Leptin, a 167 amino acid peptide, the product of the OB gene, is released mainly by adipocytes (1, 2). This peptide is involved in the regulation of food intake, body weight, and energy expenditure through a central feedback mechanism (3, 4). Leptin exerts its biological effects via specific receptor (OB-R) which have been identified in the pancreas, stomach and in a variety of other tissues (3-5).

Beside the regulation of metabolic functions of the organism leptin is implicated in the modulation of immune processes in the organism. Acute pancreatitis (AP) in humans and in the rats is associated with a marked increase of the plasma leptin level (6). In addition, the upregulation of leptin mRNA signal in the pancreas was detected in the rats after induction of pancreatitis (6). In vitro stimulation of the pancreatic acini with leptin and/or caerulein, resulted in increased of gene expression for leptin receptor (7).

Central or peripheral pretreatment with exogenous leptin protects the pancreas against damage by caerulein-induced pancreatitis (CIP) (5). Sensory nerves, calcitonin gene related peptide (CGRP) and increased generation of nitric oxide (NO) pathway are implicated in the protective effect of leptin on acute pancreatitis (5, 6). In the other side this beneficial effect of leptin was attributed to the reduction in tumor necrosis factor alpha (TNF-alpha) and to the increase in interleukin 4 (IL-4) production (6, 7).

Heat shock proteins (HSPs) have been demonstrated to protect the pancreas against its damage caused by AP. HSP70 attenuates secretagogue - induced cell injury in the pancreas by preventing intracellular trypsinogen activation (8, 9). It has been show that ischemic preconditioning (IP) reduces pancreatic damage in CIP and this effect, at least in part, depends on the pancreatic production of HSP70 and activarion of COXs (10).

The aim of this study was to investigate the changes of HSP60 mRNA signal in the pancreatic AR42J cells subjected to caerulein and leptin.


MATERIAL AND METHODS
The study was performed on AR42J cells (rat pancreatic acinar cell line) (American Type Culture Collection, Rockville, MD, USA). Cell culture was kept in the medium, containing RPMI 1640 Medium with Glutamax-I (Gibco BRL, Gaithersburg, MD, USA) and 10% fetal bovine (FBS, head-inactivated; Gibco BRL) in the ratio 1:1 and with addition of 100 U/ml Penicyline and 100 µg/ml Streptomycine (Sigma, St. Louis, MO, USA) in the standard condition (37°C and 5% CO2). Plateled cells were collected by the TRIzol REAGENT (GIBCO BRL). Caerulein (Takus) from Pharmacia GmbH, Erlangen, Germany and leptin from Sigma Co (St. Louis, MO, USA) were used for the experiments.

Experimental protocol
The study on the effects of caerulein and leptin on HSP60 gene expression in AR42J cells

In this part of the study cells were stimulated with the increasing concentrations of caerulein (10-11, 10-9 or 10-7M), whereas the others were given increasing doses of leptin (10-8 or 10-6M). Cells of both groups were incubated in presence of tested substances for: 0, 1, 3, 5, 12 or 24 hours. Subsequently, the most effective concentrations of caerulein that was 10-11M or leptin 10-8M, were selected for further experiments. Time-course experiments (data not shown) have shown that the most effective incubation time was the 3 hours and this time was used in further part of the study. The separate group of cells was stimulated by combination of caerulein (10-11M) and leptin (10-8M) during 3 hours.

Determination of gene expression for HSP60 in the AR42J cells

AR42J cells were taken (and immediately frozen in liquid nitrogen) from the control and from all experimental groups. Total RNA was extracted by a guanidinum isothiocyanat-e/phenol chlorophorm single step extraction kit from Stratagene. DNA synthesis was performed from 1 µg total cellular RNA using Promega Reverse Transcriptase System according to produced standatd procedure (Promega Corporation, USA).

Primers for HSP60 were synthetized by GIBCO BRL/Life Technologies (Eggenstein, Germany). The HSP60 sense primer was 5' TAT GGC TAT CGC TAC TGG TG, while the HSP60 antisense primer was 5' AAC CTT CAA CAC GGC TAC TC. The expected lenght of this PCR product was 300 bp. Primers were designed using programme Primer Premier 4 (Sigma, St. Louis USA). Concomitantly amplification of control rat ß actin (ClonTech, Palo Alto, CA, USA) was performed on the same sample to assess the RNA integrity. Reaction mixtures for PCR contained cDNA templates, 50 pmol of each primer, and 2.5 U of Taq DNA polymerase (Promega Co, USA) in 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 1.5 mM MgCl2, 0.5 mM dNTPs in a volume of 50 µl. To maximize the amplification specificity, hot-start PCR was performed for 29 cycles (94°C for 5 min, 94°C for 1 min, 58°C for 1 min and 72°C for 2 min). Polymerase chain reaction products were detected by electophoresis on a 1.5% agarose gels containing ethidium bromide. Then visualization under UV light was performed. To compare the level of expression of HSP60 mRNA against the reference gene (ß-actin) mRNA data, the image analysis was employed. PCR products were analyzed using program Gel-Pro analyzer (Media Cybernetics, Silver Spring, Mass, USA).


RESULTS
The study on the effects of caerulein and leptin on HSP60 gene expression in AR42J cells

HSP60 mRNA signal was detected in all samples examined (Figs 1-3). This signal was present in the control group as well as in AR42J cells stimulated by various concentrations of caerulein (10-11, 10-9 or 10-7M). Incubation of the cells with this secretagogue resulted in the significant decrease of HSP60 signal, as compared to the control value (Fig. 1).

Fig. 1. The gene expression for HSP60 measured by RT-PCR in AR42J cells incubated under basal conditions (lane 1), stimulated by caerulein at concentration of 10-11M (lane 2), 10-9M (lane 3) and10-7M (lane 4). M-molecular weight marker: HSP60 - 300bp and ß-actin 764bp.

Gene expression of HSP60 was detected in the AR42J cells after the addition of leptin (10-8 or 10-6M) to the incubation medium. The ratio of HSP60/ß-actin mRNA signal in the control group was 0.25 ± 0.03 after 3 hours of incubation. Application of leptin (10-8 or 10-6M) to AR42J cells resulted in the marked increases of this ratio to 0.78 ± 0.02 and 0.48 ± 0.02 respectively (Fig. 2).

Fig. 2. The changes of intensify of HSP60 mRNA signal detected by RT-PCR in AR42J cells incubated under basal conditions (lane 1), with addition of leptin at concentration of 10-8M (lane 2) and 10-6M (lane 3). M-molecular weight marker: HSP60 - 300bp and ß-actin 764bp.

The strongest HSP60 mRNA signal was found in AR42J cells treated with combination of leptin (10-8M) and caerulein (10-11M). In these cells the ratio of HSP60/ß-actin reached 1.3 ± 0.02 (Fig. 3).

Fig. 3. The HSP60 gene expression intensity by RT-PCR in AR42J cells incubated under basal conditions (lane 1), stimulated by caerulein at concentration of 10-11M (lane 2), or by leptin at concentration of 10-8M (lane 3), and combination of above (lane 4). M-molecular weight marker: HSP60 - 300bp and ß-actin 764bp.

DISCUSSION
Heat shock proteins (HSPs) are highly conserved molecules present in both prokaryotic and eukaryotic cells. These proteins play an important role in cellular function during stress conditions. Beside the high temperature, a variety of stressful stimuli including environmental (ultraviolet radiation or heavy metals), pathological (infections or malignancies), or physiological (growth factors or cell differentiation) factors induce a marked increase of HSP synthesis, known as the stress response (11, 12). The mechanism and the physiological significance of the HSP release are not clear.

In mammalian species, the HSP60 (chaperonin) family consists of mitochondrial Hsp60 (mt-Hsp60) and cytosolic Hsp60 (T-complex polypeptide-1) (12-14). The mt-HSP60 exists in a dynamic equilibrium among monomers, heptamers, and tetradecamers (13, 15). It dissociates into monomers at low concentrations and assembles into tetradecamers in the presence of ATP and mt-Hsp10, the cofactor of mt-Hsp60 (16). The cytosolic Hsp60 forms heterooligomeric ring structures and functions in cytosol to fold cytoskeletal protein such as actin and tubulin (17). HSP60 operated as pro- and antiapoptotic proteins (18).

Our data show, that incubation of AR42J cells in presence of caerulein leads to the significant decrease of HSP60 mRNA signal in these cells. Previous data have demonstrated that caerulein is able to reduce the HSP60 level in the pancreas of rat subjected to acute pancreatitis (19, 20). Caerulein induces apoptotic gene expression (bax, bid, p53) in pancreatic acinar AR42J cells, and it is mediated by increase of the free cystolic intracellular Ca2+ (21). In addition, caerulein stimulation resulted in the activation of NADPH oxidase. This leads to ROS- induced Ca2+-dependent, apoptosis in pancreatic acinar cells (22). Thus apoptosis linked to oxidative stress has been implicated in the pathogenesis of acute pancreatitis. In severe form of this disease apoptosis was detected not only in the pancreas but also in the liver and in the kidneys. Apoptosis could be involved in the mechanism of multiple organ dysfunction syndrome. The previous date have suggested that vascular endothelial growth factor (VEGF) via its anti-apoptotic properties prevents the organism from the organ dysfunction in the acute pancreatitis (23). Caerulein - induced interleukin 6 (IL-6) expression and apoptosis in AR42J cells, could be regulated by NF-kappaB, AP-1, and possibly by mitrogen-activated protein kinase (MAPK) in pancreatic acinar cells (24). The blockade of IL-6 suppressed STAT-3 activation in the pancreas and consequently attenuated the severity of acute pancreatitis by the promotion of pancreatic acinar cell apoptosis (25).

Leptin acts via OB receptor (OB-R) which resembles that of IL-6 and receptors of the class - I cytokine family (26). The demonstration of OB-R gene expression in the pancreatic beta cells and in the pancreatic acinar cell line AR42J, suggests that this peptide might also take part in the physiological control of pancreatic exocrine and endocrine function and signal transduction (27, 28).

Leptin protects the pancreas from the damage in the acute pancreatitis (5-7). Our results revealed that leptin is able to increase the gene expression for HSP60 in AR42J cells. The highest level of HSP60 signal has been observed after the application of leptin together with caerulein to the medium of these cells. There are quite pioneering results. Thus leptin may acts as an anti- or the proapoptotic factor. On the one hand the current study shows that the leptin deficiency is associated with impaired spermatogenesis, increased germ cell apoptosis, and upregulated expression of proapoptotic genes within the testes in mice (29). But on the other hand leptin increased viable cell number via suppression of apoptosis in isolated pancreatic islet cells under these experimental conditions. This mechanism may be responsible at least in part, for an obesity-induced increase in pancreatic beta-cell mass (30). During the lutea phase, leptin acts as an antiapoptotic factor, and at the same time, reverses antiapoptotic action of insulin like growth factor-I (IGF-I), thereby protecting the cells from excessive apoptosis and supporting retention of appropriate cell number, which is necessary for maintenance of homeostasis in developing corpora lutea (31). Leptin stimulates cell proliferation and inhibits apoptosis in OAC cells via ERK, p38 MAPK, phosphatidylinositol 3'-kinase/akt, and JAK2-dependent activation of cyclooxygenase (COX)-2 and prostaglandin E-2 (PGE-2) production. Subsequently PGE-2 mediated transactivation of the epidermal growth factor receptor and JNK activation are essential for the leptin effects. These effects may contribute to the greatly increased risk of esophageal adenocarcinoma in obesity (32).

Leptin administration to the AR42J cells could upregulate HSP60 mRNA signal, and this mechanism could be involved in the protective effect of this peptide on the pancreas, but this requires further study.


REFERENCES

1. Friedman JM, Halaas JL. Leptin and the regulation of body weight in mammals. Nature 1998; 395: 763-770.
2. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature 1994; 372: 425-432.
3. Scot J. New chapter for the fat controller. Nature 1996; 379: 113-114.
4. Campfield LA, Smith FJ, Guisez Y, Devos R, Burn P. Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks. Science 1995; 269: 546-549.
5. Jaworek J, Bonior J, Leja-Szpak A, et al. Sensory nerves in central and peripheral control of pancreatic integrity by leptin and melatonin. J Physiol Pharmacol 2002; 53: 51-74.
6. Konturek PC, Jaworek J, Maniatoglou A, et al. Leptin modulates the inflammatory response in acute pancreatitis. Digestion 2002; 65: 149-160.
7. Jaworek J, Bonior J, Pierzchalski P, et al. Leptin protects the pancreas from damage induced by caerulein overstimulation by modulating cytokine production. Pancreatology 2002; 2: 89-99.
8. Bhagat L, Singh VP, Hietaranta AJ, Agrawal S, Steer ML, Saluja AK. Heat shock protein 70 prevents secretagogue-induced cell injury in the pancreas by preventing intracellular trypsinogen activation. J Clin Invest 2000; 106: 81-89.
9. Bhagat L, Singh VP, Song AM, et al. Thermal stress-induced HSP70 mediates protection against intrapancreatic trypsinogen activation and acute pancreatitis in rats. Gastroenterology 2002; 122: 156-165.
10. Warzecha Z, Dembinski A, Ceranowicz P, et al. Ischemic preconditioning inhibits development of edematous cerulein-induced pancreatitis: involvement of cyclooxygenases and heat shock protein 70. World J Gastroenterol 2005; 11: 5958-5965.
11. Jaattela M. Heat shock proteins as cellular lifeguards. Ann Med 1999; 31: 261-271.
12. Lindquist S, Craig EA. The heat-shock proteins. Annu Rev Genet 1988; 22: 631-677.
13. Fink AL. Chaperone-mediated protein folding. Physiol Rev 1999; 79: 425-449.
14. Hartl FU, Hayer-Hartl M. Molecular chaperones in the cytosol: from nascent chain to folded protein. Science 2002; 295: 1852-1858.
15. Levy-Rimler G, Bell RE, Ben-Tal N, Azem A. Type I chaperonins: not all are created equal. FEBS Lett 2002; 529: 1-5.
16. Levy-Rimler G, Viitanen P, Weiss C, et al. The effect of nucleotides and mitochondrial chaperonin 10 on the structure and chaperone activity of mitochondrial chaperonin 60. Eur J Biochem 2001; 268: 3465-3472.
17. Llorca O, Martin-Benito J, Ritco-Vonsovici M, et al. Eukaryotic chaperonin CCT stabilizes actin and tubulin folding intermediates in open quasi-native conformations. EMBO J 2000; 19: 5971-5979.
18. Parcellier A, Gurbuxani S, Schmitt E, Solary E, Garrido C. Heat shock proteins, cellular chaperones that modulate mitochondrial cell death pathways. Biochem Biophys Res Commun 2003; 304: 505-512.
19. Otaka M, Okuyama A, Otani S, et al. Differential induction of HSP60 and HSP72 by different stress situations in rats. Correlation with cerulein-induced pancreatitis. Dig Dis Sci 1997; 42: 1473-1479.
20. Bonior J, Jaworek J, Konturek SJ, Pawlik WW. Increase of heat shock protein gene expression by melatonin in AR42J cells. J Physiol Pharmacol 2005; 56: 471-481.
21. Yu JH, Kim H, Kim KH. Calcium-dependent apoptotic gene expression in cerulean-treated AR42J cells. Ann N Y Acad Sci 2003; 1010: 66-69.
22. Yu JH, Lim JW, Kim KH, Morio T, Kim H. NADPH oxidase and apoptosis in cerulean-stimulated pancreatic acinar AR42J cells. Free Radic Biol Med 2005; 39: 590-602.
23. Ueda T, Takeyama Y, Yasuda T, et al. Vascular endothelial growth factor increases in serum and protects against the organ injuries in severe acute pancreatitis. J Surg Res 2006; 134: 223-230.
24. Lee J, Seo J, Kim H, Chung JB, Kim KH. Signal transduction of cereulein-induced cytokine expression and apoptosis in pancreatic acinar cells. Ann N Y Acad Sci 2003; 1010: 104- 108.
25. Chao KC, Chao KF, Chuang CC, Liu SH. Blocade of interleukin 6 accelerates acinar cell apoptosis and attenuates experimental acute pancreatitis in vivo. Br J Surg 2006; 93: 332-338.
26. Baumann K, Morella KK, White DW, et al. The full-lenght receptor has signaling capabilities of interleukin 6-type cytokine receptors. Proc Natl Acad Sci USA 1996; 93: 8374-8378.
27. Morton MN, Emilsson V, de Groot P, Pallet AL, Cawthorne MA. Leptin signalling in the pancreatic islets and clonal insulin-secreting cells. J Mol Endocrinol 1999; 22: 173-184.
28. Harris DM, Flannigan CL, Go VL, Wu SV. Regulation of cholecystokinin-mediated amylase secretion by leptin in rat pancreastic acinar tumor cell line AR42J. Pancreas 1999, 19: 224-230.
29. Bhat GK, Sea TL, Olatinwo MO, et al. Influence of a leptin deficiency on testicular morphology, germ cell apoptosis, and expression levels of apoptosis-related genes in the mouse. J Androl 2006; 27: 302-310.
30. Okuya S, Tanabe K, Tanizawa Y, Oka Y. Leptin increases the viability of isolated rat pancreatic islets by suppressing apoptosis. Endocrinology 2001; 142: 4827-4830.
31. Gregoraszczuk EL, Ptak A. In vitro effect of leptin on growth hormone (GH)- and insulin-like growth factor-I (IGF-I)-stimulated progesterone secretion and apoptosis in developing and mature corpora lutea of pig ovaries. J Reprod Dev 2005; 51: 727-733.
32. Ogunwobi O, Mutungi G, Beales IL. Leptin stimulates proliferation and inhibits apoptosis in Barrett's esophageal adenocarcinoma cells by cyclooxygenase-2-dependent, prostaglandin-E2-mediated transactivation of the epidermal growth factor receptor and c-Jun NH2-terminal kinase activation. Endocrinology 2006; 147: 4505-4516.