HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 17/12/1748 02-1186fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by SHANKAR, K. Articles by MEHENDALE, H. M. Search for Related Content PubMed PubMed Citation Articles by SHANKAR, K. Articles by MEHENDALE, H. M.

Activation of PPAR- in streptozotocin-induced diabetes is essential for resistance against acetaminophen toxicity¹

KARTIK SHANKAR^*, VISHAL S. VAIDYA^*, J. CHRISTOPHER CORTON, THOMAS J. BUCCI, JIE LIU, MICHAEL P. WAALKES and HARIHARA M. MEHENDALE^*,2

^* Department of Toxicology, School of Pharmacy, College of Health Sciences, The University of Louisiana at Monroe, Louisiana, USA;
ToxicoGenomics, Chapel Hill, North Carolina, USA;
Pathology Associates International, National Center for Toxicological Research, Jefferson, Arkansas, USA; and
Inorganic Carcinogenesis Section, NCI at NIEHS, Research Triangle Park, North Carolina, USA

²Correspondence: 700 University Avenue, Sugar Hall 306, Department of Toxicology, School of Pharmacy, The University of Louisiana at Monroe, Monroe, LA 71209-0495, USA. E-mail: mehendale{at}ulm.edu

SPECIFIC AIMS

Streptozotocin-induced diabetic (DB) mice are resistant to several hepatotoxicants including acetaminophen (APAP). As peroxisome proliferator pretreatment protects against hepatotoxicity, the role of peroxisome proliferator-activated receptor- (PPAR-) as a hepatoprotective mechanism in diabetes was investigated using wild-type (WT) and PPAR-^-/-–DB mice.

PRINCIPAL FINDINGS

1. Protection against APAP-induced liver injury is mediated via PPAR-
WT– and PPAR-^-/-–non-DB mice treated with APAP [600 mg/kg, intraperitoneally (ip)] died by 24 h after APAP administration. However, 70% of the WT–DB mice escaped death from the same dose of APAP. However, PPAR-^-/-–DB mice lost the protection conferred by diabetes (80% mortality). Liver injury [plasma alanine aminotransferase (ALT) and histopathology], during a time course (0–72 h) after APAP administration, was significantly higher in the WT– and PPAR-^-/-–non-DB mice compared with their DB cohorts (Fig. 1 A). Liver injury measured by plasma ALT activity and liver histopathology (Fig 1B ) in the PPAR-^-/-–DB mice increased only after 24 h and led to death of mice, showing a delayed onset and progression of liver injury.

View larger version (86K): [in this window] [in a new window]   Figure 1. A) Plasma ALT activity over a time-course after the administration of APAP. Results are expressed as means ± SE. !, Significantly lower than non-DB control at the same time-point; #, significantly higher than respective 0 h control; P 0.05. B) Representative photomicrographs of hematoxylin and eosin (H&E)-stained liver sections over a time-course after APAP administration to WT and PPAR--/- and DB and non-DB mice. a) WT–non-DB after 12 h; b) WT–DB after 12 h; c) PPAR--/-–non-DB after 12 h; d) PPAR--/-–DB after 12 h; e) WT–DB after 36 h; f) PPAR--/-–DB after 36 h. Non-DB and DB mice treated with saline (vehicle for APAP) alone showed no necrosis (data not shown). C, Central vein; P, pyknotic nuclei; N, areas of necrosis. Original magnification, 400x. C) 3H-Thymidine incorporation into hepatonuclear DNA after APAP administration to non-DB and DB and WT and PPAR--/- mice. Results are expressed as means ± SE (n=4). !, Significantly lower than non-DB control at the same time-point; #, significantly higher than the respective 0 h control; P 0.05. D) Representative photomicrographs of the proliferating cell nuclear antigen (PCNA) immunohistochemistry of liver sections from (a) WT–non-DB after 24 h; (b) WT–DB after 24 h; (c) PPAR--/-–non-DB after 24 h; (d) PPAR--/-–DB after 24 h; (e) WT–DB after 48 h; (f) PPAR--/-–DB after 48 h.

2. Compensatory cell division is substantially lower in PPAR-^-/-–DB mice
Hepatocellular S-phase DNA synthesis, assessed by ³H-thymidine pulse-labeling, was severely decreased in the non-DB mice (WT and PPAR-^-/-) after APAP treatment. Treatment with APAP resulted in higher DNA synthesis in WT–DB mice (Fig. 1C ): It was significantly higher at 36, 48, and 72 h in the WT–DB group compared with the PPAR-^-/-–DB mice (Fig. 1C ). Histomorphometric analyses of PCNA immunohistochemically stained liver sections confirmed the pulse-labeling results (Fig. 1D ).

3. Hepatic Cyp4a, 2e1, and 1a2 expression in non-DB, DB, WT, and PPAR-^-/- mice
Cyp4a induction, a well-established marker of PPAR- activation, showed twofold increase in WT–DB mice. Conversely, Cyp4a protein was decreased in the PPAR-^-/-–DB mice compared with the non-DB–PPAR-^-/- mice. No significant differences were found in microsomal Cyp2e1 and 1a1/1a2 protein levels between WT–non-DB and WT–DB mice. Cyp2e1 and 1a1/1a2 protein levels decreased in PPAR-^-/-–DB mice by 16% and 32%, respectively, compared with their non-DB counterparts.

4. Gene expression analyses via microarray and real-time-polymerse chain reaction (RT-PCR)
Using hierarchal clustering (Cluster^TM), six unique groups of genes were identified (Fig. 2 ). Gene expression of stress-response genes such as Gadd 45, Gadd 153, EGR-1, Rad50, hsp40, OSIP, and protooncogenes, like c-myc and c-fos and c-jun and jun-B, were similar in all groups (r²=0.93; Fig. 2 ). Please refer to the full manuscript for detailed results of the microarray experiments.

View larger version (31K): [in this window] [in a new window]   Figure 2. Cluster analysis of hepatic gene expression profiles in WT, PPAR--/-, non-DB, and DB mice, 12 h after APAP treatment. Forty genes that showed significant change in at least two groups were selected. Hierarchical clustering was performed using Cluster software, and data presentation was done using Treeview. Gene expression changes are depicted as intensity of color, increase (red), decrease (blue), and no change (black). On the right side of the cluster diagram, six groups of genes (A–F) are identified based on their gene expression changes, and correlations of their changes are given. Dotted red lines separate clusters.

5. Nuclear factor (NF)-B activation in WT, PPAR-^-/-, non-DB, and DB mice
Consistent with previous reports, APAP treatment inhibited NF-B. However, NF-B remained remarkably activated in the WT–DB mice at both time points. Basal expression of NF-B in PPAR-^-/-–DB mice was lower at 0 h followed by only modest activation at 4 h. PPAR-^-/-–DB mice showed considerably lower NF-B at 12 h after APAP compared with WT–DB mice. It appears that activation of NF-B in diabetes is mediated directly or indirectly via PPAR-.

6. Antimitotic intervention abolishes resistance to APAP hepatotoxicity in WT–DB mice
Lethality studies were performed to test the role of augmented tissue-repair response in the WT–DB mice. This was accomplished by blocking cell division with colchicine (CLC; 1 mg/kg, ip, in saline) at 2 and 30 h after APAP treatment. CLC antimitosis resulted in significant mortality in the WT–DB mice (WT–DB+APAP+CLC). In contrast to the WT–DB + APAP + saline group that resulted in no mortality, there was 80% mortality in the CLC-treated group of DB mice. Control groups treated with CLC alone did not show any mortality nor any adverse effects as shown previously.

CONCLUSIONS AND SIGNIFICANCE

DB mice are resistant to a normally lethal dose of APAP. Earlier investigations suggested that greater clearance of APAP might play role in DB-mediated resistance. However, three independent, indirect markers of its reactive intermediate argue that a decrease in Cyp2e1- and 1a1/1a2-mediated bioactivation is not the mechanism by which the marked hepatotoxic protection against APAP toxicity occurs in the DB mice. Several studies have shown that STZ-induced diabetes results in induction of Cyp4a mRNA and protein and also acyl-CoA oxidase protein, classical markers of PPAR- activation. Does PPAR- influence the ability of liver to respond to and repair tissue lost to injury after APAP challenge? PPAR- activation, following treatment with peroxisome proliferators, protects from toxicity of several hepatotoxicants including APAP. The protective mechanism is unrelated to decreased bioactivation or to increased detoxification by glutathione. PPAR-^-/- mice challenged with CCl₄ developed acute liver failure with 50% greater mortality compared with WT controls. The authors suggest that PPAR- may be involved in mitigating liver inflammation. Recent evidence also points to the role of PPAR- in liver regeneration and wound healing. Peak of S-phase DNA synthesis is delayed almost by 24 h in PPAR-^-/- mice after partial hepatectomy. PPAR- activation is known to increase cell proliferation and to decrease apoptosis, both of which can increase the ability of the liver to repair and restore tissue after chemical injury. These observations are consistent with the hypothesis that PPAR- activation may be a protective mechanism in DB mice. Our data from lethality experiments with WT and PPAR-^-/-–DB mice support this hypothesis. A role of HSP also appears in the DB mice after APAP challenge. Diabetes alone did not cause any major changes in gene expression of HSPs, except for a decrease in HSP 70 and increases in HSP 60 and HSP 86. Induction of HSP 70 after APAP treatment occurs only in the WT–DB mice and not in the PPAR-^-/-–DB mice, suggesting that PPAR- may be required and indirectly responsible for this response. Induced HSPs protect against liver injury of ischemic reperfusion, CCl₄, thioacetamide, bromobenzene, and APAP. The plausible involvement of PPAR- in HSP regulation is a novel hypothesis.

Several lines of evidence suggest that activation of the redox-sensitive transcription factor NF-B is important in PPAR--mediated effects. It can be speculated that PPAR--mediated increases of acyl-CoA oxidase, Cyp4A, peroxisomal ß-oxidation enzymes, and hydrogen peroxide coupled with decreases in catalase in diabetes activate oxidative stress-responsive transcription factors as NF-B. However, the question is how much does PPAR- contribute to the activation of NF-B? Our studies shed some light on this question. NF-B activation that occurs selectively in the DB mice after APAP diminishes in the PPAR-^-/-–DB mice. This is consistent with our hypothesis that enhanced oxidative stress induced indirectly via PPAR- is responsible for activation of certain cell-survival strategies (HSPs, cell-proliferation genes, etc.; Fig. 3 ). The cyclin D1 promoter contains multiple regulatory elements (AP1, E2F, Oct, SP1/SP3, NF-B) and some uncharacterized elements that play an important role in its regulation. However, the precise mechanism behind increased cyclin D1 mRNA in the DB mice remains unclear.

View larger version (21K): [in this window] [in a new window]   Figure 3. Schematic of hypothesized mechanisms involved in mediating increased cell proliferation after APAP challenge in STZ–DB mice. Diabetes leads to activation of PPAR- and consequent increased oxidative stress. Redox-sensitive transcription factor NF-B can be activated via multiple pathways, directly or indirectly via PPAR-. After APAP treatment, NF-B is activated selectively in the WT–DB mice, in a PPAR--dependent manner. Cyclin D1 gene and protein expression, which was found increased in the WT–DB mice, can be influenced directly via NF-B or other regulators such as p38 MAPK, which is known to negatively regulate cyclin D1 and is significantly decreased in diabetes. Higher cyclin D1 expression is consistent with earlier progression of hepatocytes from G0 to G1 cell-cycle phases after APAP challenge. PPRE, PPAR response element; ACO, acyl-CoA oxidase.

In conclusion, we report a novel role of diabetes-induced PPAR- as a protective mechanism. Greater HSP induction accompanied with robust regeneration in the DB mice after APAP challenge appear to be the underlying mechanisms.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1186fje; doi: 10.1096/fj.02-1186fje

This article has been cited by other articles:

				J. N. Baumgardner, K. Shankar, S. Korourian, T. M. Badger, and M. J. J. Ronis Undernutrition enhances alcohol-induced hepatocyte proliferation in the liver of rats fed via total enteral nutrition Am J Physiol Gastrointest Liver Physiol, July 1, 2007; 293(1): G355 - G364. [Abstract] [Full Text] [PDF]

				M. A. Peraza, A. D. Burdick, H. E. Marin, F. J. Gonzalez, and J. M. Peters The Toxicology of Ligands for Peroxisome Proliferator-Activated Receptors (PPAR) Toxicol. Sci., April 1, 2006; 90(2): 269 - 295. [Abstract] [Full Text] [PDF]

				S. P. Sawant, A. V. Dnyanmote, M. S. Mitra, J. Chilakapati, A. Warbritton, J. R. Latendresse, and H. M. Mehendale Protective Effect of Type 2 Diabetes on Acetaminophen-Induced Hepatotoxicity in Male Swiss-Webster Mice J. Pharmacol. Exp. Ther., February 1, 2006; 316(2): 507 - 519. [Abstract] [Full Text] [PDF]

				J. C. Corton and H. M. Brown-Borg Peroxisome Proliferator-Activated Receptor {gamma} Coactivator 1 in Caloric Restriction and Other Models of Longevity J. Gerontol. A Biol. Sci. Med. Sci., December 1, 2005; 60(12): 1494 - 1509. [Abstract] [Full Text] [PDF]

				H. M. Mehendale Tissue Repair: An Important Determinant of Final Outcome of Toxicant-Induced Injury Toxicol Pathol, January 1, 2005; 33(1): 41 - 51. [Abstract] [Full Text] [PDF]

				S. P. Anderson, P. Howroyd, J. Liu, X. Qian, R. Bahnemann, C. Swanson, M.-K. Kwak, T. W. Kensler, and J. C. Corton The Transcriptional Response to a Peroxisome Proliferator-activated Receptor {alpha} Agonist Includes Increased Expression of Proteome Maintenance Genes J. Biol. Chem., December 10, 2004; 279(50): 52390 - 52398. [Abstract] [Full Text] [PDF]

				G. L. Guo, J. S. Moffit, C. J. Nicol, J. M. Ward, L. A. Aleksunes, A. L. Slitt, S. A. Kliewer, J. E. Manautou, and F. J. Gonzalez Enhanced Acetaminophen Toxicity by Activation of the Pregnane X Receptor Toxicol. Sci., December 1, 2004; 82(2): 374 - 380. [Abstract] [Full Text] [PDF]

				J. C. Corton, U. Apte, S. P. Anderson, P. Limaye, L. Yoon, J. Latendresse, C. Dunn, J. I. Everitt, K. A. Voss, C. Swanson, et al. Mimetics of Caloric Restriction Include Agonists of Lipid-activated Nuclear Receptors J. Biol. Chem., October 29, 2004; 279(44): 46204 - 46212. [Abstract] [Full Text] [PDF]

				P. Howroyd, C. Swanson, C. Dunn, R. C. Cattley, and J. C. Corton Decreased Longevity and Enhancement of Age-Dependent Lesions in Mice Lacking the Nuclear Receptor Peroxisome Proliferator-Activated Receptor {alpha} (PPAR{alpha}) Toxicol Pathol, August 1, 2004; 32(5): 591 - 599. [Abstract] [PDF]

This Article Full Text (PDF) All Versions of this Article: 17/12/1748 02-1186fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by SHANKAR, K. Articles by MEHENDALE, H. M. Search for Related Content PubMed PubMed Citation Articles by SHANKAR, K. Articles by MEHENDALE, H. M.