Effects of Diazoxide on the Hepatic Energy Metabolism
In the control group, ATP level on POD 1 decreased from 12.76±2.24 to 8.33±1.34 μmol/g wet liver tissue. ATP levels in remnant livers were significantly higher in the diazoxide group (13.19±2.73) than the control group (9.39±1.95) on POD 2 (Fig. 4A). There was no difference on POD 1 (diazoxide; 9.07±0.99 vs. control; 8.33±1.34). 5-HD neutralized the effects of diazoxide (diazoxide; 10.31±2.14 vs. control; 10.91±0.88) (Fig. 4B).
The ratio of fluorescence intensity (F340/F380) increased by approximately twofold immediately after ATP stimulation of 50 μmol/L in nontreated hepatocytes (Fig. 5A). Similarly, in diazoxide-treated hepatocytes as well, ATP stimulation immediately increased the ratio, indicating the calcium efflux from the storage organellae. However, the increment of the ratio from baseline was significantly lower in diazoxide-treated hepatocytes than nontreated hepatocytes (Fig. 5B). The increment of the ratio from the baseline in the extracellular Ca2+ free condition was again significantly lower in diazoxide-treated hepatocytes than nontreated hepatocytes (Fig. 5C, D).
Sarcolemmal KATP channels are composed of two kinds of subunits: an inwardly rectifying potassium channel (Kir6.x) subunit (Kir6.1 or Kir6.2) consisting of four proteins and SUR subunit (SUR1, SUR2A, or SUR2B), comprising four proteins. So far, variant combinations have been known for ATP channel">KATP channel subunits, for example, Kir6.2/SUR1 in pancreatic β cells, Kir6.1/SUR2A in myocardial and skeletal muscle cells, and Kir6.1/SUR2B and Kir6.2/SUR2B in vascular smooth muscle cells (18–20). Regarding mitochondrial KATP channels, however, molecular identification has not been well defined.
In the intact rat liver, mRNA expression of Kir6.1, SUR1, and SUR2 has been reported by Malhi et al. (21); however, it was not elucidated which type of SUR, SUR1, or SUR2, actually formed the ATP channel">KATP channel in hepatocytes. They simply wrote that the abundances of Kir6.1 and SUR2 were less in the rat liver compared with the rat heart. We, therefore, first reanalyzed which subunits were expressed in hepatocytes. We confirmed the results of Malhi et al. that Kir6.1, SUR1, and SUR2B were expressed in mRNA levels. However, SUR2B protein per se was not detected by Western blotting. For this reason, it is likely that contaminated mRNA from nonparenchymal cell origin was also amplified by polymerase chain reaction process because the expression of SUR2B was much less than other subunits. To probe into the localization, as well as to verify the presence, of KATP channels in hepatocytes, we stained mitochondria immunohistochemically because Kir6.1—a partner subunit of SUR—had been already known in hepatic mitochondria (22, 23). Perfect overlap of SUR1 and Kir6.1 staining with the staining of prohibitin demonstrates that both SUR1 and Kir6.1 are located in hepatic mitochondria. This finding strongly suggests that hepatic mitochondria surely possess KATP channels composed of SUR1 and Kir6.1 subunits, although this type of combination has not been reported in the sarcolemmal ATP channel">KATP channel. Neither of these subunits was on the plasma membrane of hepatocytes, as expected from the fact that hepatocytes are not excitable cells.
Cromakalim, diazoxide, minoxidil, nicorandil, and pinacidil are known as ATP channel">KATP channel openers, and sensitivities of these openers by KATP channels are reportedly different owing to the type of SUR subunits (24). For example, nicorandil activates SUR2B subunit than SUR2A subunit, whereas pinacidil activates SUR2A and SUR2B subunits equally (25). Diazoxide is an opener of pancreatic β-cell ATP channel">KATP channel, which uses SUR1, and is known to more activate SUR1 subunit than SUR2A or 2B (26). It is also explicated that diazoxide potently opens cardiac mitoKATP channels, although the molecular identification has not yet been made. In light of the compatibility between SUR subunits and openers, the fact that diazoxide would be a strong opener of mitoKATP channels is consistent with our finding that hepatic mitoKATP channel subunits are composed of SUR1/Kir6.1, although it remains to be explored whether mitoKATP channels from different cell types equally possess SUR1 subunits.
We administered diazoxide just after hepatectomy surgery, and liver regeneration was evaluated until POD 7. Liver-to-body weight ratio was higher in the diazoxide group, peaking around POD 2, and it became equal on POD 7. This time span of enhanced liver regeneration seems reasonable as a consequence of enhanced DNA synthesis by diazoxide in cooperation with natural hepatocyte growth factor (HGF) secretion at 12 hr after partial hepatectomy (27). With respect to the HGF, Malhi et al. (21) described that mitoKATP channel openers increased DNA synthesis under recombinant human HGF stimulation. Higher PCNA labeling in the diazoxide group also would reinforce the promoted liver regeneration. An antagonistic result by 5-HD administration provides further convincing evidence that the mitoKATP channel is involved in the facilitation of liver regeneration by diazoxide. The accelerated liver weight gain in the diazoxide group during the early postoperative period—a critical period for survival—seems to be significant.
As a major consequence of MPT pore opening is dissipation of the proton motive force, the resulting uncoupling of oxidative phosphorylation leads to ATP depletion and bioenergetic failure of hepatocytes. According to Garlid et al., opening of the mitoKATP channel followed by mitochondrial K+ uptake induces matrix alkalization. It causes complex I to produce reactive oxygen species, which in turn activate protein kinase C-ε, and this inhibits the opening of the MPT pore (11). In this way, inhibition of MPT pore opening after the mitoKATP channel opening is thought to be an important process to keep the mitochondrial integrity during hazard. Although liver regeneration is not a hazardous condition similar to IR injury, maintenance of mitochondrial integrity should be critical for liver regeneration due to a tremendous energy requirement for cell proliferation. In addition, mitochondria should protect themselves from the excess induction of ROS during the hyperdynamic state of ATP production (28–32). Elevation of hepatic tissue ATP level during liver regeneration in the diazoxide group corroborates well with the better maintenance of mitochondrial function by the “opening of mitoKATP channel—inhibition of MPT pore opening” axis.
Interestingly, diazoxide decreased Ca2+ release from intracellular Ca2+ storing organellae on ATP stimulation in hepatocytes. Various studies reported that diazoxide decreased mitochondrial Ca2+ overloading in cardiomyocytes (33–36). González et al. (37) reported that diazoxide down-regulated cardiac L-type Ca2+ channel, which in turn reduced Ca2+ influx during reperfusion. Similar effects of diazoxide may be involved in the mechanism, but unlike cardiomyocytes, hepatocytes do not have an exciting membrane system using rapid Ca2+ influx and outflux. Although it is still ambiguous whether this interesting action of diazoxide to Ca2+ dynamics is the consequence of mitoKATP channel opening, we should turn our attention to the fact that intracellular Ca2+ overloading causes severe mitochondrial damage. During liver regeneration, hepatocytes are exposed to several extracellular stimuli, such as noradrenaline, vasopressin, ATP, and HGF (38). These are calcium-mobilizing agents through activation of G-protein coupling receptor. The decrease of Ca2+ release by diazoxide might lead to the protection of mitochondria and eventually to high ATP content in the remnant liver.
In conclusion, we demonstrated the existence of a mitoKATP channel in hepatocytes composed of Kir6.1 and SUR1. Diazoxide, a potent ATP channel">KATP channel opener, could enhance liver regeneration of the remnant liver by keeping a higher ATP content of the liver tissue. These results suggest that diazoxide will sustain the mitochondrial energetics through mitoKATP channel opening. Furthermore, diazoxide had an additional effect of suppressing calcium overload of the cytoplasm. Despite unanswered questions and remaining ambiguities, diazoxide may serve as a novel tool to enhance liver regeneration after partial liver transplantation including small-for-size liver grafting.
MATERIALS AND METHODS
Eight-week-old male Sprague-Dawley rats (CLEA Japan, Tokyo, Japan) were used. Rats were housed in an appropriate condition according to the guideline for animal protection. The protocols were previously approved by the Animal Research Committee of Akita University (No: 1-2105).
Isolation of Hepatocytes
Primary hepatocytes were isolated from rat livers by a two-step perfusion procedure (39). Hepatocytes were cultured onto the dish coated with collagen (Cell matrix type I-P; Nitta Gelatin, Osaka, Japan) in Dulbecco’s modified Eagle’s Medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% heat-inactivated fetal bovine serum and were maintained at 37°C under a 5% CO2 humidified atmosphere. Cell viability was counted by Trypan blue dye exclusion.
Reverse Transcription Polymerase Chain Reaction
Total mRNA was prepared as described previously (40). Total RNA of 1.5 μg was reverse transcribed using oligo (dT) primers (PrimeScript 1st strand cDNA Synthesis kit; Takara Bio, Shiga, Japan). The sequences of the specific oligonucleotides are listed in Table 1. The cycling profile was as follows: a 2-min denaturation step at 94°C, followed by 35 cycles (25 cycles for glyceraldehyde-3-phosphate dehydrogenase) of denaturation at 94°C for 45 sec, annealing for 30 sec, and extension at 72°C for 1 min. The amplified fragments were analyzed by staining with ethidium bromide on 2% agarose gels.
Western Blotting Analysis
Rat tissues were homogenized in modified radioimmunoprecipitation assay buffer (50 mmol/L Tris-HCl [pH: 8.0], 150 mmol/L NaCl, 1% Nonidet P-40, 0.1% sodium dodecylsulfate, 0.5% sodium deoxycholate, 1 mmol/L phenylmethylsulfonyl fluoride, and protease inhibitor cocktail [Roche, Mannheim, Germany;]). The antibodies used were polyclonal antibodies against Kir6.1 (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), SUR1 (1:200 dilution), and SUR2B (1:200 dilution).
Hepatocytes cultured onto cover glasses were fixed with methanol for 15 min at 4°C. For SUR1, cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with ice-cold methanol for 5 min at −20°C. Internal antigens or internal nonspecific binding proteins were blocked with 3% bovine serum albumin. Hepatocytes were incubated with polyclonal antibodies against Kir6.1 (1:50 dilution), SUR1 (1:50 dilution), SUR2B (1:50 dilution) and monoclonal antibodies against prohibitin (1:50 dilution; Calbiochem, San Diego, CA), a mitochondrial-specific protein, overnight at 4°C. After washing, cells were labeled with fluorescence-conjugated secondary antibodies for 2 hr. Alexa Fluor 488 chicken anti-mouse IgG, Alexa Fluor 594 donkey anti-goat IgG, and Alexa Fluor 594 chicken anti-rabbit IgG (1:200 dilution; Invitrogen, San Diego, CA) were used as secondary antibodies. For nuclear counterstain, 4′,6-diamidino-2-phenylindole was used. Fluorescence was visualized using confocal laser scanning microscope (LSM 510; Carl Zeiss, Göttingen, Germany).
Study Protocol In Vivo
We examined the effect of the ATP channel">KATP channel opener, diazoxide (Sigma-Aldrich), on liver regeneration. Rats were divided into two groups, diazoxide group (n=24) and control group (n=24). All rats were anesthetized with sodium pentobarbital (40 mg/kg, intraperitoneally) and were subjected to 70% partial hepatectomy (41). In the diazoxide group, diazoxide dissolved in dimethyl sulfoxide (2.5 mg/kg) was administered intraperitoneally immediately after surgery. The control group received only vehicle. Rats were killed and their livers collected on PODs 1, 2, 3, and 7 (n=6, each time point). Liver tissues were weighed followed by a fixation in 20% formalin or frozen by freeze cramp for further analysis. All animals survived until that time after surgery. For the antagonizing experiment, we used 5-HD (Biomol Research Laboratories, Plymouth Meeting, PA) dissolved in 0.9% saline as the ATP channel">KATP channel blocker. Rats were subjected to 70% partial hepatectomy in the same way as previous experiments. 5-HD (5 mg/kg) was administered from the tail vein immediately after surgery. After 10 min, diazoxide (n=6) or vehicle (n=6) was administered. We killed the rats and collected livers on POD 2.
Hepatocyte proliferation was evaluated by immunohistochemical staining for PCNA. To prevent nonspecific binding, the sections were blocked with Avidin/Biotin blocking kit (Vector Laboratories, Burlingame, CA). Subsequently, the sections were incubated with mouse monoclonal antibodies against PCNA (1:100 dilution; DakoCytomation, Glostrup, Denmark) overnight at 4°C. The immunoreaction was detected using Vectastain ABC kit (Vector Laboratories). The sections were visualized with diaminobenzidine (Nacalai Tesque, Kyoto, Japan) and counterstained with hematoxylin. The numbers of positive nuclei and hepatocytes were counted in 10 high-power fields in each six rats. Percentage of positive nuclei was calculated as PCNA labeling index.
Hepatic tissue ATP levels were measured using ENLITEN ATP Assay kit (Promega, Madison, WI). Frozen liver samples were weighed and homogenized in 100 volumes of ice-cold buffer containing 0.25 mmol/L sucrose and 10 mmol/L HEPES-NaOH (pH: 7.4). The lysates were centrifuged at 1000×g for 10 min, and ATP was extracted from the supernatant by adding equal amount of Tris-Acetate buffer (pH: 7.75) containing 4% trichloroacetic acid (final concentration; 2%). The samples were aliquoted and stored at −80°C until the experiments. The rL/L reagent and diluted ATP standard or diluted samples were added in Nunc 96 Well Optical Bottom Plates (Thermo Fischer Scientific, Waltham, MA). Luminescences were measured using Infinite M200 (Tecan trading AG, Männedolf, Switzerland). An individual standard curve of ATP dilution was performed in each assay.
Calcium Dynamics in Hepatocyte
Primary hepatocytes were cultured onto cover glasses. Twenty-four hours later, hepatocytes were incubated with Ca2+ indicator, Fura 2-AM (15 μM; Dojindo Labolatories, Kumamoto, Japan), for 45 min at 37°C in Dulbecco’s modified Eagle’s Medium supplemented with 10% fetal bovine serum. Loaded cells were rinsed in Tyrode’s solution (137 mmol/L NaCl, 2.7 mmol/L KCl, 1 mmol/L MgCl2, 1.8 mmol/L CaCl2, 12 mmol/L NaHCO3, 0.45 mmol/L NaH2PO4, 5.5 mmol/L glucose, and 5 mmol/L HEPES [pH: 7.0–7.2]) (21). Fluorescence images of the cells were recorded and analyzed using Aquacosmos imaging system (Hamamatsu Photonics, Shizuoka, Japan) under stimulation of ATP (Cytoskeleton, Denver, CO). To analyze the effect of diazoxide, hepatocytes were exposed to 100 μmol/L diazoxide for 15 min. Fluorescence intensity at an emission wavelength of 510 nm was sequentially analyzed in nontreated hepatocytes or diazoxide-treated hepatocytes by alternating excitation at 340 and 380 nm every 4 sec. Each fluorescence intensity was recorded as F340 and F380, and the ratio (F340/F380) was identified as the level of intracellular Ca2+. For Ca2+-free environment, CaCl2 was replaced with 1 mmol/L ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′- tetraacetic acid in Tyrode’s solution.
Data are presented as mean±standard deviation. Statistical analysis was performed by Student’s t test. A P value of less than 0.05 was considered significantly different.
1. Sekiya S, Suzuki A. Glycogen synthase kinase 3β
-dependent Snail degradation directs hepatocyte proliferation in normal liver regeneration
. Proc Natl Acad Sci USA 2011; 108: 1 1175.
2. Dahm F, Georgiev P, Clavien PA. Small-for-size syndrome after partial liver
transplantation: Definition, mechanisms of disease and clinical implications. Am J Transplant 2005; 5: 2605.
3. Kiuchi T, Onishi Y, Nakamura T. Small-for-size graft: Not defined solely by being small for size. Liver
Transpl 2010; 16: 815.
4. Yellon DM, Baxter GF, Garcia-Dorado D, et al.. Ischemic preconditioning: Present position and future directions. Cardiovasc Res 1998; 37: 21.
5. Kume M, Yamamoto Y, Saad S, et al.. Ischemic preconditioning of the liver
in rats: Implications of heat shock protein induction to increase tolerance of ischemia-reperfusion injury. J Lab Clin Med 1996; 128: 251.
6. Nakayama H, Yamamoto Y, Kume M, et al.. Pharmacologic stimulation of adenosine A2
receptor supplants ischemic preconditioning in providing ischemic tolerance in rat livers. Surgery 1999; 126: 945.
7. Peralta C, Hotter G, Closa D, et al.. The protective role of adenosine in including nitric oxide synthesis in rat liver
ischemia preconditioning is mediated by activation of adenosine A2
receptors. Hepatology 1999; 29: 126.
8. Minor T, Akbar S, Yamamoto Y. Adenosine A2 receptor stimulation protects the predamaged liver
from cold preservation through activation of cyclic adenosine monophosphate-protein kinase A pathway. Liver
Transpl 2000; 6: 196.
9. Dawson VL, Dawson TM. Neuronal ischaemic preconditioning. Trends Pharmacol Sci 2000; 21: 423.
10. Heurteaux C, Lauritzen I, Widmann C, et al.. Essential role of adenosine, adenosine A1 receptors, and ATP-sensitive K+
channels in cerebral ischemic preconditioning. Proc Natl Acad Sci USA 1995; 92: 4666.
11. Garlid KD, Costa AD, Quinlan CL, et al.. Cardioprotective signaling to mitochondria
. J Mol Cell Cardiol 2009; 46: 858.
12. Garlid KD, Paucek P, Yarov-Yarovoy V, et al.. Cardioprotective effect of diazoxide
and its interaction with mitochondrial ATP-sensitive K+
channels. Possible mechanism of cardioprotection. Circ Res 1997; 81: 1072.
13. Nishida H, Sato T, Ogura T, et al.. New aspects for the treatment of cardiac diseases based on the diversity of functional controls on cardiac muscles: Mitochondrial ion channels and cardioprotection. J Pharmacol Sci 2009; 109: 341.
14. Wang L, Kinnear C, Hammel JM, et al.. Preservation of mitochondrial structure and function after cardioplegic arrest in the neonate using a selective mitochondrial ATP channel">KATP channel
opener. Ann Thorac Surg 2006; 81: 1817.
15. Liu Y, Sato T, O’Rourke B, et al.. Mitochondrial ATP-dependent potassium channels: Novel effectors of cardioprotection? Circulation 1998; 97: 2463.
16. Wang Y, Hirai K, Ashraf M. Activation of mitochondrial ATP-sensitive K+
channel for cardiac protection against ischemic injury is dependent on protein kinase C activity. Circ Res 1999; 85: 731.
17. Iwai T, Tanonaka K, Koshimizu M, et al.. Preservation of mitochondrial function by diazoxide
during sustained ischemia in the rat heart. Br J Pharmacol 2000; 129: 1219.
18. Babenko AP, Aguilar-Bryan L, Bryan J. A view of SUR/KIR
channels. Annu Rev Physiol 1998; 60: 667.
19. Isomoto S, Kondo C, Yamada M, et al.. A novel sulfonylurea receptor forms with BIR (Kir6.2) a smooth muscle type ATP-sensitive K+
channel. J Biol Chem 1996; 271: 24321.
20. Yamada M, Isomoto S, Matsumoto S, et al.. Sulphonylurea receptor 2B and Kir6.1 form a sulphonylurea-sensitive but ATP-insensitive K+
channel. J Physiol 1997; 499: 715.
21. Malhi H, Irani AN, Rajvanshi P, et al.. KATP
channels regulate mitogenically induced proliferation in primary rat hepatocytes and human liver
cell lines. Implications for liver
growth control and potential therapeutic targeting. J Biol Chem 2000; 275: 26050.
22. Suzuki M, Kotake K, Fujikura K, et al.. Kir6.1: A possible subunit of ATP-sensitive K+
channels in mitochondria
. Biochem Biophys Res Commun 1997; 241: 693.
23. Foster DB, Rucker JJ, Marbán E. Is Kir6.1 a subunit of mitoKATP
? Biochem Biophys Res Commun 2008; 366: 649.
24. Ashcroft FM, Gribble FM. New windows on the mechanism of action of ATP channel">KATP channel
openers. Trends Pharmacol Sci 2000; 21: 439.
25. Shindo T, Yamada M, Isomoto S, et al.. SUR2 subtype (A and B)-dependent differential activation of the cloned ATP-sensitive K+
channels by pinacidil and nicorandil. Br J Pharmacol 1998; 124: 985.
26. Liu Y, Ren G, O’Rourke B, et al.. Pharmacological comparison of native mitochondrial KATP
channels with molecularly defined surface KATP
channels. Mol Pharmacol 2001; 59: 225.
27. Zarnegar R, DeFrances MC, Kost DP, et al.. Expression of hepatocyte growth factor mRNA in regenerating rat liver
after partial hepatectomy. Biohem Biophys Res Commun 1991; 177: 559.
28. Holley AK, Dhar SK, St Clair DK. Manganese superoxide dismutase vs. p53: Regulation of mitochondrial ROS. Mitochondrion 2010; 10: 649.
29. Ozawa K, Honjo I. Control of phosphorylative activity in human liver mitochondria
through changes in respiratory enzyme contents. Clin Sci Mol Med 1975; 48: 75.
30. Kamiyama Y, Ozawa K, Honjo I. Changes in mitochondrial phosphorylative activity and adenylate energy charge of regenerating rabbit liver
. J Biochem 1976; 80: 875.
31. Ozawa K, Yamada T, Ukikusa M, et al.. Mitochondrial phosphorylative activity and DNA synthesis in regenerating liver
of diabetic rats. J Surg Res 1981; 31: 38.
32. Nishihira T, Tanaka J, Nishikawa K, et al.. Biological significance of enhanced mitochondrial membrane potential in regenerating liver
. Hepatology 1986; 6: 220.
33. Murata M, Akao M, O’Rourke B, et al.. Mitochondrial ATP-sensitive potassium channels attenuate matrix Ca2+
overload during simulated ischemia and reperfusion: Possible mechanism of cardioprotection. Circ Res 2001; 89: 891.
34. Holmuhamedov EL, Wang L, Terzic A. ATP-sensitive K+
channel openers prevent Ca2+
overload in rat cardiac mitochondria
. J Physiol 1999; 519: 347.
35. Wang L, Cherednichenko G, Hernandez L, et al.. Preconditioning limits mitochondrial Ca2+
during ischemia in rat hearts: Role of KATP
channels. Am J Physiol Heart Circ Physiol 2001; 280: H2321.
36. Eaton M, Hernandez LA, Schaefer S. Ischemic preconditioning and diazoxide
limit mitochondrial Ca2+
overload during ischemia/reperfusion: Role of reactive oxygen species. Exp Clin Cardiol 2005; 10: 96.
37. González G, Zaldívar D, Carrillo ED, et al.. Pharmacological preconditioning by diazoxide
downregulates cardiac L-type Ca2+
channels. Br J Pharmacol 2010; 161: 1172.
38. Michalopoulos GK, DeFrances MC. Liver regeneration
. Science 1997; 276: 60.
39. Seglen PO. Preparation of rat liver
cells: III. Enzymatic requirements for tissue dispersion. Exp Cell Res 1973; 82: 391.
40. Kikuchi I, Uchinami H, Nanjo H, et al.. Clinical and prognostic significance of urinary trypsin inhibitor in patients with hepatocellular carcinoma after hepatectomy. Ann Surg Oncol 2009; 16: 2805.
41. Higgins GM, Anderson RM. Experimental pathology of the liver
: I. Restoration of the liver
of the white rat following partial surgical removal. Arch Pathol 1931; 12: 186.
Keywords:© 2012 Lippincott Williams & Wilkins, Inc.
ATP channel">KATP channel; Diazoxide; Mitochondria; Liver; Regeneration