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To determine if the stress experienced during streptozotocin. (STZ)-induced diabetes altered the heat shock response, male Sprague-Dawley rats (n.
Cell Stress & Chaperones (2007) 12 (4), 342–352 䊚 Cell Stress Society International 2007 Article no. csac. 2007.CSC-292R

Altered heat stress response following streptozotocin-induced diabetes Eva Najemnikova, Carol D. Rodgers, and Marius Locke Faculty of Physical Education and Health, University of Toronto, 55 Harbord Street, Toronto, Ontario, Canada, M5S 2W6

Abstract The heat shock response involves activation of heat shock transcription factor 1 (Hsf1) followed by the rapid synthesis of the protective heat shock proteins (Hsps). To determine if the stress experienced during streptozotocin (STZ)-induced diabetes altered the heat shock response, male Sprague-Dawley rats (n ⫽ 33; 280–300 g) were assigned to 4 groups: (1) control, (2) diabetic (30 days after 55 mg/kg STZ i.v.), (3) heat stressed (42⬚C for 15 minutes), and (4) diabetic heat-stressed group (heat stressed 42⬚C for 15 minutes, 30 days after 55 mg/kg STZ i.v.). The content of Hsp72, Hsp25, and Hsf1 in skeletal muscles, heart, kidney, and liver was assessed by Western blotting, while electrophoretic mobility shift gel analysis was used to assess Hsf activation. Without heat stress, the constitutive expression of Hsp25, Hsp72, and Hsf1 in tissues from diabetic animals and controls was similar. However, 24 hours following heat stress, the heart, kidney, and liver from diabetic animals showed an increased Hsp72 and Hsp25 content compared to the same tissues from heat-stressed nondiabetic animals (P ⬍ 0.05). The white gastrocnemius and plantaris muscles from heat-stressed animals (diabetic and nondiabetic) both showed significant and similar elevations in Hsp72 content. Interestingly, while all muscles from nondiabetic animals showed significant (P ⬍ 0.05) increase in Hsp25 content after heat stress, no increase in Hsp25 content was detected in muscles from heat-stressed diabetic animals. As expected, Hsf activation was undetectable in all tissues from non–heat-stressed animals but was detectable in tissues from both diabetic and nondiabetic animals following heat stress with the exception of diabetic skeletal muscle, where it was attenuated. Hsf1 content was unaltered in all tissues examined except in the white gastrocnemius muscles from heat-stressed diabetic animals. where it was undetectable. These results suggest that when tissues from STZ-induced diabetic animals are heat stressed, the Hsp/stress response is altered in a tissue-specific manner. This impaired ability to activate the stress response may explain, at least in part, the selective atrophy of certain muscles or muscle fiber types during diabetes.

INTRODUCTION

Cells respond to pathophysiological and environmental stressors by rapidly synthesizing protective proteins known as ‘‘heat shock’’ (Hsps) or ‘‘stress proteins.’’ Hsp72 and Hsp25 are Hsps that appear to confer cellular protection against protein damaging stressors by acting as molecular chaperones and thereby reducing or minimizing protein denaturation, aggregation, and/or incorrect folding (Martin et al 1997; Aufricht et al 1998; Baek et al 2000; Smolka et al 2000; Fauconneau et al 2002). The

Correspondence to: Marius Locke, Tel: 416 978-7055; Fax: 416 946-5310; E-mail: [email protected]. Received 25 April 2007; Revised 12 June 2007; Accepted 18 June 2007.

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regulation of Hsps during episodes of acute stress is mediated by a heat shock transcription factor (Hsf1). When activated, Hsf1 forms a trimer capable of binding to a specific sequence known as the heat shock element (HSE) located in the promoter region of the Hsp genes (Pirkkala et al 2001; Trinklein et al 2004). The term ‘‘Hsf activation’’ refers to the trimerization and HSE binding that precedes the rapid transcription of the protective Hsp genes. While it is well established that Hsps play important roles in minimizing protein aggregation during an acute exposure to stress, their role during exposures to chronic stressors has not been extensively investigated. Given that certain disease conditions, including diabetes, have been linked with incorrect protein folding (Thomas et al 1995; Hayden et al 2005), Hsps may play important roles in

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minimizing protein damage that may occur from the stressful conditions created by the disease. The conditions created during diabetes are known to increase oxidative stress as well as downregulate the cellular antioxidant defense systems (Muchova et al 1999; Kumar et al 2007). Thus, the increased levels of reactive oxygen species and/ or free radicals (Matkovics et al 1982; Sano et al 1998) may modify and/or structurally alter proteins, and this may contribute to protein aggregation and possibly alter cell function. In view of this, Hsp content or the ability to increase Hsp content may be altered during certain chronic stress conditions and warrants further investigation. In addition to limiting proteotoxicity during the disease state, any impaired ability to mount a normal Hsp response may also render tissues more susceptible to certain stressors. Thus, maintaining the ability to induce Hsps may also be an important aspect of adapting to or coping with certain disease states. Research investigating the constitutive or inducible expression of Hsp in the diabetic state is limited and equivocal. The constitutive expression of Hsps in diabetics has been reported to be decreased (Swiecki et al 2003; Atalay et al 2004) or unchanged (Joyeux et al 1999; Yamagishi et al 2001; Chen et al 2005). The stress-inducible expression of Hsps in the diabetic state has also yielded equivocal results. For example, the heat-induced expression of Hsp72 in the liver and adrenal glands from diabetic rats has been reported to be significantly reduced (Yamagishi et al 2001), suggesting the stress/heat shock response may be compromised in the diabetic state. In contrast, others (Joyeux et al 1999; Swiecki et al 2003) have shown the accumulation of myocardial Hsp72 after heat stress to be similar between diabetics and nondiabetics. These discrepancies may be the result of tissue-specific differences, the duration and/or severity of diabetes, or other factors. At present, no study has provided a comprehensive investigation into the tissues known to be affected during diabetes. Thus, the purpose of this study was two fold. First, to determine if the constitutive expression of Hsps in organs and skeletal muscles is altered by diabetes and, second, to determine whether organs and skeletal muscles from diabetic animals demonstrated any alterations in their ability to activate the stress response and accumulate Hsps following an acute heat stress. To do this, rats were made diabetic using the well established method of streptozotocin (STZ) injection (Armstrong et al 1975; Pain et al 1983; Ward et al 2001). Thirty days after STZ and the development of diabetes, a subset of animals was heat stressed (15 minutes at 42⬚C), allowed to recover for various times, and assessed for Hsp72, Hsp25, and Hsf1 content or Hsf activation.

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MATERIALS AND METHODS Animals, STZ injection, and heat stress

Thirty-three male Sprague-Dawley rats (280–300 g; Charles River, Quebec) were maintained on a 12-hour dark/light cycle, housed at 20 ⫾ 1⬚C, 50% relative humidity, and provided with food and water ad libitum. Animals were randomly assigned to one of 4 groups: a control group (C; n ⫽ 5), an STZ-treated group hereafter termed diabetic (D; n ⫽ 5), a heat-stressed group (HS; n ⫽ 10), and an STZ-induced diabetic heat-stressed group hereafter termed diabetic heat stressed (DHS; n ⫽ 13). Diabetes was induced by a single tail vein injection of STZ (55 mg/kg body weight, prepared in 0.1 M NaCl buffer). Thirty days after STZ injection, animals were anesthetized with isoflurane (2%–5% with 1.0 L/min O2) and either euthanized or subjected to heat stress. Animals subjected to heat stress were placed on the surface of a Fisher Standard Isoelectric Focusing System (Fisher Scientific, Unionville, ON, Canada) and core temperature raised to 42⬚C for 15 minutes. Rectal temperature was measured before and during heat stress using a Thermistor TSD 102CA Probe (Santa Barbara, CA, USA) connected to a Biopac data acquisition system. At the appropriate time after heat stress (0, 1, or 24 hours), animals were again anesthetized and various tissues (plantaris, soleus, gastrocnemius, heart, liver, and kidney) removed, quickly weighed, and frozen in liquid nitrogen. Samples were maintained at ⫺70⬚C until processed for either Western blotting or gel shift analyses. Physical characteristics and blood glucose

Body mass and blood glucose concentration of each animal were measured weekly. Blood samples were acquired from a tail vein, and blood glucose concentration was measured using One Touch Basic Blood Glucose Monitoring System (Lifescan Canada Ltd, Burnaby, BC, Canada) and One Touch test strips (Lifescan Canada; range ⫽ 0–600 mg/dl). Electrophoretic mobility shift assay

Portions of muscle and organ tissue (⬃50 mg) were homogenized in 15 volumes of extraction buffer, and analyses of Hsf-HSE binding in extracts were performed using electrophoretic mobility shift gel analysis (EMSA) according to the procedure described by Locke (2000). Protein extracts (50 ␮g) from tissues or muscles were incubated with a 32P-labeled, self-complementary, ideal HSE oligonucleotide (5⬘-CTA GAA GCT TCT AGA AGC TTC TAG-3⬘) in binding buffer (10% glycerol, 50 mM NaCl, 1.0 mM ethylenediamine tetraacetic acid [pH 8.0], 20 mM Tris [pH 8.0], 1.0 mM dithiothreitol (DTT), 0.3 Cell Stress & Chaperones (2007) 12 (4), 342–352

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mg/ml bovine serum albumin) with approximately 0.1 ng (50 000 cpm) of 32P-labeled oligonucleotide and 5.0 ␮g poly (dI dC) (Pharmacia Fine Chemicals, Piscataway, NJ, USA) for at least 30 minutes at room temperature. Samples were electrophoresed on 4% acrylamide gel (5⫻ gel running buffer [pH 8.5], 30% acrylamide, 2% bis-acrylamide, 50% glycerol, 30% APS, Temed) at 200 V for 2–3 hours. Gels were exposed to radiographic film (Amersham-ECL, Mississauga, ON, Canada) for 1 or 2 days at ⫺20⬚C. The correct Hsf-HSE interaction was confirmed by the absence of Hsf activation with the addition of an excess of nonlabeled HSE oligonucleotides as previously described (Locke 2000). Polyacrylamide gel electrophoresis and Western blotting

Muscle and organ portions of ⬃0.05 g were homogenized in 10⫻ volume of 600 mM NaCl and 15 mM Tris (pH 7.5), and protein concentration was determined by the method described by Lowry et al (1951). One-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method described by Laemmli (1970). Following electrophoretic separation, proteins were transferred to nitrocellulose membranes (0.22-␮m pore size; Bio-Rad Laboratories, Mississauga, ON, Canada) as described by Towbin et al. (1979) using the Bio-Rad mini-protean II gel transfer system at a voltage of 45 V for 3 hours. Following protein transfer, nitrocellulose membranes were reacted with polyclonal antibody specific for either Hsp72 (#SPA812, Stress-Gen, Victoria, BC, Canada) or Hsp25 (#SPA801, Stress-Gen) diluted in a 1:2000 dilution in TTBS with 2% nonfat dried skim milk powder as described by Locke (2000). Immunoblots or autoradiograms were scanned using an Agfa Arcus II scanner, and quantification of bands from immunoblots was performed using Kodak 1-D Image Analysis Software, version 2.1 (Kodak Scientific Imaging Systems, New Haven, CT, USA). Heat shock factor 1 (Hsf1) protein content was determined by reacting blots with a polyclonal antibody (Hsf1; #SPA-901, Stress-Gen), a goat anti-rabbit horseradish peroxidase–conjugated secondary antibody was used (Dako Envision #K4000, DakoCytomation, Mississauga, ON, Canada), and the protein was visualized using an enhanced chemiluminescence technique with Biomax-MR Kodak X-ray film (CAT #8701302). Statistical analyses

Two-way analysis of variance was used to test the effect of STZ injection and heat stress. Tuckey’s post hoc comparisons were used to assess significant differences between the treatments. Student’s t-tests were used to comCell Stress & Chaperones (2007) 12 (4), 342–352

Fig 1. Blood glucose and body mass changes after streptozotocin (STZ) injection. (A) Blood glucose levels following STZ injection. Blood samples were collected at 0, 7, 14, 22, and 30 days after STZ (55 mg/kg, i.v.) administration and analyzed for glucose content as described in Materials and Methods. (B) Total body mass following STZ injection. Body mass was measured at 0, 7, 14, 22, and 30 days after STZ administration. Values are provided as mean ⫾ SEM for groups of control (n ⫽ 11) and diabetic (n ⫽ 14) rats. An asterisk (*) signifies a statistical significance (P ⬍ 0.05) between control and diabetic animals.

pare glucose levels, body mass, organ, and muscle mass between control and diabetic groups. Differences between groups were considered statistically significant when P ⬍ 0.05. Data are expressed as means ⫾ SEM. RESULTS Blood glucose and body mass

Initially, diabetic animals showed blood glucose levels (102 ⫾ 1 mg/dL) similar to controls (101 ⫾ 1 mg/dL; Fig 1A). However, 1 week after the injection, STZ-treated animals demonstrated a significant increase (P ⬍ 0.05) in blood glucose levels such that they were 3-fold higher than controls (325 ⫾ 16 vs 101 ⫾ 1 mg/dL). Two weeks after injection and thereafter, blood glucose levels from

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Table 1 Relative weights for organ and muscles 30 days after streptozotocin (STZ) injectiona Muscle/organ Soleus Plantaris Gastrocnemius Heart Kidney Liver

Control (g/kg bw) 0.401 0.915 4.976 2.688 3.406 34.59

⫾ ⫾ ⫾ ⫾ ⫾ ⫾

STZ injected (g/kg bw)

0.01 0.02 0.07 0.05 0.08 0.53

0.411 0.875 4.529 2.934 5.581 47.23

⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.01 0.02 0.08* 0.06* 0.14* 1.61*

a Values are means ⫾ SEM; n ⫽ 5 for both control and STZ treated. An asterisk (*) indicates a significant difference (P ⬍ 0.05) from control.

the diabetic animals were approximately 4-fold higher (P ⬍ 0.05) than control animals (week 2: 420 ⫾ 17 vs 100 ⫾ 1 mg/dL). Additional support for animals experiencing changes associated with the development of diabetes is evident by the observed changes in body mass (Fig 1B). Prior to STZ injection, there were no differences in body mass between the animals; however, 1 week after STZ injection, there was a significantly reduced body mass observed in the diabetic animals compared to nontreated controls (347.0 ⫾ 6.1 vs 370.1 ⫾ 2.1 g). Thereafter, there was a progressive increase in the body mass of both control and diabetic animals. However, the diabetic animals showed a slower rate of body mass gain such that 30 days after STZ injection, diabetic animals were only 78% of the controls. Taken together, these data suggest that the diabetic state created by STZ resulted in conditions similar to that observed during type I diabetes. Organ and muscle mass

Given the lower body mass observed after in diabetic animals, muscle and organ mass might also be expected to be lower. Thus, it was necessary to normalize organ and muscle masses to body mass (Table 1). When the relative muscle and organ masses are expressed per unit body mass, heart, kidney, and liver values were significantly higher (P ⬍ 0.05) in the diabetic animals when compared to controls. The relative mass of the soleus and plantaris muscles were not significantly different between diabetic and control animals, but the relative mass of the gastrocnemius muscles was significantly lower (P ⬍ 0.05) in diabetics vs controls (4.57 ⫾ 0.07 vs 4.97 ⫾ 0.07 mg/g body mass). Thus, STZ-induced diabetes increased the mass of the heart, kidney, and liver, while it decreased the mass of the gastrocnemius muscle. Thermal responses during heat stress

In order to assess the heat stress response during the diabetic state, animals were heat stressed by raising their core temperature to 42⬚C for 15 minutes. Rectal temper-

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ature before the heat stress was not significantly different between diabetic and nondiabetic animals (37.4 ⫾ 0.1 and 37.6 ⫾ 0.2, respectively). Similarly, there were no significant differences observed between diabetic and nondiabetic animals during the 15-minute, 42⬚C heat stress for mean rectal temperatures (42.0 ⫾ 0.1 vs 42.1 ⫾ 0.1⬚C), peak heat stress temperature (42.4 ⫾ 0.2 vs 42.4 ⫾ 0.1⬚C), and heating rate (0.3 ⫾ 0.1 vs 0.3 ⫾ 0.1⬚C per minute). These data confirm that all animals were exposed to a similar heat stress. Hsp72 accumulation in organs following heat stress

To determine whether STZ injection and/or heat stress altered heart, liver, and kidney Hsp72 content, protein homogenates from organs were subjected to SDS-PAGE followed by Western blotting. Portions of representative Western blots showing Hsp72 content from either control (C), diabetic (D), heat stressed (HS), or diabetic with heat stress (DHS) are presented in Figure 2A, visual inspection of which shows that Hsp72 content in the heart, kidney, and liver was not different between control and diabetic animals (lane 1 vs 2). When bands from these and other blots were quantified by densitometric scanning, organ Hsp72 content was not statistically different between the control and diabetic (both non–heat-stressed) animals (Fig 2B). To determine if the heat-induced expression of Hsp72 was altered in organs from both nondiabetic and diabetic animals, tissues were assessed for Hsp72 content 24 hours after a 15-minute 42⬚C heat stress. As expected, the organs from heat-stressed (nondiabetic) animals showed an increased Hsp72 content when compared to organs from controls (Figure 2A, lane 3 vs 1). There was also an increased Hsp72 content in the organs from heatstressed diabetic animals when compared to non–heatstressed diabetic controls (Fig 2B, lane 4 vs 2). When quantified, Hsp72 content in all organs from heatstressed diabetic animals was significantly elevated (P ⬍ 0.05) above that observed in heat-stressed (nondiabetic) animals (Fig 2B). The highest increase in organ Hsp72 content was observed in the liver (30-fold in DHS group vs 20-fold in HS group) followed by the heart (10-fold in DHS vs 5-fold in HS) and the kidney (5-fold in DHS vs 2-fold in HS). These data suggest that organs from diabetic animals are capable of mounting an Hsp72 response to heat stress and may slightly overexpress Hsp72. Hsp25 accumulation in organs following heat stress

To provide an assessment of other protective Hsps, the content of Hsp25 was also examined in organs from diabetic animals both with and without heat stress. Portions of representative Western blots showing the content of Hsp25 in organs from control and diabetic animals are Cell Stress & Chaperones (2007) 12 (4), 342–352

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Fig 2. Hsp72 accumulates in diabetic organs after heat stress. (A) Western blots reacted with Hsp72 antibody. Organs are identified in the left column and experimental treatments across the top. Proteins from control, diabetic, heat-stressed, and heat-stressed diabetic organs were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose, and reacted with Hsp72-specific antibody as described in Materials and Methods. Lane 1: control (C); lane 2: diabetic (D); lane 3: heat stressed (HS); lane 4: heat-stressed diabetic (DHS). (B) Graphical representation of Hsp72 content from blots for heart, kidney, and liver. Values for Hsp72 were obtained by densitometric scanning of bands on Western blots and are expressed as a percentage of control (mean ⫾ SEM). Statistical significance (P ⬍ 0.05) between control and heatstressed groups is indicated by an asterisk (*) and between heatstressed groups by a number sign (#).

shown in Figure 3A, lanes 1 and 2. Similar to what was observed for Hsp72, Hsp25 content remained unchanged in all organs from diabetic animals. Quantification of bands by densitometric scanning showed no statistically significant differences in heart, kidney, and liver Hsp25 content between the control and diabetic animals (Fig Cell Stress & Chaperones (2007) 12 (4), 342–352

Fig 3. Hsp25 accumulates in diabetic organs after heat stress. (A) The bands from Western blots reacted with Hsp25 antibody. Organs are identified in the left column and experimental treatments across the top. Proteins from control, diabetic, heat-stressed control, and heat-stressed diabetic organs were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose, and reacted with Hsp25-specific antibody as described in Materials and Methods. Lane 1: control (C); lane 2: diabetic (D); lane 3: heat stressed (HS); lane 4: heat-stressed diabetic (DHS). (B) Graphical representation of Hsp25 content from blots for heart, kidney, and liver. Values for Hsp25 were obtained by densitometric scanning of bands on Western blots and are expressed as a percentage of control (mean ⫾ SEM). Statistical significance (P ⬍ 0.05) between control and heat-stressed groups is indicated by an asterisk (*) and between heat-stressed groups by a number sign (#).

3B). These results suggest that the diabetic conditions experienced over the 30 days did not provide a sufficient stress to elevate Hsp25 content in these organs. As expected, when nondiabetic animals were heat stressed and allowed to recover for 24 hours, Hsp25 content was increased in all organs examined compared to unstressed (nondiabetic) controls (Fig 3A, lane 3 vs 1). Similarly, 24 hours after exposing the diabetic animals to heat stress, all organs examined also demonstrated an increased Hsp25 content relative to non–heat-stressed dia-

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betic animals (Fig 3A, lane 4 vs 2). Similar to what was observed for Hsp72, after heat stress, there was also a significantly greater increase in organ Hsp25 content (P ⬍ 0.05) from diabetic animals when compared to the organs from heat-stressed nondiabetic animals (Fig 3B). Again, the highest accumulation of Hsp25 was observed in liver (28-fold in the DHS group vs 17-fold in the HS group), while kidney Hsp25 content showed a 10-fold increase if animals were diabetic and heat stressed but only a 5-fold increase with heat stress alone. Similarly, a 2.5fold increase in heart Hsp25 content was detected in heatstressed (nondiabetic) animals, but the amount doubled if the animals were diabetic and heat stressed. The greater heat-induced accumulation of Hsp25 in the organs from diabetics suggests that the conditions experienced during diabetes may predispose organs to be more responsive to certain stressors. Hsp72 accumulation in skeletal muscles following heat stress

To determine whether STZ injection and the subsequent development of diabetes altered the expression of Hsp72 in muscles of the rat hindlimb, muscle proteins (WG, plantaris, and soleus) from control and diabetic animals were assessed for Hsp72 content by Western blotting. Portions of representative Western blots showing the content of Hsp72 in skeletal muscles from control, diabetic, heatstressed, and diabetic/heat-stressed animals are presented in Figure 4A. Similar to what was observed for organs, Hsp72 content in muscles was not elevated by diabetes alone (Fig 4A, lane 1 vs 2). As expected, muscle Hsp72 content was significantly increased in the WG and plantaris muscles (P ⬍ 0.05) but not in the soleus muscles from heat-stressed (nondiabetic) animals when compared unstressed (control) animals. A similar pattern of expression was observed in the muscles from heat-stressed diabetic animals (Fig 4B). However, in contrast to what was observed for organs, muscle Hsp72 content following heat stress was similar between diabetic animals and nondiabetic animals. These results suggest the metabolic alterations experienced over 30 days of diabetes does not cause an accumulation of Hsp72 in rat skeletal muscles and that muscles from diabetic animals respond to heat stress in a similar manner as nondiabetic animals. Hsp25 accumulation in skeletal muscles following heat stress

The content of Hsp25 in muscles from control and diabetic animals prior to and after heat stress are presented in Figure 5A. Hsp25 content in WG, plantaris, and soleus muscles was similar between control and diabetic animals (Fig 5A, lane 1 vs 2). Quantification of bands from

Fig 4. Hsp72 accumulation is similar between control and diabetic muscles after heat stress. (A) The bands from Western blots reacted with Hsp72 antibody. Muscles are identified in the left column and experimental treatments across the top. Proteins from control, diabetic, heat-stressed control and heat-stressed diabetic muscles were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose, and reacted with Hsp72-specific antibody as described in Materials and Methods. Lane 1: control (C); lane 2: diabetic (D); lane 3: heat-stressed (HS); lane 4: heat-stressed diabetic (DHS). (B) Graphical representation of Hsp72 content from blots for muscles. Values for Hsp72 were obtained by densitometric scanning of bands and are expressed as a percentage of control (mean ⫾ SEM). Statistical significance (P ⬍ 0.05) from the respective control is indicated by an asterisk (*).

blots showed no statistically significant differences in muscle Hsp25 content between the control and diabetic animals (Fig 5B), suggesting that diabetes alone was an insufficient stressor to alter the constitutive expression of Hsp25 in muscle. When control and diabetic animals were subjected to heat stress and allowed to recover for 24 hours, the skeletal muscles from heat-stressed (nondiabetic) animals demonstrated an increased Hsp25 content (Fig 5A, lane 3 vs 1). As expected, robust increases were observed for the WG and the plantaris muscles, while a lesser but still significant increase was also observed in the soleus musCell Stress & Chaperones (2007) 12 (4), 342–352

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Fig 5. Hsp25 does not accumulate in diabetic muscles after heat stress. (A) The bands from Western blots reacted with Hsp25 antibody. Muscles are identified in the left column and experimental treatments across the top. Proteins from control, diabetic, heatstressed control, and heat-stressed diabetic muscles were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose, and reacted with Hsp25-specific antibody as described in Materials and Methods. Lane 1: control (C); lane 2: diabetic (D); lane 3: heat stressed (HS); lane 4: heat-stressed diabetic (DHS). (B) Graphical representation of Hsp25 content from WG, plantaris, and soleus muscles. Values for Hsp25 were obtained by densitometric scanning of bands on Western blots and are expressed as a percentage of control (mean ⫾ SEM). Statistical significance (P ⬍ 0.05) between control and heat stress is indicated by an asterisk (*) and between heat-stressed groups by a number sign (#).

cles. Quantification showed Hsp25 content was significantly (P ⬍ 0.05) increased in all muscles from heatstressed animals when compared to the non–heatstressed controls (Fig 5B). When diabetic animals were subjected to heat stress and allowed to recover for 24 hours, no increase in muscle Hsp25 content was observed in any of the muscles examined, and Hsp25 content in the muscles from heat-stressed diabetic animals was not significantly different compared to their respective controls (non–heat-stressed diabetic animals). These results Cell Stress & Chaperones (2007) 12 (4), 342–352

Fig 6. Hsf activation in organs and muscles. (A) Organs. (B) Skeletal muscles. Protein extracts from organs or muscles were assessed by mobility shift electrophoresis as described in Materials and Methods. Lane 1: free probe (FP) no extract added; lane 2: control (C); lane 3: heat stressed (immediately after heat stress; HS); lane 4: heat stressed (1 hour after heat stress; HS1); lane 5: diabetic (D); lane 6: diabetic heat stressed (immediately after heat stress; DHS); lane 7: diabetic heat stressed (1 hour after heat stress; DHS1); lane 8: pure Hsf protein (P).

suggest an altered heat stress response with respect to Hsp25 in skeletal muscles from diabetic animals. Hsf activation in organs from diabetic animals following heat stress

Since diabetes resulted in an altered accumulation of Hsps, the main regulatory protein of the heat stress response was examined by assessing Hsf activation (trimerization and DNA binding ability) by EMSA in heart, kidney, and liver extracts from control and diabetic animals (Fig 6A). When either no extract (lane 1), extracts from unstressed (control) organs (lane 2), or extracts from organs from diabetic animals (lane 5) were incubated with a 32P-labeled HSE and assessed by EMSA, no evidence of Hsf activation was observed (lanes 1, 2, and 5, respectively). However, when organ extracts from heat-

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stressed (nondiabetic) animals and heat-stressed diabetic animals were assessed directly after the heat stress, Hsf activation was detected (lanes 3 and 6, respectively). When both nontreated and diabetic animals were allowed to recover for 1 hour after heat stress, a lower level of Hsf activation was observed (lanes 4 and 7, respectively). A positive control (lane 8) using purified Hsf demonstrates the validity of the assay. Hsf activation in skeletal muscles from diabetic animals following heat stress

Hsf activation was also assessed in skeletal muscles from control (nondiabetic) and diabetic animals with and without heat stress (Fig 6B). No Hsf activation was detected in muscle extracts (WG, plantaris, and soleus) from both control (lane 2) and diabetic (lane 5) animals that were not heat stressed. As expected, Hsf activation was detected in all muscle extracts examined from heat-stressed nondiabetic animals (lane 3). In contrast, although there was a detectable level of Hsf activation directly after heat stress in diabetic animals (lane 6), it appeared slightly reduced when compared to nondiabetic heat-stressed animals. A recovery of 1 hour after the heat stress showed no Hsf activation in any muscle extracts examined (lanes 4 and 7, respectively). These results suggest that Hsf activation may be reduced in muscles from diabetic animals. Hsf1 content in control, heat-stressed, and STZ-treated animals

Since diabetes altered Hsp accumulation after heat stress in certain tissues and the level of Hsf activation also appeared to be reduced, the content of Hsf1, the transcription factor that mediates the stress-induced expression of the Hsps, was assessed in organs and muscles from control, heat-stressed, diabetic, and heat-stressed/diabetic animals (Fig 7). Western blot analyses of organs (Fig 7A) shows organ-specific differences in Hsf1 content in heart, kidney, and liver. However, there were no detectable differences between diabetic animals and controls (Fig 7A, lane 1 vs 2). Hsf1 content was also similar between controls and diabetic animals that were subjected to heat stress and allowed to recover for 24 hours (Fig 7A, lanes 3 and 4). Similar to what was observed with the organs, skeletal muscle Hsf1 content was also detectable in all muscles examined (Fig 7B), and no differences in muscle Hsf1 content were detected between controls and diabetic animals (Fig 7B, lanes 1 and 2). When both nondiabetic and diabetic animals were heat stressed and allowed to recover for 24 hours, no changes in Hsf1 content were observed in the plantaris and soleus muscles; however, Hsf1

Fig 7. Diabetes does not alter organ Hsf1 content, but following heat stress, Hsf1 content is decreased in the WG. Organs (A) and skeletal muscles (B) show that bands from Western blots reacted with Hsf1 antibody. Proteins from control, diabetic, heat-stressed, and heat-stressed diabetic tissues were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose, and reacted with Hsf1-specific antibody as described in Materials and Methods. Lane 1: control (C); lane 2: diabetic (D); lane 3: heat stressed (HS); lane 4: heat-stressed diabetic (DHS).

was undetectable in the WG muscles from heat-stressed diabetic animals. This result was consistent for all heatstressed/diabetic animals (n ⫽ 9) examined. These results suggest that when certain muscles from diabetic animals are subjected to a heat stress and allowed to recover, Hsf1 content may be altered. DISCUSSION

The stress of diabetes eventually damages certain tissues and often leads to serious pathologies. In view of this, at some point in the process, cells and tissues may be rendered more susceptible to certain stressors. The aim of the present study was to determine if tissues from diabetic animals demonstrated an altered constitutive or Cell Stress & Chaperones (2007) 12 (4), 342–352

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heat-induced Hsp expression. To do this, the well-established method of pancreatic beta cell destruction via STZ injection was used to create a diabetic state in rodents. After 30 days, the STZ-treated animals demonstrated elevated blood glucose levels, a reduced body mass, and muscle atrophy. These data are consistent with other studies using STZ injection (Armstrong et al 1975; Pain et al 1983; Ward et al 2001) and suggest that the STZtreated animals in the present study experienced conditions similar to those known to occur during type I diabetes. There are 4 novel findings of this study. First, the diabetic state by created by STZ injection did not alter the constitutive expression of Hsp72 or Hsp25 in organs or skeletal muscles. Second, when diabetic animals were heat stressed and allowed to recover, all organs (heart, liver, and kidney) demonstrated a greater accumulation of Hsp72 and Hsp25 compared to the same tissues from heat-stressed nondiabetic animals. Third, skeletal muscles from heat-stressed diabetic animals failed to show an increased Hsp25 accumulation after heat stress. Lastly, Hsf1 content was unchanged in tissues regardless of treatments except in the white portion of the gastrocnemius muscles from heat-stressed diabetic animals, where Hsf1 was undetectable 24 hours after heat stress. Taken together, these data suggest that the stress associated with diabetes may be insufficient to damage/denature proteins and increase Hsp content but that when tissues from diabetic animals are challenged with heat stress, the stress/ heat shock response may be altered in a tissue-specific manner.

from diabetic animals suggests that 4 weeks of diabetes was insufficient to generate a level of proteotoxicity (protein damage) required to constitutively increase Hsp content.

STZ-induced diabetes does not alter the constitutive expression of Hsp72 or Hsp25

Heat stress fails to increase skeletal muscle Hsp25 content in diabetic animals

In the present study, no differences were observed in the ‘‘basal’’ or constitutive expression of either Hsp25 or Hsp72 in organs or muscles from animals 30 days after STZ injection. These findings are in agreement with others showing the constitutive Hsp72 levels from diabetic animals to be unchanged in liver (Swiecki et al 2003; Atalay et al 2004) and heart (Joyeux et al 1999; Chen et al 2005; Shinohara et al 2006). Similarly, the constitutive expression of myocardial Hsp27 has also been reported to be unchanged in the diabetic state (Chen et al 2005). In contrast, Atalay et al (2004) examined the constitutive expression of Hsp72 in STZ-induced diabetic rats and observed Hsp72 to be decreased in heart, liver, and vastus lateralis muscles but unchanged in the red portion of gastrocnemius muscles. The exact reason for these discrepancies remains unclear, but they may reflect animal age or differences in the duration and/or severity of the diabetes experienced. Our findings showing an unaltered constitutive expression of Hsp72 and Hsp25 in tissues

The present study also investigated the heat induction of Hsps in skeletal muscles from diabetic and nondiabetic animals. As expected and by design, Hsp25 content was elevated in all muscles from heat-stressed (nondiabetic) animals. However, when diabetic animals were heat stressed and allowed to recover for 24 hours, no increase in Hsp25 content was observed in any of the skeletal muscles examined. The reason for the lack of increase in Hsp25 in skeletal muscles from diabetic animals following heat stress remains unknown and may be related to several factors. First, it may reflect the level of Hsf1 activation in skeletal muscles since heat-stressed diabetic animals appeared to show a reduced Hsf activation when compared to muscles from nondiabetic animals (Fig 6). Thus, diabetes may repress Hsf1 activation and thereby alter the heat-induced Hsp25 expression. In support of this idea, the content and activity of glycogen synthase kinase 3 (GSK-3), a protein known to suppress Hsf1 activity (He et al 1998), is elevated in skeletal muscles from

Cell Stress & Chaperones (2007) 12 (4), 342–352

STZ-induced diabetes augments heat-induced Hsp accumulation in organs

When animals were heat stressed and allowed to recover for 24 hours, all organs showed increases in both Hsp25 and Hsp72 content. However, organs from heat-stressed diabetic animals showed a greater Hsp accumulation when compared to heat-stressed nondiabetic animals. These findings are similar to Swiecki et al (2003), where the ratios of liver Hsp72 to actin were greater in heatstressed diabetics when compared to heat-stressed nondiabetics. Since the heat-stressed diabetic organs responded with a greater Hsp accumulation, it suggests that diabetes may create an environment that may prime or sensitize cells to a subsequent stressor. Since diabetes is known to increase the oxidative stress and reduce the expression of antioxidant enzymes (Muchova et al 1999; Kumar et al 2007), it follows that when challenged with the additional stress of hyperthermia, organs from diabetics might experience a relatively greater overall level of stress, thus necessitating greater Hsps levels. Whether the greater Hsp content observed in the diabetic tissues after heat stress stems from an increased requirement for Hsps to cope with the diabetic conditions, an impaired Hsp regulation, or from some other factors remains to be determined.

Diabetes and Hsp expression

diabetic patients (Nikoulina et al 2000). Thus, any increase in GSK-3 content or activity resulting from the diabetic state may enhance Hsf1 inactivation and thereby influence Hsp expression. Second, MAPKAP kinase 2 (MK2) has also been shown to be a potent inhibitor of Hsf1 (Wang et al 2006). Diabetes is known to activate the p38 MAP kinase pathway in certain tissues (Igarashi et al 1999; Wilmer et al 2001), and thus activation of MK2 may also possibly explain the reduced Hsp25 expression in diabetics after heat stress. The reason why Hsp72 expression was unaffected in the diabetic state remains unclear but may reflect a specific requirement for certain Hsps. Hsf1 was undetectable in WG muscles from diabetic animals

Hsf1 was detectable and unchanged in all tissues regardless of treatment with the exception of the white portion of the gastrocnemius muscle from heat-stressed diabetic animals. The exact reason why only the WG muscles from heat-stressed diabetic animals showed no evidence of Hsf1 expression remains unclear, but it was a consistent finding that was observed in all samples (n ⫽ 9) examined. While it may seem contradictory that Hsf1 activation was detectable yet the Hsf1 protein was undetectable, it should be noted that Hsf1 activation was detected in tissues removed from animals either directly after or at 1 hour after heat stress (Fig 6), while Hsf content was measured 24 hours after the heat stress (Fig 7). This suggests that following heat stress, either the existing Hsf1 proteins were degraded and/or the synthesis of new Hsf1 proteins was blunted in the diabetic WG muscles. A common consequence of diabetes is muscle atrophy (Armstrong et al 1975; Pain et al 1983; Ward et al 2001) which is thought to be caused primarily from increased protein degradation (Pain et al 1983). In support of this, the transcription factor NF-kB has been shown to become activated during hyperglycemia (Nishikawa et al 2000) and involved in muscle atrophy (Hunter et al 2002; Dongsheng et al 2004). Thus, it may be the case that after heat stress, Hsf1 was degraded because of the enhanced muscle protein degradation occurring in the WG muscle fibers. This may be an important step in the mechanism(s) that regulate diabetes-induced muscle atrophy. In the present study, all muscles from diabetic animals showed a reduced muscle mass; however, when muscles were normalized to body mass, only the gastrocnemius muscles continued to show a relative reduction in muscle mass or atrophy. This selective muscle (and possibly fiber-type) atrophy has been reported previously, as Armstrong et al (1975) showed that STZ treatment had little effect of slow-twitch muscle fibers but significantly de-

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creased the size of fast-twitch skeletal muscle fibers. One plausible reason for these observed muscle (and possibly fiber-type) differences may be the constitutive level of certain Hsps. The soleus muscles are comprised of predominantly type I (slow) fibers and are known to express high constitutive levels of Hsp72 (Locke and Tanguay 1996). In contrast, the gastrocnemius muscles, at least the white portion, are comprised primarily of type II (fast) fibers, which are known to constitutively express a low Hsp content. Thus, the constitutive Hsp content may a role in minimizing muscle loss. In agreement with this, the damaging effects of NF-kB in heart are minimized by Hsp72 (Chen et al 2004), and skeletal muscle atrophy is minimized following heat stress and the concurrent induction of Hsp72 (Naito et al 2000). This suggests a possible protective role for Hsps in muscle against the deleterious effects of chronic diseases and that an impaired stress response may be involved in the etiology of the selective muscle atrophy known to occur during diabetes. In conclusion, the results of this study suggest that the diabetic state itself did not cause alterations in the Hsp content; however, the Hsp response was altered in both the muscles and the organs from diabetic animals following heat stress. The heat-induced accumulation of Hsp25 and Hsp72 was augmented in all the organs from diabetic animals. In contrast, the heat-induced accumulation of Hsp25 was downregulated in skeletal muscles. Taken together, these data suggest that when tissues from STZinduced diabetic animals are heat stressed, the Hsp/ stress response is altered in a tissue-specific manner. An impaired ability of certain tissues (gastrocnemius muscles) from diabetic animals to activate the stress response may explain, at least in part, the selective atrophy observed in certain muscles or muscle fiber types. Thus, the diabetic state may alter the cellular heat shock or stress response such that the diabetic animal may be at greater risk when challenged with an acute stress. REFERENCES Armstrong RB, Gollnick, PD, Ianuzzo CD. 1975. Histochemical properties of skeletal muscle fibers in streptozotocin diabetic rats. Cell Tissue Res 162: 387–394. Atalay M, Oksala NKJ, Laaksonen DE, et al. 2004. Exercise training modulates heat shock protein response in diabetic rats. J Appl Physiol 97: 605–611. Aufricht C, Ardito T, Thulin G, Kashgarian M, Siegel NJ, Van Why SK. 1998. Heat-shock protein 25 induction and redistribution during actin reorganization after renal ischemia. Am J Physiol 274: F215–F222. Baek SH, Min JN, Park EM, Han MY, Lee YS, Lee YS, Park YM. 2000. Role of small heat shock protein Hsp25 in radioresistance and glutathione-redox cycle. J Cell Physiol 183: 100–107. Chen Y, Arrigo AP, Currie RW. 2004. Heat shock treatment suppresses angiotensin II-induced activation of NF-kappaB pathway and heart inflammation: a role for IKK depletion by heat shock. Am J Physiol 287: H1104–H1114.

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