The effect of dietary lipid manipulation on murine splenic lymphocytes apoptosis and heat shock protein over expression

In this study, we kept BALB/c mice on a hyperlipidic diet for 120 days and then assessed the predisposition to apoptosis and the appearance of heat shock protein (Hsp) on splenic lymphocytes. By immunoblot analysis, bands corresponding to Hsp 60 and Hsp 70 in cells from mice kept on a saturated fatty acid diet showed a greater expression already after 1 month while two other bands, which correspond to Hsp 25 and Hsp 27, were slightly present after 1 month of treatment. In cells from mice kept on a diet rich in unsaturated fatty acid, there was a marked expression of Hsp 25 and Hsp 27 after only 30 days of treatment, which was maintained constant for up to 4 months; while for bands corresponding to Hsp 60 and Hsp 70, a significant minor signal was only detectable after 2-4 months from the beginning of the treatment. Splenic lymphocytes from animals kept on a lipidic diet containing saturated fatty acids were more susceptible to death by apoptosis, while cells of animals treated with unsaturated fatty acid were shown to be more resistant.     

Cell signaling and heat shock protein expression

Exposure of cells and organs to heat shock is associated with numerous changes in various cellular metabolic parameters and overexpression of proteins collectively known as heat shock proteins (HSP). In this communication we review the cell-signaling events that are altered in response to heat shock as they relate to the subsequent induction of HSP 70 kd (HSP-70) expression. We also review the mechanisms by which HSP-70 is involved in conferring cytoprotective effects. The possibility of altering HSP expression through manipulations of the cell-signal process has clinical importance.The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of the Army or Department of Defense.     

Dobutamine mediates cytoprotection by induction of heat shock protein 70 in vitro

Aims

Dobutamine is cytoprotective when applied before a subsequent stress. However, the underlying molecular mechanism is unknown. Dobutamine also inhibits nuclear factor (NF)-κB in human T lymphocytes. Other inhibitors of NF-κB induce a so-called heat shock response. We hypothesized that dobutamine mediates protection from apoptotic cell death by the induction of a heat shock response.

Main methods

Jurkat T lymphoma cells were preincubated with dobutamine (0.1, 0.5 mM) before the induction of apoptosis (staurosporine, 2 μM). DNA-binding of heat shock factor (HSF)-1 was analyzed by electrophoretic mobility shift assay, mRNA-expression of heat shock protein (hsp)70 and hsp90 by Northern Blot, activity of caspase-3 by fluorogenic caspase activity assay and cleavage of pro-caspase-3 by Western Blot. Apoptosis was assessed by flow cytometry after annexin V-fluorescein isothiocyanate staining. Hsp70 and hsp90 were inhibited using N-formyl-3,4-methylenedioxy-benzylidene-gamma-butyrolaetam and 17-allylamino-17-demethoxygeldana-mycin, respectively. All data are given as median and 25/75% percentile.

Key findings

Pre-incubation with dobutamine inhibited staurosporine-induced annexin V-fluorescence (28 [20–32] % vs. 12 [9–15] % for dobutamine 0.1 mM and 7 [5–12] % for dobutamine 0.5 mM, p < 0.001), cleavage of pro-caspase-3 as well as caspase-3-like activity (0.46 [0.40–0.48] vs. 0.32 [0.27–0.39] for Dobutamine 0.1 mM and 0.20 [0.19–0.23] for Dobutamine 0.5 mM, p < 0.01). Dobutamine induced DNA-binding of HSF-1 and mRNA-expression of hsp70 and hsp90. While inhibition of Hsp90 had no effect, inhibition of Hsp70 increased the number of annexin V-positive cells (33 [32–36] % vs. 18 [16–24] %) and caspase-3-like activity (0.21 [0.19–0.23] vs. 0.16 [0.13–0.17], p < 0.05).

Significance

Dobutamine protects from apoptotic cell death via the induction of Hsp70.     

Geranylgeranylaceton induces heat shock protein 72 in skeletal muscle cells

Effects of an antiulcer drug, geranylgeranylaceton (GGA), and/or heat-stress on 72 kDa heat shock protein (HSP72) expression and protein content in cultured skeletal muscle cells were studied. Mouse skeletal muscle cells (C(2)C(12)) were subjected to either 1) control (cultured at 37 degrees C without GGA), 2) GGA administration (10(-11) - 10(-8) M), 3) heat-stress at 41 degrees C for 60 min, or 4) GGA administration combined with heat-stress. Expression of HSP72 was up-regulated by GGA administration. Heat-stress further enhanced the GGA-related up-regulation of HSP72. Administration of GGA caused an increase of muscular protein content as a dose-dependent manner. Protein synthesis was also stimulated by heat-stress alone in myotubes. It was suggested that GGA stimulates the differentiation of myoblasts and protein synthesis. These observations may also suggest that the administration of GGA could be one of the useful tools to gain muscular mass not only in athletes, but also in patients during rehabilitation.     

Different effect of glutamine on macrophage tumor necrosis factor-alpha release and heat shock protein 72 expression in vitro and in vivo

Macrophage plays a vital role in sepsis. However, the modulatory effect of glutamine （Gin） on macrophage/ monocyte-mediate cytokines release is still controversial. Thus, we investigated the effect of Gin on macro- phage tumor necrosis factor （TNF）-α release and heat shock protein （HSP） 72 expression in vivo and in vitro. Data from our study indicated that the increase of HSP72 expression was significant at 8 mM of Gin 4 h after lipopolysaccharide （LPS） stimulation and became independent of Gin concentrations at 24 h, whereas TNF-α release was dose-and time-dependent on Gin. Heat stress （HS） induced more HSP72 and less TNF-α production compared with the non-HS group. However, the production of TNF-α in cells pretreated with HS was increased with increasing concentrations of Gin. Treatment with various concentrations of Gin for 1 h and then 0.5mM Gin for 4h led to an increase in HSP72 expression, but not in TNF-α production. In sepsis model mice, Gin treatment led to a significantly lower intracellular TNF-α level and an increase in HSP72 expression in mouse peritoneal macrophages. Our results demonstrate that Gin directly increases TNF-α release of LPS-stimulated RAW264.7 macro- phages in a dose-dependent manner, and also decreases mouse peritoneal macrophages TNF-α release in the sepsis model. Taken together, our data suggest that there may be more additional pathways by which Gin modulates cytokine production besides HSP72 expression in macrophage during sepsis.     

Calcineurin and heat shock protein 72 in functionally overloaded rat plantaris muscle

The involvement of calcineurin (CaN) and heat shock protein (Hsp) 72 in the regulation of fiber size and/or phenotype in response to functional overload (FO) was investigated. In one FO group, the plantaris muscle was overloaded by cutting the distal tendons (5-10 mm length) of the soleus and gastrocnemius of 3-week-old male Wistar rats. Cyclosporin A (CsA), a CaN inhibitor, was injected daily (5 mg/kg body weight, i.p.) in a second group of FO rats (FO+CsA group) for a 2-week period. Compared to age-matched controls (Con), the absolute and relative plantaris weights were increased in both FO groups: the hypertrophic response was attenuated in FO+CsA rats. The mean cross-sectional area of each fiber type was increased (approximately 2.0-fold) in the plantaris of FO rats: CsA treatment attenuated this effect, although the fibers were still larger than in Con rats. The percent composition of myosin heavy chain (MHC) IIb decreased from 54% in Con to 19% in FO rats, whereas types I, IIa, and IIx MHC increased in the FO rats. CsA treatment blunted the shifts in MHC isoforms: the FO+CsA group showed a smaller decrease in type IIb and a smaller increase in type IIx MHC than the FO group. The levels of CaN-A and -B proteins were higher (approximately 2.5-fold) in FO than Con rats, whereas these values were similar in Con and FO+CsA rats. Hsp72 protein levels were higher in FO (3.6-fold) and FO+CsA (5.2-fold) than Con rats, with the values being significantly higher in the FO+CsA than FO rats. CsA treatment in Con rats had no effects on muscle mass, fiber size, MHC composition, and Hsp72 or CaN levels. Combined, these results suggest that CaN levels are related to changes in both fiber size and phenotype, and that Hsp72 levels are more related to the levels of stress added to the muscle rather than to increases in the slow fiber phenotype in functionally overloaded rat plantaris muscles.     

The acute antinociceptive effect of hyperbaric oxygen is not accompanied by an increase in markers of oxidative stress

Aims

Exposure to hyperbaric oxygen (HBO₂) causes an antinociceptive response in mice. However, breathing oxygen (O₂) at an elevated pressure can potentially cause oxygen toxicity. The aim of this study was to identify the determinants of HBO₂ antinociception and the toxicity profile of HBO₂.

Main methods

Male NIH Swiss mice were assessed for acute antinociceptive responsiveness under room air or 100% O₂ at 1.0 or 3.5 atmospheres absolute (ATA), using the acetic acid-induced abdominal constriction test. For the oxygen toxicity test, mice were exposed to 3.5 ATA oxygen for 11 min, 60 min, and 60 min daily for 2 days (120 min) or 60 min daily for 4 days (240 min), then assessed by analyzing the levels of two oxidative stress markers, MDA (malondialdehyde) and protein carbonyl in brain, spinal cord and lung.

Key findings

Only the combination of 100% O₂ and 3.5 ATA caused significant antinociception. The antinociceptive effect of 100% O₂ was pressure-dependent up to 3.5 ATA. In the oxygen toxicity test, mice exposed to HBO₂ for different time intervals had levels of brain, spinal cord and lung MDA and protein carbonyl that were comparable to that of control animals exposed to room air.

Significance

Treatment with 100% O₂ evokes a pressure-dependent antinociceptive effect. Since there was no significant increase in levels of the oxidative stress markers in the tested tissues, it is concluded that HBO₂ at 3.5 ATA produces antinociception in the absence of oxidative stress in mice.     

Simulated diving after heat stress potentiates the induction of heat shock protein 70 and elevates glutathione in human endothelial cells

Heat stress prior to diving has been shown to confer protection against endothelial damage due to decompression sickness. Several lines of evidence indicate a relation between such protection and the heat shock protein (HSP)70 and HSP90 and the major cellular red-ox determinant, glutathione (GSH). The present study has used human endothelial cells as a model system to investigate how heat stress and simulated diving affect these central cellular defense molecules. The results demonstrated for the first time that a simulated dive at 2.6 MPa (26 bar) had a potentiating effect on the heat-induced expression of HSP70, increasing the HSP70 concentration on average 54 times above control level. In contrast, a simulated dive had no significant potentiating effect on the HSP90 level, which might be due to the higher baseline level of HSP90. Both 2 and 24-h dive had similar effects on the HSP70 and HSP90, suggesting that the observed effects were independent of duration of the dive. The rapid HSP response following a 2-h dive with a decompression time of 5 min might suggest that the effects were due to compression or pressure per se rather than decompression and may involve posttranslational processing of HSP. The exposure order seemed to be critical for the HSP70 response supporting the suggestion that the potentiating effect of dive was not due to de novo synthesis of HSP70. Neither heat shock nor a simulated dive had any significant effect on the intracellular GSH level while a heat shock and a subsequent dive increased the total GSH level approximately 62%. Neither of these conditions seemed to have any effect on the GSH red-ox status.     

The maximal cytoprotective function of the heat shock protein 27 is dependent on heat shock protein 70

Endogenous heat shock proteins (HSPs) 70 and 25/27 are induced in renal cells by injury from energy depletion. Transfected over-expression of HSPs 70 or 27 (human analogue of HSP25), provide protection against renal cell injury from ATP deprivation. This study examines whether over-expressed HSP27 depends on induction of endogenous HSPs, in particular HSP70, to afford protection against cell injury. LLC-PK1 cells transfected with HSP27 (27OE cells) were injured by ATP depletion for 2 h and recovered for 4 h in the presence of HSF decoy, HSP70 specific siRNA (siRNA-70) and their respective controls. Injury in the presence of HSF decoy, a synthetic oligonucleotide identical to the heat shock element, the nuclear binding site of HSF, decreased HSP70 induction by 80% without affecting the over-expression of transfected HSP27. The HSP70 stress response was completely ablated in the presence of siRNA-70. Protection against injury, provided by over-expression of HSP27, was reduced by treatment with HSF decoy and abolished by treatment with siRNA-70. Immunoprecipitation studies demonstrated association of HSP27 with actin that was not affected by either treatment with HSF decoy or siRNA. Therefore, HSP27 is dependent on HSP70 to provide its maximal cytoprotective effect, but not for its interaction with actin. This study suggests that, while it has specific action on the cytoskeleton, HSP 25/27 must have coordinated activity with other HSP classes, especially HSP70, to provide the full extent of resistance to injury from energy depletion.     

Effects of prolonged stanozolol treatment on antioxidant enzyme activities, oxidative stress markers, and heat shock protein HSP72 levels in rat liver

The abuse of anabolic-androgenic steroids (AAS) to enhance physical performance is widespread in sport communities despite their reported side effects. Since the biochemical bases for the hepatotoxic effects of these compounds are largely unknown, this investigation was aimed at testing whether prolonged (8 weeks) treatment with high doses (2 mg kg⁻¹ body weight; 5 d wk⁻¹) of stanozolol (ST), either alone or in conjunction with treadmill-exercise training, induced changes in oxidative stress biomarker levels and antioxidant defence systems in rat liver. After ST oral administration, the mean values of serum parameters related to hepatic function were within normal ranges. No changes in protein carbonyl content and in the reduced to oxidized glutathione (GSH/GSSG) ratio were detected in liver homogenates of ST-treated rats, whereas thiobarbituric acid-reactive substances (TBARS) levels resulted increased (P<0.05). Total superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) activities were higher (P<0.05) in the liver of treated rats but mitochondrial SOD and glutathione reductase (GR) activities, and the 72 kDa heat shock protein (HSP72) level were not modified. Chronic exercise alone did not change any of the above parameters except for a remarkable enhancement of HSP72 expression; in no case training modified the effects of ST treatment. The present data show that 8 wk ingestion of ST, either with or without concurrent exercise training, can induce oxidative stress in rat liver despite the up-regulation of enzymatic antioxidant activities.     

Modulation of protein phosphorylation and stress protein expression by okadaic acid on heat shock cells

We have demonstrated that pretreatment but not post-treatment with okadaic acid (OA) can aggravate cytotoxicity as well as alter the kinetics of stress protein expression and protein phosphorylation in heat shocked cells. Compared to heat shock, cells recovering from 1 hr pretreatment of OA at 200 nM and cotreated with heat shock at 45°C for the last 15 min of incubation (OA→HS treatment) exhibited enhanced induction of heat shock proteins (HSPs) 70 and 110. In addition to enhanced expression, the attenuation of HSC70 and HSP90 after the induction peaks was also delayed in OA→HS-treated cells. The above treatment also resulted in the rapid induction of the 78 kDa glucose-regulated protein (GRP78), which expression remained constant in cells recovering from treatment with 200 nM OA for 1 hr, heat shocked at 45°C for 15 min, or in combined treatment in reversed order (HS→OA treatment). Enhanced phosphorylation of vimentin and proteins with molecular weights of 65, 40, and 33 kDa and decreased phosphorylation of a protein with a molecular weight of 29 kDa were also observed in cells recovering from OA→HS treatment. Again, protein phosphorylation in cells recovering from HS→OA treatment did not differ from those in cells treated only with heat shock. Since the alteration in the kinetics of stress protein expression and protein phosphorylation was tightly correlated, we concluded that there is a critical link between induction of the stress proteins and phosphorylation of specific proteins. Furthermore, the rapid induction of GRP78 under the experimental condition offered a novel avenue for studying the regulation of its expression. © 1996 Wiley-Liss, Inc.     

Identification and expression analysis of four heat shock protein genes associated with thermal stress in rice weevil, Sitophilus oryzae

In order to explore the function of heat shock proteins during thermal stress in rice weevil, Sitophilus oryzae, four heat shock protein genes were cloned and characterized. These heat shock protein genes (hsps) were named as Sohsp70–1, Sohsp70–2, Sohsc70, and Sohsp90, respectively. These hsps showed high sequence conservation with the maximum identity with hsps of Tribolium castaneum and other insects. All the four genes showed the highest mRNA expression in pupal stage and the lowest levels in larval stage. The induced expression of the two Sohsp70s (Sohsp70–1 and Sohsp70–2) were reached to the highest levels (15.59-fold and 12.66-fold) after 2?h of incubation at 37?°C, respectively. Expression of Sohsp90 not only was significantly elevated by heat stress but also by cold stress. Whereas, expression level of Sohsc70 was not induced either by heat or cold stress. Furthermore, for rapid heat hardening, the expression levels of Sohsp70–1, Sohsp70–2, Sohsc70 and Sohsp90 were observed as 2.57, 2.53, 3.33 and 2.33-fold higher than control, respectively; for rapid cold hardening, the expression levels of Sohsp70–1, Sohsp70–2, Sohsc70 and Sohsp90 were reported as 2.27, 3.02, 3.37 and 2.23-fold higher than control, respectively. Hence, our results revealed that the four Sohsps were associated with temperature adaption under rapid heat or cold hardening.