.) except HSP30 which was provided by Dr. Peter Candido (Department of Biochemistry, University of British Columbia). These antibodies have been demonstrated to cross-react with either chicken (HSP60, 72/73, 90; StressGen) or osprey (HSP30, 72/73; Hoekstra et al., unpublished observations) tissues. Proteins were visualized using either goat anti-rabbit or goat anti-mouse alkaline-phosphatase Ig secondary antibody (Life Technologies), followed by application of alkaline phosphatase buffer (100mM Tris, 100mM NaCI, 50mM MgCI2), nitro blue tetrazolium, and 5-bromo-4-chloro-3-indolyl phosphate (Sigma Chemical Co.). 20 2.2.5 DENSITOMETRIC QUANTIFICATION OF HSP LEVELS Optical density was quantified by a digital image acquisition system (Biolmage, AnnArbor, Ml). During image collection, each image was adjusted to a background grey level of 200 ± 5 grey scale units (300=black, 0=white). Optical density was calculated by taking the area (in pixels) intensity comprising each band. An average of five intensity measurements was obtained from each band and was taken as representative of the entire band. Only bands on the same Western blot were used for intensity measurement comparisons. 2.2.6. STATISTICAL ANALYSIS The band intensities were analyzed by a one-way analysis of variance (ANOVA) and the Dunn's comparison test was used for mean separation. The body weights and core temperature changes were analyzed using the paired t-test (Sigma Stat, 1994). The before and after treatment Tl durations for each of the eight treatments were compared separately using a paired t-test (Sigma Stat, 1994). Another analysis comparing the 8 treatments was conducted with the help of PROC GLM (1985) using the following statistical model: 21 YIJk =u+ T, + Dj+ TDU +B,j + E i J k where Y = the after treatment Tl duration of a particular individual, U = the population mean, 7~ = the effect of a particular treatment, D = the effect of the duration of stress exposure (5 or 10-day sessions), TD = the interaction between treatment and exposure, and B = the before treatment Tl duration of the same individual as a covariate, and E = the error term. 2.3 RESULTS The body weights of experimental birds taken before and after the stress sessions did not show significant differences in any of the 8 experimental groups. The declines in mean core temperatures (°C) for individuals in Treatment group 5 after the cold exposures were: 1.24 ±0.71, 1.98 ±0.41, 1.00 ±0.16, 1.55 ± 0.34, and 1.42 ± 0.71 for the 5 or 10-day stress sessions. Except for one bird in the 5-day session exposure, the magnitude of temperature declines for each individual was highly consistent from session to session (Fig. 2.1). 2.3.1 HSP EXPRESSION 2.3.1.1. HSP70 Heart tissue from birds in Treatment 1 (control), as well as in Treatment groups 3, 7, and 8, expressed a single band at the 70-kDa marker position (Lanes 1, 2 in Figs. 2.2-2.5). However, in Treatments 2, 4, 5 and 6, (Lanes 3-6 in Figs. 2.2-2.5) a more intense band was observed at the 70-kDa marker position. The level of HSP70 expression in these treatments was confirmed semi-quantitatively as the optical density of the bands were significantly (PO.001) higher than that of the controls (Figs. 2.6-2.9). With Treatment 5 (restraint in cold), there was a very high correlation (r= 0.87) between individual optical density and the magnitude of its mean core temperature decline (Fig. 2.1). No increased expression of HSP70 was observed in any of the other tissues examined (liver, lung, gonad or kidney). 2.3.1.2 HSP30, 60, AND 90 No significant increases in the expression of these HSPs were detected in any of the tissues examined from control or stressed birds. 23 Figure 2.1 The relationship between HSP70 expression and mean core temperature decline after whole body exposure of Japanese quail to cold temperature stress. The core temperature was measured before and after exposure to cold stress. Mycocardium samples were collected and analyzed for HSP. The band intensity of HSP70 as determined by light transmission was quantified by densitometry and compared to the respective bird's mean core temperature decline. Lower values of band intensity indicate a more intense band and higher level of protein concentration. 24 Figure 2.2 Expression of HSP70 in heart tissue following isolation in darkness stress. Representative Western blot of HSP70 expression (25p.g per lane) of cardiac tissue harvested from individual Japanese quail. Lane 0, molecular weight standard; Lane 1, 5 day control; Lane 2, 10 day control; Lane 3-4, 5 day stressed; Lanes 5-6, 10 days stressed. k D a 90=> 70=> 3 4 5 6 Figure 2.3 Expression of HSP70 in heart tissue following noise stress. Representative Western blot of HSP70 expression (25u.g per lane) of cardiac tissue harvested from individual Japanese quail. Lane 0, molecular weight standard Lane 1, 5 day control; Lane 2, 10 day control; Lane 3-4, 5 day stressed; Lanes 5-6, 10 days stressed. k D a 90=> 70=> 1 2 3 4 5 6 Figure 2.4 Expression of HSP70 in heart tissue following cold temperature stress. Representative Western blot of HSP70 expression (25u.g per lane) of cardiac tissue harvested from individual Japanese quail. Lane 0, molecular weight standard; Lane 1, 5 day control; Lane 2, 10 day control; Lane 3-4, 5 day stressed; Lanes 5-6, 10 days stressed. 27 k D a 90=> 70=> Figure 2.5 Expression of HSP70 in heart tissue following inescapable irritation. Representative Western blot of HSP70 expression (25|ig per lane) of cardiac tissue harvested from individual Japanese quail. Lane 0, molecular weight standard; Lane 1, 5 day control; Lane 2, 10 day control; Lane 3-4, 5 day stressed; Lanes 5-6, 10 days stressed. 28 300 250 £l 200 00 LU Q _l < O h-D_ O 150 100 50 H 0 T T C5 C10 S5 S5 S10 S10 Figure 2.6 Effects of isolation in darkness stress on HSP70 expression in heart tissue harvested from groups of 5 (C5) and 10 (C10) day control birds and 5 (S5) and 10 (S10) day stressed birds. The results are expressed as the mean+SEM optical density for five animals per group. *P<0.01 as analyzed by a one-way analysis of variance and the Dunn's comparison test. 29 3 0 0 Figure 2.7 Effects of noise stress on HSP70 expression in heart tissue harvested from groups of 5 (C5) and 10 (C10) day control birds and 5 (S5) and 10 (S10) stressed birds. The results are expressed as the mean±SEM relative optical density for five animals per group. *P<0.01 as analyzed by a one-way analysis of variance and the Dunn's comparison test. 30 350 300 H CO z LU Q 0_ O 250 200 H O 150 -I 100 50 H X * _ x _ T 1 r C5 C10 S5 S5 S10 S10 Figure 2.8 Effects of cold temperature stress on HSP70 expression in heart tissue harvested from groups of 5 (C5) and 10 (C10) day control birds and 5 (S5) and 10 (S10) stressed birds. The results are expressed as the mean+SEM relative optical density for five animals per group. *P<0.01 as analyzed by a one-way analysis of variance and the Dunn's comparison test. 31 Figure 2.9 Effects of inescapable irritation on HSP70 expression in heart tissue harvested from groups of 5 (C510 ) and 10 (C10) day control birds and 5 (S5) and (S10) stressed birds. The results are expressed as the mean±SEM relative optical density for five animals per group. *P<0.01 as analyzed by a one-way analysis of variance and the Dunn's comparison test. 32 2.3.2. Tl DURATION Only in Treatment 4 (noise) was Tl duration significantly different from the control (Treatment 1; 50.18 sec). The after treatment Tl was significantly prolonged for 10-day exposures (600 sec, P<0.001) exposures compared to the before treatment Tl (25.17 sec). The frequency of exposure, two-way interaction and the covariate effects were not significant. 2.4 DISCUSSION This study revealed that an increased accumulation of HSP70 occurred in the heart, but not in the liver, kidney, lungs or gonads of Japanese quail, following exposure to a variety of stressors. This finding shows that whole animal stress can elicit HSP70 expression and this shows a high degree of tissue selectivity. The present investigation was designed to explore the types of stressful stimuli that can cause in vivo induction of HSP in birds with hopes to eventually study the interplay of stress and hypercholesterolemia Previously, it has been demonstrated that heat shock induces a tissue-specific HSP70 response in rats (Flanagen et al., 1995; Skidmore et al., 1995) and it has also been reported that 33 surgery or mild restraint induces the HSP70 mRNA transcription in the adrenals and vasculature (but not in the heart) of rats (Holbrook and Udelsman, 1994; Udelsman et al., 1993). The present study in birds also showed that, as in rats, mild restraint (Treatment 3) did not result in increased HSP70 expression in the heart tissue. In addition, we have shown that other stressors, such as isolation in darkness (Treatment 2), loud noise (Treatment 4), inescapable irritation (Treatment 6) or cold temperature and darkness (Treatment 5) did cause a tissue-specific HSP70 response in the heart tissue of Japanese quail. It seems probable that the stressor in Treatment 4 was noise rather than restraint because restraint alone (Treatment 3) did not cause increased HSP expression. While we cannot eliminate hypoxia as a possible stress factor, it is more likely that the stressor in Treatment 5 was the cold temperature because there was a very high correlation between the magnitude of mean core temperature decline and the level of HSP70 expression in these birds. It is assumed that the birds in Treatment 6 were stressed by inescapable irritation because the birds reacted violently to the tape around their neck by jumping up and down, flipping up-side-down and shaking their head, due to their inability to remove the object with their beak or their claws. Social stressors, such as being placed in the territories of strange males (Treatment 8), or being swamped by strangers in an unfamiliar environment (Treatment 7), did not produce any detectable differences in HSP70 expression relative to the control. Holbrook and Udelsman (1994) reported that longer periods of restraint led to a waning in the HSP70 induction response. 34 Since we did not collect tissues for analyses until about 20 h after the 5th or the 10th consecutive exposure, it is possible that in some treatments (e.g. 7 and 8) adaptation to the stressor may have occurred rapidly, such that alterations in HSP expression were not detectable. Under our experimental conditions, the Tl trials did not induce any HSP expression. Increased fear response only was detected in birds repeatedly exposed to loud noise (Treatment 4). While it has been shown that Japanese quail with high plasma corticosterone responses to restraint had significantly longer Tl duration than quail with low plasma corticosterone responses (Satterlee and Jones, 1995), the correlation may not be sufficiently strong to allow the use of Tl as a good predictor of plasma corticosterone response to stress. In general, it might be anticipated that regular handling of birds would reduce their fear response and suppress their Tl duration (Jones and Faure, 1981). However, suppression of Tl duration was not detected in any of our treatment groups, suggesting that our handling of the birds perse during the treatment period did not alter their fear response or modify their stress response (Cheng et al., 1990; Jones, 1986; Jones and Faure, 1981). Changing housing conditions or social environments reportedly can cause increased fear response in chickens (Jones, 1986). In our Treatments 7 and 8, however, the changes in social environment did not result in increased Tl duration in Japanese quail. In conclusion, the data suggest that a number of stressors can induce preferential in vivo expression of tissue- and class-specific HSPs in the 35 Japanese quail. HSP70 may be used as indicator of stress under certain situations. Blake et al. (1990) suggests that the in vivo HSP response to stress differs from the in vitro response in the kinetics of induction. Tissue cultures, for example, do not have the complexity of whole-animal where multiple systems may be required to increase HSP expression (Blake et al., 1990; Fortan et al., 1989). The HSP induction characteristics of a whole organism, therefore, may differ considerably from a cellular system exposed to the same stressor (Welch et al., 1983). Although in vivo induction characteristics and mechanisms need to be studied before further evaluation of HSPs as a biomonitoring tool can be undertaken, the expression or activation of stress proteins may respond to all the requirements (i.e. accurate, reliable, reproducible, dose-dependent, and sensitive) for molecular biomarkers in environmental medicine. 36 CHAPTER THREE THE EFFECTS OF RESTRAINT STRESS ON HEAT SHOCK PROTEIN EXPRESSION IN THE DIABETIC RAT 3.1 INTRODUCTION Heat shock (or stress) proteins are expressed as part of the cellular response to different physiological, psychological and environmental stresses (Linquist and Craig, 1988). This stress response appears to provide protection against what might otherwise be detrimental changes in the cellular environment. In addition to heat, other stressors that induce HSP synthesis include heavy metals (Morimoto et al., 1994), hypoxia (Iwaki et al., 1993), ischemia (Gralinski et al., 1996), hemodynamic overload (Gralinski et al., 1996), and energy depletion (Skidmore et al., 1995). Current evidence suggests that HSPs provide a protective and/or adaptive response to cellular stressors by mechanisms involving interactions between HSPs and other metabolically important cellular proteins (Blake et al., 1993). So, while the exact function of HSPs remains unclear, they are known to bind to other cellular proteins and may facilitate their translocation across 37 membranes of cytoplasmic organelles or protect them from damage incurred due to the presence of a stressor (Blake et al., 1993). However, restraint is one type of stressor that should not cause appreciable damage to cells. For example, it has been shown in rats that HSPs induced by restraint in a polyvinylchloride tube is mediated by physiological alterations in several pituitary hormones (Udelsman et al., 1994). All these hormones produce a variety of cellular effects, including induction of several regulatory proteins (Udelsman et al., 1994). Therefore, HSPs may be co-induced by these hormones to insure that all newly synthesized hormone-inducible proteins are properly transported to their correct cellular location, suggesting that a close relationship may exist between the physiological activation of neuroendocrine stress response systems and the cellular activation of the heat shock response (Morimoto, 1996). Diabetes mellitus is a disorder in the control of carbohydrate metabolism that can be vastly affected by a number of variables such as weight, diet and exercise (Surwit et al., 1992). Diabetes is commonly classified as either the reduction of insulin secretion (insulin-dependent diabetes mellitus, IDDM-Type 1) or in the insulin sensitivity of target tissues (non-insulin dependent diabetes mellitus, NIDDM-Type 2) (Surwit et al., 1992). Both forms are manifested by chronic hyperglycemia. Chronic hyperglycemia is widely believed to be a key factor in the development of nephropathy and neuropathy (Sowers and Epstein, 1995). It is associated with retinopathy, hypertension and atherosclerotic disease as well (Sowers and Epstein, 1995). An increasing body of evidence suggests that stress can play a permissive role in the pathophysiology of this disease in both animals and humans (Surwit et al., 1992; Gelber et al., 1994; Sowers and Epstein, 1995). Stress has long been shown to have major effects on metabolic activity. Energy mobilization is a primary result of the "fight or flight" response. Stress hormones (i.e. adrenaline and Cortisol) released as a result of stress can result in elevated blood glucose levels (Surwit et al., 1993). The adaptive benefit of stress-induced energy mobilization in healthy, nondiabetic animals is obvious. However, in diabetes where there is a relative or absolute lack of insulin, stress-induced increases in glucose can be counterproductive. Therefore, in view of current evidence supporting the idea that a variety of neuroendocrine hormones are released in response to stress in the diabetic animal (Surwit et al., 1992), the cellular activation of the heat shock response may also occur. Previous studies have shown differences in HSP expression between various organs of animals under stress (Flanagan et al., 1995). Therefore, it was hypothesized that restraint stress in normal and diabetic animals might result in an intensification of diabetes-related changes by stress or vice versa in terms of HSP expression. 3.2 METHODS 3.2.1 TREATMENT PROTOCOL 39 The stress and diabetic treatment protocols as described in this section were performed by Philip Toleikis as part of his Ph.D thesis research in the Department of Pharmacology and Therapeutics (Toleikis, 1995). All procedures were performed according to protocols approved by the University of British Columbia Animal Care Committee. Tissues (heart, liver, kidney, and lung) from the experimental animals were obtained for the present study. Male Wistar rats were obtained from the breeding colony of the University of British Columbia Animal Care Centre and housed individually for a 1-week adaptation period prior to the experimental protocol. They were maintained on a 12-hour light/dark schedule with standard laboratory chow and water fed ad libitum. Six animals were randomly assigned to one of six treatments: Treatment 1 (Control); - the rats were left in their home cages except for weighing and injection with saline as the sham procedure for induction of diabetes (see below). Treatment 2 (Stressed); - The animals were injected with saline as sham induction of diabetes. After two weeks following injection of saline, rats were exposed to a stress protocol (Toleikis and Godin, 1995) for the next 14 days (see below). Treatment 3 (Short-term diabetic); - Diabetes was induced in each rat as outlined below. The rats were diabetic for four weeks in this treatment group before being sampled. 40 Treatment 4 (Chronic diabetic);-- Diabetes was induced in each rat as outlined below. The rats were diabetic for 12 weeks in this treatment group before being sampled. Treatment 5 (Short-term diabetic + stressed);- Diabetes was induced in each rat as outlined below. The rats were diabetic for four weeks in this treatment group and were exposed to the stress protocol (see below) during the last two weeks. Treatment 6 (Chronic diabetic + stressed); ~ Diabetes was induced in each rat as outlined below. The rats were diabetic for 12 weeks in this treatment group and were exposed to the stress protocol (see below) during the last two weeks The stress protocol was performed by exposing the rats to one of seven different restraint stressors twice daily for 1-h intervals. The sequence of the stressors was randomized for the first seven twice daily sessions. The morning and afternoon series was exchanged for the next seven days to minimize habituation. The stressors used are as follows: a) towel wrap secured with tape; b) the latter, with animals placed in a supine position; c) restraint in a plastic box with lid; d) restraint in a polyvinylchloride tube closed at either end; e) immobilization on a board with tape; f) the latter, with animals placed in a supine position; and g) restraint in a metal bar cage. These restraint stressors do not result in physical harm to the rat but do produce marked elevations in plasma corticosterone and catecholamine levels (Toleikis and Godin, in preparation). 41 For treatments 3 to 6, each rat subsequently underwent a standardized induction of diabetes or injection of saline as described in Toleikis and Godin (1995). Briefly, the animals were anesthetized with halothane and diabetes was induced by a single injection of streptozotocin (STZ, 60mg/kg). Animals in treatment groups 1 and 2 received the same volume of saline (sham). Each animal was weighed weekly and the rate of weigh gain was recorded. Animals with blood glucose values above 300 mg/dl at end of the study were considered diabetic. Animals with lower blood glucose values were excluded from the experiment. 3.2.2. TISSUE PREPARATION Following either the 4-week or 12-week experimental period, the six animals from each treatment group were removed from their holding facility and quickly killed by decapitation. Trunk blood was collected and centrifuged at 3000 rpm for 5 min at 4°C and plasma was stored at -70° C for subsequent biochemical assays. Heart, liver, kidney, and lung were excised, placed in homogenizing buffer (50mM Tris-0.1 mM ethylendiamine tetraacetic acid, pH=7.6) and were homogenized (10% w/v) on ice for two 15-s bursts (Brinkmann Polytron). After homogenization, the aliquots were stored at -70° C for subsequent Western blot analysis and densitometric evaluation as described earlier (see Sections 2.2.4 42 and 2.2.5). In addition to a prestained molecular weight standard (Life Technologies), a purified HSP70 (StressGen, Victoria, BC) was used as a positive control. 3.2.3. STATISTICAL ANALYSIS The band intensities were analyzed by a one-way analysis of variance (ANOVA) and the Dunn's comparison test was used for mean separation. 3.3 RESULTS A light band at 70-kDa corresponding to constitutive expressed HSP70 was observed in all tissues examined except lung (for summary see Table 1). No basal HSP expression was observed for HSP30, 60, or 90 in control rats. Table 1. Increased heat shock protein (HSP) expression in heart, liver, and kidney tissue of control, stressed, diabetic, and diabetic-stressed rats. (* = not detectable) HEART LIVER KIDNEY Treatment 1 Control A * * Treatment 2 Stress Only HSP30, HSP70 * * Treatment 3 Diabetic 4 wks HSP30, HSP70 HSP70 * Treatment 4 Diabetic 14 wks HSP30, HSP70 HSP70 HSP70 Treatment 5 Diabetic 4 wks & Stress HSP30, HSP70 HSP70 * Treatment 6 Diabetic 14 wks & Stress HSP30, HSP70 HSP70 HSP70 43 3.3.1 HEART TISSUE No increases in HSP60 or 90 were detected in the heart tissue for any of the treatment groups. Increased expression of HSP70, as expressed by a more intense band at the 70-kDa marker position, was found in the heart tissue of individual rats in Treatments 2, 3, 4, 5, and 6 (Figs. 3.1 and 3.3). Densitometric analysis of the heart tissue demonstrates that animals in Treatments 2 (Figs. 3.2 and 3.4), 3 (Fig. 3.2), 4 (Fig. 3.2), 5 (Fig. 3.4), and 6 (Fig. 3.4), accumulated significantly (P<0.01) greater amounts of HSP70 compared to controls in Treatment 1 (Figs. 3.2 and 3.4). Even basal levels of HSP30 were not detectable in the heart/vascular tissue of controls from Treatment 1 (Figs. 3.5 and 3.7). Yet, all treatments (2, 3, 4, 5, and 6) resulted in increased HSP30 expression (Figs. 3.5, 3.7). Densitometric analysis confirmed a significantly (P<0.01) greater amount of HSP30 in Treatments 2, 4, 5, and 6 as compared to Treatment 1 (Figs. 3.6 and 3.8). Treatment 3 (Fig. 3.6) resulted in a milder, but still significant (PO.05) expression of HSP30 as compared to Treatment 1. Figure 3.1 Expression of HSP70 in the heart tissue of rats following restraint and/or induction of diabetes. Representative Western blot analysis of cardiac tissue harvested from individual male Wistar rat. Lane 1-2, controls; Lanes 3-4, restraint stress; Lanes 5-6, 4 wks diabetes; Lanes 7-8, 4 wks diabetes including 2 wks restraint stress; Lane 9, purified HSP70. 45 250 200 >-^ 150 H LU Q < o h-O 100 50 RS D B 4 D R 4 Figure 3.2 Summary of results comparing heart HSP70 expression in groups of control (C), restraint stress (RS), 4 wks diabetes (DB4), and 4 wks diabetes, including 2 wks restraint stress (DR4). Results are expressed as the mean ± SEM optical density for six animals per group. * P<0.01 as compared to control group Figure 3.3 Expression of HSP70 in the heart tissue of rats following restraint and/or induction of diabetes. A representative Western blot analysis of cardiac tissue harvested from individual male Wistar rat. Lane 0, molecular weight standard; Lanes 1-2, controls; Lanes 3-4, restraint stress; Lanes 5-6, 12 wks diabetes; Lanes 7-8, 12 wks diabetes including 2 wks restraint stress. 47 250 200 H CO z LU Q _l < p 100 Q_ o 150 50 Figure 3.4 Summary of results comparing heart HSP70 expression in groups of control (C), restraint stress (RS), 12 wks diabetes (DB12), and 12 wks diabetes, including 2 wks restraint stress (DR12). Results are expressed as the mean ± SEM optical density for six animals per group. * P<0.01 as compared to control group Figure 3.5 Expression of HSP30 in the heart tissue of rats following restraint and/or induction of diabetes. Representative Western blot analysis of cardiac tissue harvested from individual male Wistar rat. Lane 0, molecular weight standard; Lane 1-2, controls; Lanes 3-4, restraint stress; Lanes 5-6, 4 wks diabetes; Lanes 7-8, 4 wks diabetes, including 2 wks restraint stress. 49 200 > -CO z UJ Q _ l < O I -D_ o Figure 3.6 Summary of results comparing heart tissue HSP30 expression in groups of control (C), restraint stress (RS), 4 wks diabetes (DB4) and 4 wks diabetes, including 2 wks restraint stress (DR4). Results are expressed as the mean ± SEM optical density for six animals per group. *P<0.05, **P<0.01 as compared to control group Figure 3.7 Expression of HSP30 in the heart tissue of rats following restraint restraint and/or induction of diabetes. Representative Western blot analysis of cardiac tissue harvested from individual male Wistar rat. Lane 0, molecular weight standard; Lane 1-2, controls; Lanes 3-4, restraint stress; Lanes 5-6, 12 wks diabetes; Lanes 7-8, 12 wks diabetes including 2 wks restraint stress. 51 >-CO -z. UJ Q _ J < O h-Q. o RS DB12 DR12 Figure 3.8 Summary of results comparing heart tissue HSP30 expression in groups of control (C), restraint stress (RS), 12 wks diabetes (DB12), and 12 wks diabetes, including 2 wks restraint stress (DR12). Results are expressed as the mean ± SEM optical density for six animals per group. *P<0.01. 52 3.3.2 L I V E R T I S S U E No increase in HSP30, 60, or 90 was detected in the liver in any of the treatment groups. The liver tissue did demonstrate increased amounts of HSP70. Animals from Treatments 3, 4, 5, and 6 showed increased expression of HSP70 (Figs. 3.9 and 3.11). Densitometric analysis confirmed that diabetic rats (Treatments 3, 4, 5, and 6) accumulated greater (PO.01) amounts (Figs. 3.10 and 3.12) of HSP70 compared to Treatment 1 (Figs. 3.10 and 3.12). 3.3.3 K I D N E Y T I S S U E The kidney showed an increased expression of HSP70 in rats from Treatments 4 and 6 (Fig. 3.13). Again, densitometric analysis confirmed that chronically diabetic rats (Treatments 4 and 6) accumulated significantly (P<0.01) greater amounts of HSP70 as compared to the controls (Fig. 3.14). Short-term (4 wks) restrained diabetic rats did not exhibit increased HSP70 expression (not shown). No increase in HSP30, 60 or 90 was detected in the kidney for any of the treatment groups. Figure 3.9 Expression of HSP70 protein in liver tissue of rats after induction of diabetes. Representative Western blot of HSP70 expression (25ug per lane) of hepatic tissue harvested from individual male Wistar rat. Lane 0, molecular weight standard; Lanes 1-2, control; Lane 3-4, restraint stressed; Lanes 5-6, 4 wks after induction of diabetes; Lanes 7-8, 4 wks diabetes including 2 wks of restraint stress; Lane 9, purified HSP70. 54 Figure 3.10 Summary of results comparing HSP70 expression in liver tissue of control (C), restraint stressed (RS), four wk diabetics (DB4) and 4wk diabetics, including 2 wk restraint stressed rats (DR4). The results are expressed as the mean ± SEM optical density for six animals per group. * P<0.01 as compared to control and restraint stressed groups. 55 Figure 3.11 Expression of HSP70 protein in liver tissue of rats after induction of diabetes. Representative Western blot of HSP70 expression (25ug per lane) of hepatic tissue harvested from individual male Wistar rat. Lanes 1, control; Lane 2-3, restraint stressed; Lanes 4-5, 12 wks after induction of diabetes; Lanes 6-7, 12 wks after induction of diabetes including 2 wks of restraint stress; Lane 8, purified HSP70. 56 250 200 7* 150 ioo -\ 50 A DB12 DR12 Figure 3.12 Summary of results comparing HSP70 expression in liver tissue of control (C), restraint stressed (RS), 12 wks diabetic (DB12) and 12 wks diabetic, including 2 wks restraint stressed rats (DR12). The results are expressed as the mean ± SEM optical density for six animals per group. * P<0.01 as compared to control and restraint stressed groups. Figure 3.13 Expression of HSP70 in kidney tissue of rats after induction of diabetes. Representative Western blot of HSP70 expression (25ug per lane) of renal tissue harvested from individual male Wistar rat. Lane 0, molecular weight standard; Lanes 1, control; Lane 2, restraint; Lane 3-4, 4 wks after induction of diabetes; Lanes 5-6, 12 wks induction diabetes; Lanes 7-8, 12 wks after induction of diabetes, including 2 wks restraint; Lane 9, purified HSP70. 58 200 >-00 UJ Q _ i < O h-Q_ O 150 H 100 H RS DB4 DB12 DR12 Figure 3.14 Summary of results comparing kidney tissue HSP70 expression in control (C), restraint stressed (RS), 4 wk diabetic rats (DB4), 12 wk diabetic rats (DB12), and 12 wks diabetic, including 2 wks restraint stressed (DR12). Results are expressed as the mean ± S E M optical density for six animals per group. * P<0.01 as compared to control, restraint stressed, and DB4 groups. 59 3.3.4 L U N G T I S S U E The lung tissue did not demonstrate any increased expression of HSP 30, 60, or 90 in any of the treatment groups. 3.4 D I S C U S S I O N Udelsman et al. (1993) have reported an increase in the expression of HSP27 and HSP70 mRNA and protein in the aorta of rats following restraint stress. Following in situ hybridization, expression of HSP70 was localized to the smooth muscle layers in the media with minimal expression in the endothelial cell layer in the intima (Udelsman et al., 1993). Of particular interest, HSP27 has been implicated in mediating sustained gastrointestinal smooth muscle contraction in response to the neuropeptide bombesin (Bitar et al., 1991). This suggests the possibility that HSPs serve a similar contractile function in the smooth muscle of the aorta (Udelsman et al., 1993). After further experiments using cti - and (3-adrenergic agonists and antagonists, it was determined that increased aortic HSP expression was mediated by activation of the ai -adrenergic receptor (Udelsman et al., 1994b). The agonist binding activates phospholipase C, which leads to the cleavage of phosphatidyl 4,5-biphosphate, 60 resulting in the generation of inositol triphosphate and diacyglycerol (Udelsman et al., 1994b). These second messengers mobilize intracellular calcium and activate protein kinase C, which leads to the phosphorylation and subsequent activation of intracellular proteins (Udelsman et al., 1994b). The authors suggest that an interaction of HSPs is likely at any of the intermediate steps after cti -receptor activation (Udelsman et al., 1994b). Others have shown that dopamine agonists and catecholamine reuptake inhibitors share an ability with restraint to induce HSP70 expression in the aorta (Blake et al., 1995). These investigations provide evidence that stress-induced expression of HSP70 in aorta results from catecholamine neurotransmission after activation of the sympathetic nervous system (SNS). In the concurrent study, a similar restraint stress protocol has been shown to activate the SNS to produce marked elevations in plasma corticosterone and catecholamines (Toleikis and Godin, in preparation). In addition, rats subjected to the restraint stress protocol showed increased expression of HSP30 and HSP70 in the heart. This confirms previous findings (Udelsman et al., 1994b; Blake et al., 1995) that SNS stimulation can cause up-regulation of these HSPs. Although the mechanism as of the up-regulation of HSP expression under these restraint stress protocols remains to be determined, their expression in these experiments confirms that HSPs play a fundamental role in maintaining homeostasis, specifically in the heart when exposed to restraint stress. 61 Previous studies have demonstrated that the inducibility of HSP70 is greater in hypertensive rats (Hamet et al., 1990a; 1990b). That is, genetically hypertensive animals display a greater increase in heat-induced HSP70 expression than do normotensive control animals. This hypertension-dependent supersensitivity to heat-induced HSP70 expression was demonstrated in vascular smooth muscle cell cultures (Hamet et al., 1990a) and several tissues of in vivo heat-stressed animals (Hamet et al., 1990b). Further reports showed that this hypersensitivity results from enhanced activation of heat-shock transcriptional regulatory factors in genetically hypertensive animals (Hashimoto et al., 1991) and this may be linked to a polymorphism of the HSP70 gene (Hamet et al., 1992). In attempts to elevate blood pressure in normal rats that do not have hereditary hypertension, Blake et al. (1995) compared the effects of acute and chronic restraint and restraint in combination with a randomized air jet on the development of hypertension and the induction of HSP70 in the aorta and adrenal gland. They found that hypertension developed in normal rats in response to repeated exposure to restraint in combination with intermittent air jet. No hypertension developed in rats exposed only to repeated restraint (Blake et al., 1995). Despite these differences between air jet-induced hypertension and genetic models of hypertension, an increase in HSP70 response in the aorta was observed (Blake et al., 1995). The authors concluded that an increased sensitivity of the HSP response occurs concomitantly with hypertension, 62 regardless of the underlying mechanism responsible for generating hypertension (Blake etal., 1995). It is well accepted that diabetic individuals develop hypertension through disease progression (Sowers and Epstein, 1995). It has been suggested that diabetic hypertension often antedates and may contribute to the development of nephropathy and cardiovascular disease in many diabetic individuals (Sowers and Epstein, 1995). In the present study, the increased HSP70 expression in the heart and liver of rats with short term and chronic diabetes and in the kidney tissue of chronic diabetic rats may be a direct or indirect consequence of hypertension due to diabetes. While not statistically significant, the level of HSP70 expression in the heart tissue of stressed diabetic rats was consistently higher than that of diabetic non-stressed and stressed non-diabetic rats. Whether or not stress and diabetes have additive effects on hypertension and resulting complications remains to be determined; however, it was demonstrated that restraint stress alone did not cause a detectable increase in HSP expression in the liver or kidney. Heat shock proteins have also been observed in many inflammatory and autoimmune diseases, such as arthritis and diabetes (Lamb et al., 1989; Young, 1990; Cohen, 1991; Feige and Cohen, 1991; Gelberetal., 1994). For example, HSP60 has received specific attention for its role as an antigen in (3-cell destruction in IDDM. Jones et al. (1990) have shown that HSP60 is present at low levels in the rat insulinoma cell line. After heat stress or cytokine treatment 63 (eg. TNF-a , IFN-y), increased synthesis of HSP60 was detected (Jones et al., 1990). The antibody reactivity to HSP60 in sera of patients with IDDM was found to be significantly higher in newly diagnosed human patients and in those with long-standing IDDM than those with NIDDM and control subjects (Jones et al., 1990, 1991). Next, the proliferative T-cell responses to human HSP60 in IDDM were measured (Jones et al., 1990, 1991; Jones and Armstrong, 1996). In this assay, reactive CD+ T cells are stimulated by antigen and the proliferative response is measured on the basis of thymidine uptake. In 22 patients with IDDM, 20 showed positive proliferative responses to IDDM whereas only 4 of 17 nondiabetic subjects showed proliferative responses. This demonstration of increased antibody reactivity to human HSP60 in IDDM subjects established HSP60 as a possible antigen in the p-cell of IDDM. Insulin-dependant diabetes mellitus is a complex multifactorial disease, in which genetic susceptibility and environmental conditions each have a distinct role. To date, HSPs have been extensively studied in the p-cells of the pancreatic islets. In the present study, other tissues (i.e. heart, liver, kidney, and lung) of STZ-diabetic rats were examined for HSP expression. Increased HSP70 expression was found in the heart, liver and kidney of diabetic rats. To my knowledge, this is the first reported evidence that HSPs other than HSP60 are expressed in heart, liver and kidney tissues of STZ-diabetic rats. Previously, HSP70 has been examined in pancreatic p-cells (Pugliese et al., 1992). However, there are no reports of increased HSP70 expression in IDDM and 64 expression of HSP70 does not prevent insulitis or IDDM in the nonobese diabetic (NOD) mouse (Pugliese et al., 1992). The diabetic rats in my experiments exhibited significant elevations of plasma glucose (Toleikis and Godin, 1995). It has been suggested that the hyperglycemia associated with diabetes eventually may contribute to hypertension, vascular disease, and nephropathy in the diabetic individual. Hyperglycemia may have direct toxic effects on major organ systems (Sowers and Epstein, 1995). In the heart, the toxic effects of glucose may lead to decreased endothelium-mediated vascular relaxation, increased vasoconstriction and promotion of vascular smooth muscle cell (VSMC) hyperplasia resulting in vascular remodeling and the possibility of atherosclerotic events (Sowers et al., 1994). In the kidney, hyperglycemia results in an increase in the thickness of the glomerular basement membrane (i.e. mesangial expansion) resulting in an increased volume of the glomerulus compared with that of nondiabetic subjects (Sowers and Epstein, 1992, 1995). Basement membranes are composed specialized regions of extracellular matrix composed of type IV collagen, larninin, entactiun/nidogen and proteoglycans which together form a complex mesh-like structure (Myers, 1990). Nonenzymatic glycosylation of long-lived proteins, such as the basement membrane components type IV collagen and larninin and cross-link formation of these components appear to lead to modification of basement membrane ultrastructure and loss of permeability (Myers, 1990). Previous studies have identified a group of "prompt" 65 heat shock proteins (P-SP) which are translocated during heat shock, associate with the nuclear matrix-intermediate filament and regulate both chromatin conformation and the expression of late-appearing, classical HSPs (Saah and Hahn, 1992). Recently, Henle et al. (1993) provided evidence for the prompt heat shock response during protein glycosylation. Therefore, the expression of HSPs may be a direct consequence of glycosylation-induced heat shock response. Alternatively, the mesangial cells in the glomerulus synthesize several growth factors such as platelet-derived growth factor (PDGF), interleukin -1 (IL-1) and endothelin which when released can alter capillary flow, pressure, or both, in the glomerulus (Sowers and Epstein, 1992, 1995). Satoh and Kim (1995) have shown that treatment of astrocytes with PDGF, IL-1, endothelin and other cytokines and growth factors leads to an increased expression of HSP27, suggesting that HSPs are key cellular substrates by which signaling events are mediated. The increased activity of these growth factors and cytokines results in mesangial cell proliferation and matrix overproduction. Hyperglycemia may result in the structural damage of proteins due to free radicals and hydrogen peroxide slowly produced by glucose oxidation (Wolff et al., 1991). These free radical products function as signaling devices which at elevated levels cause cellular toxicity. At these elevated concentrations, reactive oxygen species lead to oxidative stress, and may damage almost all cellular components. The gene products induced after the heat shock response play an established role in survival against reactive oxygen species and oxidative stress (Becker et al., 1990). Thus, HSPs may protect against free radical damage and reactive oxygen species through mechanisms not completely understood (Becker et al., 1990). With the myriad of tissue insults that occur in short-term and chronic diabetes, it is not surprising that increased HSP expression may be a direct consequence of the disease process. In conclusion, an intensification of diabetes-related changes by stress or vice versa has resulted in increased expression in HSP70 in heart, liver, and kidney and HSP30 in heart tissue. 67 C H A P T E R F O U R GENERAL SUMMARY AND CONCLUSIONS The overall objectives of this thesis were to investigate and compare the effects of different stressors on HSP expression in normal and diabetic animals. The findings presented in the previous chapters indicate that a physical stressor and diabetes result in a HSP response. This conclusion is supported by the following observations. 1) Japanese quail, chronically exposed to one of seven stressors for 5 or 10 days, showed increased HSP70 expression only in the heart tissue following either isolation in darkness, noise, cold temperature or inescapable irritation stresses (Chapter 2). 2) Rats which underwent a series of chronic physical restraint stressors exhibited increased expression HSP30 and HSP70 only in the heart tissue (Chapter 3). 3) Rats with short-term diabetes (4 wks) showed specific expression of increased HSP30 and HSP70 in heart and HSP70 in liver tissue (Chapter 3). 4) . Rats with chronic diabetes (12 wks) exhibited specific expression of increased HSP30 and HSP70 in heart, HSP70 in the kidney and liver tissue (Chapter 3). 68 5) The combination of diabetes and restraint stress exhibited identical results as previous conclusion (observation 4). (Chapter 3). While it is clear that many cell-damaging stressors such as heat shock lead to the increased expression of HSPs, we are only beginning to appreciate that many other stressors which are not sufficiently severe to directly affect the viability of tissues may also elicit a stress protein response. Studies have established a link between neurohormonal stress and the activation of heat shock factor-1 in certain select tissues which demonstrates that molecules such as A C T H can initiate a cell type-specific stress response (Udelsman et al., 1993). In diabetes, HSPs have been expressed in p-cells of the pancreas and in the present study, expressed in heart, liver, and kidney tissues. While stressed and diabetic rats increased HSP expression in heart, liver, and kidney tissues, a combination of stress and diabetes failed to further increase HSP expression. 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