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Effect of Amorphophallus Konjac oligosaccharides on STZ-induced diabetes model of isolated islets
Life Sciences 72 (2002) 711 – 719 www.elsevier.com/locate/lifescie
Effect of Amorphophallus Konjac oligosaccharides on STZ-induced diabetes model of isolated islets Xiu-Ju Lu *, Xiu-Min Chen, De-Xian Fu, Wei Cong, Fan Ouyang National Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, P.O. Box 353, Beijing 100080, PR China Received 25 January 2002; accepted 27 August 2002
Abstract Three oligosaccharide fractions from the root of Amorphophallus Konjac, which was reported with hypoglycemic effects on diabetes subjects, were isolated and studied using the STZ-treated diabetes model. Among them, one fraction named as KOS-A, was found with nitric oxide (NO) free radical regulation effect, while the other two were not. At concentrations less than 1.5 mM, KOS-A positively decreased STZ-induced NO level of islets, but normal NO release for non-STZ-treated islets was not affected within the range. At 15 mM, KOS-A played a contrary role and increased NO level for islets both with and without STZ-treatment. Islets insulin secretion changed corresponding to NO level in the assay. Increased insulin secretion appeared parallel to the decrease of NO, and normal insulin release was not affected by KOS-A less than 1.5 mM. Structure determination of KOS-A shows that it is a tetrasaccharide with Mw of 666 Da and reductive end of a-D-mannose. These results indicate that low dosage of KOS-A, with its function on attenuating STZ-induced NO level, doesn’t alter normal NO and insulin secretion pathways of isolated islets. The NO attenuation function of KOS-A on the diabetes model is mainly resulted from environmental free radical scavenging by the oligosaccharide. Present results also imply the mechanism of clinical Amorphophallus Konjac hypoglycemic function maybe related with free radical attenuation and lower risks of islets damage from NO radical. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Diabetes model; Streptozotocin; Konjac oligosaccharide; Nitric oxide
* Corresponding author. Tel.: +86-10-62574303; fax: +86-10-62561822. E-mail address: [email protected] (X.-J. Lu). 0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 2 3 0 3 - 2
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Introduction Numerous studies have shown that while different indices of free-radical damage increase, there is a decrease in the concentration of various individual antioxidant substances, indicating the presence of oxidative stress in diabetes [1–8]. Streptozotocin (STZ) caused a selective destruction of islet h cells associated with generation of free radicals [9], which has been used in inducing diabetes in experimental model [10]. Acute exposure of isolated islets to STZ in vitro results in a series of damages in h-cell metabolism and function, and STZ-treated islets was also used as a diabetes model for new drug discovery [11]. Amorphophallus Konjac has long been used in Traditional Chinese Drugs (TCD) as an immunoregulating and health-care food. Konjac Oligosaccharide (KOS) is the basic components of Amorphophallus Konjac composed of h-D-glucose and h-D-mannose with the ratio of 1:1.5 or 1:1.6 [12]. Recent studies demonstrate that Konjac functions in reactive oxygen species (ROS) related diseases, such as lowering fast plasma glucose in subjects with type 2 diabetes mellitus (T2DM) [13,14], preventing the senescence of cranial nerve cells and cardiac muscle cells in mice [15,16], and inhibiting tumor genesis and metastasis [17]. However, there is still no report of KOS effects on islets, though it maybe related with detail mechanisms of Konjac on diabetes. Based on above information, we evaluated the function of KOS on a diabetes model of isolated islets to find details of the OS on pathogenesis of diabetes. In order to exclude the effects of different fractions of KOS on the model, crude KOS was first separated and purified by a mixing column chromatography. Different components were collected when eluted at ethanol concentrations from 5% to 20%. Here the STZ treated islets were used as the model imitating islet h-cell damages in vitro. Nitric oxide (NO) radical and insulin secretion were assayed for evaluation KOS effects on islets. The results show that one component of KOS consisted of tetrasaccharide could regulate NO level on STZ-treated islets in a dose-dependent manner.
Materials and Methods Materials Radioimmunoassay (RIA) kits for insulin test were from Chinese Institute of Atomic Energy (Beijing, China). All other chemicals and biochemicals were of analytical grade and purchased from Sigma Chem. Co. (St. Louis, MO) or Life Technologies Inc. (Beijing, China). Crude KOS was kindly provided by Zhaotong Pharmaceutical Co. (Yunnan, China). Pancreatic islets were isolated from 3 weeks old mice of either sex by collagenase digestion and density gradient centrifugation [18]. STZ was dissolved as 0.1 M stock in citrate buffer (10 mM, pH4.5) immediately before use, and sterilized by 0.22 Am microfiltration. Then the stock was diluted in Krebs medium (10 mM Hepes, pH7.4, 115 mM NaCl, 24 mM NaHCO3, 5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 11.1 mM D-glucose, 0.1% Bovine serum albumin) to final concentrations of 0.5–5 mM. Separation and purification of Konjac oligosaccharides KOS crude solution in deionized (D.I.) water was purified through a mixing column filled with activated charcoal and diatomite with the ratio of 1:1. Eluted parts with step-wise ethanol solutions were
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collected after being first washed with D.I. water to get rid of salt and monosaccharide. Here three fractions corresponding to ethanol concentration elutes of 5%, 10% and 20% were used in following experiments, termed KOS-A, KOS-B and KOS-C, respectively. Nitric oxide release Nitrite (NO2 ), which is a stable oxidized end product of NO, was measured by a colorimetric assay based on the Griess reaction [19]. Briefly, 100 Al culture supernatants were incubated for 10 min at room temperature with 100 Al Griess reagent (0.5% sulfanilamide, 0.05% naphthylethylenediamine dihydrochloride, and 2.5% H3PO4). The absorbance at 540 nm was determined by a spectrophotometer. Nitrite concentrations (from 6 separate experiments) were then interpreted from a NaNO2 standard curve (1 AM–1 M). Due to the islet-related variations, NO level here was expressed as a ratio of the value recorded in basal experiments (100%), i.e. in the absence of KOS or STZ in presence of Krebs medium. Insulin assay Insulin secretion was also expressed as a ratio of the value recorded in basal experiments (100%: no added KOS or STZ and presence of Krebs). The insulin content was assayed by RIA methods [20]. The medium taken from wells was kept at 20 jC before being tested. Statistical analysis Results are presented as mean F S.E. Statistical analysis of differences between mean values is carried out with student’s t-test. Differences are considered as statistically significant at P < 0.05 or P < 0.01.
Results and discussion The current study showed that, among the three components, KOS-A had dose-related effects on STZ-induced NO level of islets. Under 1.5 mM, KOS-A could positively down-regulate STZ-induced NO formation, but did not affect normal NO release of islets without STZ-treatment. At 15 mM, KOSA enhanced NO level for islets both with and without STZ treatment. Insulin secretion increased corresponding to the decrease of STZ-induced NO level. The normal insulin secretion of islets in absence of STZ was not affected by KOS-A at concentrations below 1.5 mM, but decreased at 15 mM KOS-A with the increase of NO level. Structure determination of KOS-A showed that it is a kind of tetrasaccharide with the terminal reductive residue of a-D-mannose and molecular weight of 666 Da. Concentration-dependence of NO formation, insulin release with STZ on isolated islets STZ is a selective toxin to pancreatic h-cells associated with generation of NO free radicals. NO permeates cell membrane, contains an unpaired electron, and readily oxidizes ferrous atom into ferrite, or forms secondary radicals with superoxide radical [21]. Too much NO in cells inactivates iron-containing enzymes and the decomposition products of secondary radical induce peroxide of lipids, oxidize
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Fig. 1. The effects of STZ on isolated islets NO formation and insulin secretion. Islets were incubated for 2 hrs at 37 jC under 95% air/5% CO2 with or without STZ (0.5 – 5 mM) and then medium nitrite content and insulin level was measured as described in Materials and Methods.
methionine and SH residues in proteins, deplete antioxidants and cause DNA damage [22]. Because the activity of anti-oxidative enzymes is lower in islet cells than other tissues [23], islets are very vulnerable to free radical attacks. The pathogenesis of diabetes is intimately related to free radicals, especially NO attacks [24]. The effect of STZ concentration on isolated islets, mainly as NO formation and insulin release, is shown in Fig. 1. Incubation of isolated islets with STZ (0.5–5 mM) induced a positive concentrationdependent accumulation of nitrite in medium that significantly exceeded that of control (the same medium without STZ), and an inverse dose-response of insulin release in parallel. This means islets function, expressed as insulin secretion is tightly correlated with NO level in medium. For the potential damages of free radical and the dose-dependent NO formation of STZ on islets, the STZ-treated islets could be used as the model for the study. There were two sources of NO in the STZ-treated islet model, which were NO release from islets themselves and decomposition from STZ. In order to determine the reason for dramatic NO increase in the assay system, NO level in STZ solution both with islets and not were assayed during the 2 hrs incubation at 5 mM STZ and 1 mM STZ. As shown in Table 1, from the variation of NO level in the absence and presence of islets, we could see the significant NO increment of STZ-treated islets was almost completely caused by STZ decomposition, but not from islets release.
Table 1 NO formation of STZ solution in the absence or presence of islets NO level without islets (AM) NO level with islets (AM) NO from islets (AM)
No STZ
1.0 mM STZ
5.0 mM STZ
0 31.82 F 0.95 31.82
73.75 F 17.41 108.48 F 4.91 34.73
378.47 F 15.26 411.05 F 7.56 32.58
STZ solution (100 Al) was incubated in 95% CO2/5% O2 incubator at 37 jC for 2 hrs in the absence or presence of islets (about 30islets/well). Results are means F S.E. for 3 experiments.
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Table 2 Effects of different KOS fractions on STZ-induced NO formation
KOS-A KOS-B KOS-C
1.0 mg/ml KOS + 5 mM STZ
0.1 mg/ml KOS + 5 mM STZ
Control no KOS + 5 mM STZ
8.05 F 0.62** 10.86 F 0.25 11.77 F 0.43
8.65 F 0.56** 11.07 F 0.32 11.81 F 0.49
10.81 F 0.32
NO content is expressed as the times of basal NO value (NO release during 2 hrs incubation in Krebs medium). Results are means F S.E. for 6 independent experiments. Statistics: ** P < 0.01 for difference from control (student’s t-test).
Effect of KOS fractions on STZ-induced NO formation The three fractions, KOS-A, B and C, were respectively dissolved in Krebs medium for two final concentrations of 1.0 mg/ml and 0.1 mg/ml. Freshly isolated islets was first cultured in 96 well microplate for 24 hrs in RPMI-1640 medium for fully adherence to the wall. At the end of incubation, islets were exposed to Krebs medium for 2 hrs, and NO level in this medium was taken as the basal value for following experiments. After washed with Krebs twice, each well was added medium mixed with different KOS fractions and 5 mM STZ, then further incubated for 2 hrs. Control is medium with 5 mM STZ and no KOS. When the incubation ended, medium was collected for NO test. Results are shown in Table 2. In this table, the component KOS-A has a significant effect in reducing NO level, while KOS-B and C does not. Compared with control, the NO value is reduced by 20% and 25% at 0.1 and 1.0 mg/ml of KOS-A, correspondingly. This indicates that KOS-A is positive in attenuating NO level in STZ-treated islets, but KOS-B and C hardly change NO level. Dose-response of KOS on NO release and insulin secretion In order to further understand the relationship of KOS-A concentrations on islets function, KOS-A ranged from 1.5 10 4 mM to 15 mM were used in dose-response experiments with the same method illustrated above. The results are shown in Fig. 2, and each sample is mean value of 9 independent
Fig. 2. Dose-response of KOS-A on NO formation under 5 mM STZ-treatment. Control is islets incubated in Krebs with 5 mM STZ and no KOS-A. Statistics: * P < 0.05 and ** P < 0.01 for difference from control of 9 independent experiments.
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experiments. Interestingly, from the experiments, it could be seen that at concentrations less than 1.5 mM, KOS-A down-regulated NO formation, NO level was decreased with the increasing of KOS-A dosages. While as KOS-A was increased to 15 mM, NO level became higher than control instead of decrease. These data indicates a threshold for the OS in attenuating NO formation in isolated islets. At high concentration up to 15 mM, KOS-A functions as NO formation elicitor, this may suggest a kind of cytotoxicity. The same dose-response assay of KOS-B and C showed that they had no effect on islets function (Data not shown). Endogenous NO is synthesized from L-arginine and it facilitates vasorelaxation and synaptic transmission in normal physical conditions [21]. In order to find whether KOS-A affects normal physical NO release in islets, further experiments were carried out on islets without STZ treatment, and the result is shown in Fig. 3. Compared with control, where no KOS-A in medium, no difference of NO formation was observed at KOS-A concentrations ranging from 1.5 10 4 mM to 1.5 mM. Yet at 15 mM, a dramatic increase of NO release, more than 3 times of control could be seen. In comparison with Fig. 2, these data demonstrate that in concentration range where STZ-induced NO level is attenuated, islets normal NO release is not affected by KOS-A. But at 15 mM, a significant radical formation is induced by KOS-A on both islets with STZ-treatment or not. These results imply that, at low concentrations, KOS-A can scavenge environmental free radical, but doesn’t alter islets normal NO release pathway. While at high concentration, the OS influences islets NO pathway, and makes NO release increase. Insulin secretion in above experiments with or without STZ treatment is shown in Table 3. For STZtreated islets, incubation with KOS-A ameliorated insulin secretion at concentrations ranged from 1.5 10 4 mM to 1.5 mM. While at 15 mM, adding KOS-A made insulin release decrease about 21% by comparison of control. For islets without STZ treatment, there was no difference of insulin secretion at KOS-A concentrations less than 1.5 mM. But at 15 mM, insulin secretion was decreased by 15% compared with control. These data confirm above deduction of KOS-A effects on NO release. In general, elevated environmental NO level causes insulin secretion reduction (as shown in Fig. 1), and
Fig. 3. The effects of KOS-A on NO release of non STZ-treated islets. Control is islets cultured at the same Krebs buffer with no KOS-A added. ** P < 0.01 for difference from control.
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Table 3 Effects of KOS-A on insulin secretion in pancreatic islets with and without STZ-treatment KOS-A (mM)
1.5 10
4
1.5 10
3
1.5 10
2
1.5 10
1
1.5
15
Control #
STZ-treated 0.47 F 0.09 0.48 F 0.06 0.52 F 0.10 0.64 F 0.06* 0.86 F 0.10* 0.35 F 0.02* 0.44 F 0.03 non STZ treated 1.02 F 0.10 0.96 F 0.06 0.99 F 0.04 0.97 F 0.09 1.01 F 0.07 0.84 F 0.03* 0.99 F 0.05 Islets were cultured in the presence or absence of STZ (5 mM) for 2 hrs. Insulin secretion is presented as the ratio of basal insulin value (2 hrs insulin secretion in Krebs medium). Data are means F S.E. for 7 to 9 separate experiments. # Control for STZ-treated islets is Krebs medium with 5 mM STZ without KOS-A, and for non STZ-treated islets is Krebs medium without KOS-A. * P < 0.05 versus control islets.
this is the direct-consequence of NO damage on islet function, maybe related with destruction of specific enzymes, proteins or DNA. At concentrations less than 1.5 mM, KOS-A protects islets from damage of STZ by attenuating NO level in medium, and insulin secretion increases with the dosage of KOS-A, correspondingly. At 15 mM, insulin secretion decreases for both islets with and without STZ because of the dramatic NO increase in this circumstance. The strict correlativity of insulin secretion to NO level demonstrates that islet insulin secretion variation is resulted from NO level changing, and the OS does not dramatically influence insulin secretion pathway in isolated islets within the operating range. The chemical structure of KOS-A is analyzed by Frontier Transformed Infrared Remoter (FT-IR), Element Analysis, Mass Spectrum (MS), Nuclear Magnetic Resonance (NMR) and High Pressure Liquid Chromatography (HPLC). Primary result shows it is a tetrascaaharide composed of h-D-glucose and h-D-mannose by h-glycoside bond, with reductive terminal of a-D-mannose. Its molecular weight (Mw) is 666 Da. Other study has shown Konjac prevented the senescence of cranial nerve cells and cardiac muscle cells in mice [15,16], and inhibited tumor genesis and metastasis [17]. It is well known that cell senescence and tumor genesis are intimately related with free radical damages. Then does it mean there is something in Konjac with radical scavenging function? The result in this work provides some hints. Because the pathogenesis of T2DM has been proved related with free radicals damages on islets [25–27], according to the study, it provides the possibility that the clinical hypoglycemic function of Konjac [13,14] is related with free radical attenuation mechanism. KOS, mainly referred to KOS-A, scavenges environmental nitric oxide level in certain dosage range, thus lowers the risks of radical attack on islets. Attenuated free radicals environment helps islets recover normal insulin secretion and keep plasma glucose balance, consequently. For all these results, a special phenomenon should not be ignored, that is at high concentration of 15 mM, KOS-A alters NO pathway and increases free radical release. Related work for the further study of this mechanism is being undertaken in our lab.
Conclusions All above research of Amorphophallus Konjac oligosaccharides on NO release and insulin secretion demonstrates that KOS-A is a potential free radical scavenger for isolated islets. It attenuates environmental NO level, but does not affect islets normal NO release and insulin secretion pathway at low
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concentrations. The tetrasaccharide KOS-A with its regulating effects on STZ-induced NO indicates the potential usage of the OS in diabetes or antioxidant drug exploration. References [1] Oblerley LW. Free radicals and diabetes. Free Radical Biological Medicine 1988;5:113 – 24. [2] Abdella N, Al Awadi F, Salman A, Armstrong D. Thiobarbituric acid test as a measure of lipid peroxidation in Arab patients with NIDDM. Diabetes Research 1990;15:173 – 7. [3] Strain JJ. Disturbances of micronutrient and antioxidant status in diabetes. Proceeding of Nutrition Society 1991; 50:591 – 604. [4] Jain SK, Levine SN, Duett J, Hollier B. Reduced vitamin E and increased lipofuscin products in erythrocytes of diabetic rats. Diabetes 1991;40:1241 – 4. [5] Nourooz-Zadeh J, Tajaddini-sarmadi J, McCarthy S, Betteridge DJ, Wolff SP. Evaluated levels of authentic plasma hydroperoxides in NIDDM. Diabetes 1995;44:1054 – 8. [6] Galvan AQ, Muscelli E, Catalano C, Natali A, Sanna G, Masoni A, Bernardini B, Barsacchi R, Ferrannini E. Insulin decreases circulating vitamin E levels in humans. Metabolism 1996;45:998 – 1003. [7] Paolisso G, Giugliano D. Oxidative stress and insulin action: Is there a relationship? Diabetologia 1996;39:357 – 63. [8] Santini SA, Marra G, Giardina B, Cotroneo P, Mordente A, Martorana GE, Manto A, Ghirlanda G. Defective plasma antioxidant defenses and enhanced susceptibility to lipid peroxidation in uncomplicated IDDM. Diabetes 1997; 46:1853 – 8. [9] Thomas G, Ramwell PW. Streptozotocin: a nitric oxide-carrying molecule and its effects on vasodiation. European Journal of Pharmaceutical 1989;261:279 – 80. [10] Shafrir E. Animal models of non-insulin-dependent diabetes. Diabetes Metabolism Review 1992;8:179 – 208. [11] Kim SY, Kang SK, Lee DG, Park YG, Lee YC, Chung JC, Kim CH. Effect of Jindangwan on streptozotocin-induced diabetes. Life Sciences 2000;67:1251 – 63. [12] Ai ZM, Xu L. Research progress of chemical components of Traditional Chinese Drugs. Chinese Traditional and Herbal Drugs 1999;30(6):468 – 70. [13] Wang XG, Zhao J, Du QP. Effects of compound Konjac granules on plasma glucose level of type 2 diabetes subjects. Journal of Jining Medical College (Chinese) 1995;18(3):29 – 30. [14] Mao CP, Gu ZG. Hypoglycemic effect of puncturevine caltrap (Amorphophallus Konjac). The Chinese Pharmaceutical Association 1999;16(6):14 – 5. [15] Peng SS, Zhang MY, Zhang YZ. Effects of Amorphophallus Konjac on the senescence of cranial nerve cells, liver cell and cardiac muscle cells in rats. Acta Nutrimenta Sinica 1994;16(3):280 – 4. [16] Gu YD, Shi JX, Hu ZY. Anti-senescence effect of Amorphophallus Konjac polysaccharide. Chinese Traditional and Herbal Drugs 1999;2(2):127 – 8. [17] Luo DY, Li YQ. Inhibition effects of Konjac on genesis of MNNG-induced lung tumor in mice. Chinese Journal of Oncology 1992;14(1):48 – 50. [18] Ramanadham S, Bohrer A, Mueller M, Jett P, Gross R, Turk J. Mass spectrometric identification and quantitation of Arachidonate-containing phospholipids in pancreatic islets: prominence of plasmenylethanolamine molecular species. Biochemistry 1993;32(1):5339 – 51. [19] Ridnour LA, Sim JE, Hayward MA, Wink DA, Martin SM, Buettner GR, Spitz DR. A spectrophotometric method for the direct detection and quantitation of nitric oxide, nitrite and nitrate in cell culture media. Analytical Biochemistry 2000;281(2):223 – 9. [20] Starr JI, Horwitz DL, Rubenstein AH, Mako ME. Insulin, Proinsulin and C-peptide. In: Jaffe BM, Behrman HR, editors. Methods of Hormone Radioimmunoassay. London: Academic Press Inc.; 1978. p. 613 – 33. [21] Moncada S, Palmer R, Higgs E. Nitric oxide: physiology, pathophysiology and pharmacology. Pharmaceutical Review 1991;43(2):109 – 42. [22] Beckman JS, Chen J, Ischiropoulos H, Grow JP. Oxidative chemistry of peroxinitrite. Methods in Enzymology 1994;233:229 – 40. [23] Gandy SE, Galbrath RA, Crouch RK, Buse MG, Galbraich GM. Superoxide dismutase in human islets of Langerhans. The New England Journal of Medicine 1981;304:1547 – 53.
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[24] Corbett JA, Kwon G, McDaniel ML. Role of nitric oxide in h-cell destruction. Diabetes 1995;44:39 – 46. [25] Opara EC, Abdel-Rahman E, Soliman S, Kamel WA, Souba S, Lowe JE, Abdel-Aleem S. Depletion of total antioxidant capacity in type 2 diabetes. Metabolism 1999;48(11):1414 – 7. [26] Rehman A, Nourooz-Zadeh J, Moller W, Tritschler H, Halliwell B. Increased oxidative damage to all DNA bases in patients with type 2 diabetes mellitus. FEBS letters 1999;448:120 – 2. [27] Zykova S, Jenssen T, Berdal M, Olsen R, Myklebust R, Seljelid R. Altered cytokine and nitric oxide secretion in vitro by macrophages from diabetic type 2-like db/db mice. Diabetes 2000;49:1451 – 8.
Effect of Amorphophallus Konjac oligosaccharides on STZ-induced diabetes model of isolated islets Xiu-Ju Lu *, Xiu-Min Chen, De-Xian Fu, Wei Cong, Fan Ouyang National Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, P.O. Box 353, Beijing 100080, PR China Received 25 January 2002; accepted 27 August 2002
Abstract Three oligosaccharide fractions from the root of Amorphophallus Konjac, which was reported with hypoglycemic effects on diabetes subjects, were isolated and studied using the STZ-treated diabetes model. Among them, one fraction named as KOS-A, was found with nitric oxide (NO) free radical regulation effect, while the other two were not. At concentrations less than 1.5 mM, KOS-A positively decreased STZ-induced NO level of islets, but normal NO release for non-STZ-treated islets was not affected within the range. At 15 mM, KOS-A played a contrary role and increased NO level for islets both with and without STZ-treatment. Islets insulin secretion changed corresponding to NO level in the assay. Increased insulin secretion appeared parallel to the decrease of NO, and normal insulin release was not affected by KOS-A less than 1.5 mM. Structure determination of KOS-A shows that it is a tetrasaccharide with Mw of 666 Da and reductive end of a-D-mannose. These results indicate that low dosage of KOS-A, with its function on attenuating STZ-induced NO level, doesn’t alter normal NO and insulin secretion pathways of isolated islets. The NO attenuation function of KOS-A on the diabetes model is mainly resulted from environmental free radical scavenging by the oligosaccharide. Present results also imply the mechanism of clinical Amorphophallus Konjac hypoglycemic function maybe related with free radical attenuation and lower risks of islets damage from NO radical. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Diabetes model; Streptozotocin; Konjac oligosaccharide; Nitric oxide
* Corresponding author. Tel.: +86-10-62574303; fax: +86-10-62561822. E-mail address: [email protected] (X.-J. Lu). 0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 2 3 0 3 - 2
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X.-J. Lu et al. / Life Sciences 72 (2002) 711–719
Introduction Numerous studies have shown that while different indices of free-radical damage increase, there is a decrease in the concentration of various individual antioxidant substances, indicating the presence of oxidative stress in diabetes [1–8]. Streptozotocin (STZ) caused a selective destruction of islet h cells associated with generation of free radicals [9], which has been used in inducing diabetes in experimental model [10]. Acute exposure of isolated islets to STZ in vitro results in a series of damages in h-cell metabolism and function, and STZ-treated islets was also used as a diabetes model for new drug discovery [11]. Amorphophallus Konjac has long been used in Traditional Chinese Drugs (TCD) as an immunoregulating and health-care food. Konjac Oligosaccharide (KOS) is the basic components of Amorphophallus Konjac composed of h-D-glucose and h-D-mannose with the ratio of 1:1.5 or 1:1.6 [12]. Recent studies demonstrate that Konjac functions in reactive oxygen species (ROS) related diseases, such as lowering fast plasma glucose in subjects with type 2 diabetes mellitus (T2DM) [13,14], preventing the senescence of cranial nerve cells and cardiac muscle cells in mice [15,16], and inhibiting tumor genesis and metastasis [17]. However, there is still no report of KOS effects on islets, though it maybe related with detail mechanisms of Konjac on diabetes. Based on above information, we evaluated the function of KOS on a diabetes model of isolated islets to find details of the OS on pathogenesis of diabetes. In order to exclude the effects of different fractions of KOS on the model, crude KOS was first separated and purified by a mixing column chromatography. Different components were collected when eluted at ethanol concentrations from 5% to 20%. Here the STZ treated islets were used as the model imitating islet h-cell damages in vitro. Nitric oxide (NO) radical and insulin secretion were assayed for evaluation KOS effects on islets. The results show that one component of KOS consisted of tetrasaccharide could regulate NO level on STZ-treated islets in a dose-dependent manner.
Materials and Methods Materials Radioimmunoassay (RIA) kits for insulin test were from Chinese Institute of Atomic Energy (Beijing, China). All other chemicals and biochemicals were of analytical grade and purchased from Sigma Chem. Co. (St. Louis, MO) or Life Technologies Inc. (Beijing, China). Crude KOS was kindly provided by Zhaotong Pharmaceutical Co. (Yunnan, China). Pancreatic islets were isolated from 3 weeks old mice of either sex by collagenase digestion and density gradient centrifugation [18]. STZ was dissolved as 0.1 M stock in citrate buffer (10 mM, pH4.5) immediately before use, and sterilized by 0.22 Am microfiltration. Then the stock was diluted in Krebs medium (10 mM Hepes, pH7.4, 115 mM NaCl, 24 mM NaHCO3, 5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 11.1 mM D-glucose, 0.1% Bovine serum albumin) to final concentrations of 0.5–5 mM. Separation and purification of Konjac oligosaccharides KOS crude solution in deionized (D.I.) water was purified through a mixing column filled with activated charcoal and diatomite with the ratio of 1:1. Eluted parts with step-wise ethanol solutions were
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collected after being first washed with D.I. water to get rid of salt and monosaccharide. Here three fractions corresponding to ethanol concentration elutes of 5%, 10% and 20% were used in following experiments, termed KOS-A, KOS-B and KOS-C, respectively. Nitric oxide release Nitrite (NO2 ), which is a stable oxidized end product of NO, was measured by a colorimetric assay based on the Griess reaction [19]. Briefly, 100 Al culture supernatants were incubated for 10 min at room temperature with 100 Al Griess reagent (0.5% sulfanilamide, 0.05% naphthylethylenediamine dihydrochloride, and 2.5% H3PO4). The absorbance at 540 nm was determined by a spectrophotometer. Nitrite concentrations (from 6 separate experiments) were then interpreted from a NaNO2 standard curve (1 AM–1 M). Due to the islet-related variations, NO level here was expressed as a ratio of the value recorded in basal experiments (100%), i.e. in the absence of KOS or STZ in presence of Krebs medium. Insulin assay Insulin secretion was also expressed as a ratio of the value recorded in basal experiments (100%: no added KOS or STZ and presence of Krebs). The insulin content was assayed by RIA methods [20]. The medium taken from wells was kept at 20 jC before being tested. Statistical analysis Results are presented as mean F S.E. Statistical analysis of differences between mean values is carried out with student’s t-test. Differences are considered as statistically significant at P < 0.05 or P < 0.01.
Results and discussion The current study showed that, among the three components, KOS-A had dose-related effects on STZ-induced NO level of islets. Under 1.5 mM, KOS-A could positively down-regulate STZ-induced NO formation, but did not affect normal NO release of islets without STZ-treatment. At 15 mM, KOSA enhanced NO level for islets both with and without STZ treatment. Insulin secretion increased corresponding to the decrease of STZ-induced NO level. The normal insulin secretion of islets in absence of STZ was not affected by KOS-A at concentrations below 1.5 mM, but decreased at 15 mM KOS-A with the increase of NO level. Structure determination of KOS-A showed that it is a kind of tetrasaccharide with the terminal reductive residue of a-D-mannose and molecular weight of 666 Da. Concentration-dependence of NO formation, insulin release with STZ on isolated islets STZ is a selective toxin to pancreatic h-cells associated with generation of NO free radicals. NO permeates cell membrane, contains an unpaired electron, and readily oxidizes ferrous atom into ferrite, or forms secondary radicals with superoxide radical [21]. Too much NO in cells inactivates iron-containing enzymes and the decomposition products of secondary radical induce peroxide of lipids, oxidize
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Fig. 1. The effects of STZ on isolated islets NO formation and insulin secretion. Islets were incubated for 2 hrs at 37 jC under 95% air/5% CO2 with or without STZ (0.5 – 5 mM) and then medium nitrite content and insulin level was measured as described in Materials and Methods.
methionine and SH residues in proteins, deplete antioxidants and cause DNA damage [22]. Because the activity of anti-oxidative enzymes is lower in islet cells than other tissues [23], islets are very vulnerable to free radical attacks. The pathogenesis of diabetes is intimately related to free radicals, especially NO attacks [24]. The effect of STZ concentration on isolated islets, mainly as NO formation and insulin release, is shown in Fig. 1. Incubation of isolated islets with STZ (0.5–5 mM) induced a positive concentrationdependent accumulation of nitrite in medium that significantly exceeded that of control (the same medium without STZ), and an inverse dose-response of insulin release in parallel. This means islets function, expressed as insulin secretion is tightly correlated with NO level in medium. For the potential damages of free radical and the dose-dependent NO formation of STZ on islets, the STZ-treated islets could be used as the model for the study. There were two sources of NO in the STZ-treated islet model, which were NO release from islets themselves and decomposition from STZ. In order to determine the reason for dramatic NO increase in the assay system, NO level in STZ solution both with islets and not were assayed during the 2 hrs incubation at 5 mM STZ and 1 mM STZ. As shown in Table 1, from the variation of NO level in the absence and presence of islets, we could see the significant NO increment of STZ-treated islets was almost completely caused by STZ decomposition, but not from islets release.
Table 1 NO formation of STZ solution in the absence or presence of islets NO level without islets (AM) NO level with islets (AM) NO from islets (AM)
No STZ
1.0 mM STZ
5.0 mM STZ
0 31.82 F 0.95 31.82
73.75 F 17.41 108.48 F 4.91 34.73
378.47 F 15.26 411.05 F 7.56 32.58
STZ solution (100 Al) was incubated in 95% CO2/5% O2 incubator at 37 jC for 2 hrs in the absence or presence of islets (about 30islets/well). Results are means F S.E. for 3 experiments.
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Table 2 Effects of different KOS fractions on STZ-induced NO formation
KOS-A KOS-B KOS-C
1.0 mg/ml KOS + 5 mM STZ
0.1 mg/ml KOS + 5 mM STZ
Control no KOS + 5 mM STZ
8.05 F 0.62** 10.86 F 0.25 11.77 F 0.43
8.65 F 0.56** 11.07 F 0.32 11.81 F 0.49
10.81 F 0.32
NO content is expressed as the times of basal NO value (NO release during 2 hrs incubation in Krebs medium). Results are means F S.E. for 6 independent experiments. Statistics: ** P < 0.01 for difference from control (student’s t-test).
Effect of KOS fractions on STZ-induced NO formation The three fractions, KOS-A, B and C, were respectively dissolved in Krebs medium for two final concentrations of 1.0 mg/ml and 0.1 mg/ml. Freshly isolated islets was first cultured in 96 well microplate for 24 hrs in RPMI-1640 medium for fully adherence to the wall. At the end of incubation, islets were exposed to Krebs medium for 2 hrs, and NO level in this medium was taken as the basal value for following experiments. After washed with Krebs twice, each well was added medium mixed with different KOS fractions and 5 mM STZ, then further incubated for 2 hrs. Control is medium with 5 mM STZ and no KOS. When the incubation ended, medium was collected for NO test. Results are shown in Table 2. In this table, the component KOS-A has a significant effect in reducing NO level, while KOS-B and C does not. Compared with control, the NO value is reduced by 20% and 25% at 0.1 and 1.0 mg/ml of KOS-A, correspondingly. This indicates that KOS-A is positive in attenuating NO level in STZ-treated islets, but KOS-B and C hardly change NO level. Dose-response of KOS on NO release and insulin secretion In order to further understand the relationship of KOS-A concentrations on islets function, KOS-A ranged from 1.5 10 4 mM to 15 mM were used in dose-response experiments with the same method illustrated above. The results are shown in Fig. 2, and each sample is mean value of 9 independent
Fig. 2. Dose-response of KOS-A on NO formation under 5 mM STZ-treatment. Control is islets incubated in Krebs with 5 mM STZ and no KOS-A. Statistics: * P < 0.05 and ** P < 0.01 for difference from control of 9 independent experiments.
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experiments. Interestingly, from the experiments, it could be seen that at concentrations less than 1.5 mM, KOS-A down-regulated NO formation, NO level was decreased with the increasing of KOS-A dosages. While as KOS-A was increased to 15 mM, NO level became higher than control instead of decrease. These data indicates a threshold for the OS in attenuating NO formation in isolated islets. At high concentration up to 15 mM, KOS-A functions as NO formation elicitor, this may suggest a kind of cytotoxicity. The same dose-response assay of KOS-B and C showed that they had no effect on islets function (Data not shown). Endogenous NO is synthesized from L-arginine and it facilitates vasorelaxation and synaptic transmission in normal physical conditions [21]. In order to find whether KOS-A affects normal physical NO release in islets, further experiments were carried out on islets without STZ treatment, and the result is shown in Fig. 3. Compared with control, where no KOS-A in medium, no difference of NO formation was observed at KOS-A concentrations ranging from 1.5 10 4 mM to 1.5 mM. Yet at 15 mM, a dramatic increase of NO release, more than 3 times of control could be seen. In comparison with Fig. 2, these data demonstrate that in concentration range where STZ-induced NO level is attenuated, islets normal NO release is not affected by KOS-A. But at 15 mM, a significant radical formation is induced by KOS-A on both islets with STZ-treatment or not. These results imply that, at low concentrations, KOS-A can scavenge environmental free radical, but doesn’t alter islets normal NO release pathway. While at high concentration, the OS influences islets NO pathway, and makes NO release increase. Insulin secretion in above experiments with or without STZ treatment is shown in Table 3. For STZtreated islets, incubation with KOS-A ameliorated insulin secretion at concentrations ranged from 1.5 10 4 mM to 1.5 mM. While at 15 mM, adding KOS-A made insulin release decrease about 21% by comparison of control. For islets without STZ treatment, there was no difference of insulin secretion at KOS-A concentrations less than 1.5 mM. But at 15 mM, insulin secretion was decreased by 15% compared with control. These data confirm above deduction of KOS-A effects on NO release. In general, elevated environmental NO level causes insulin secretion reduction (as shown in Fig. 1), and
Fig. 3. The effects of KOS-A on NO release of non STZ-treated islets. Control is islets cultured at the same Krebs buffer with no KOS-A added. ** P < 0.01 for difference from control.
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Table 3 Effects of KOS-A on insulin secretion in pancreatic islets with and without STZ-treatment KOS-A (mM)
1.5 10
4
1.5 10
3
1.5 10
2
1.5 10
1
1.5
15
Control #
STZ-treated 0.47 F 0.09 0.48 F 0.06 0.52 F 0.10 0.64 F 0.06* 0.86 F 0.10* 0.35 F 0.02* 0.44 F 0.03 non STZ treated 1.02 F 0.10 0.96 F 0.06 0.99 F 0.04 0.97 F 0.09 1.01 F 0.07 0.84 F 0.03* 0.99 F 0.05 Islets were cultured in the presence or absence of STZ (5 mM) for 2 hrs. Insulin secretion is presented as the ratio of basal insulin value (2 hrs insulin secretion in Krebs medium). Data are means F S.E. for 7 to 9 separate experiments. # Control for STZ-treated islets is Krebs medium with 5 mM STZ without KOS-A, and for non STZ-treated islets is Krebs medium without KOS-A. * P < 0.05 versus control islets.
this is the direct-consequence of NO damage on islet function, maybe related with destruction of specific enzymes, proteins or DNA. At concentrations less than 1.5 mM, KOS-A protects islets from damage of STZ by attenuating NO level in medium, and insulin secretion increases with the dosage of KOS-A, correspondingly. At 15 mM, insulin secretion decreases for both islets with and without STZ because of the dramatic NO increase in this circumstance. The strict correlativity of insulin secretion to NO level demonstrates that islet insulin secretion variation is resulted from NO level changing, and the OS does not dramatically influence insulin secretion pathway in isolated islets within the operating range. The chemical structure of KOS-A is analyzed by Frontier Transformed Infrared Remoter (FT-IR), Element Analysis, Mass Spectrum (MS), Nuclear Magnetic Resonance (NMR) and High Pressure Liquid Chromatography (HPLC). Primary result shows it is a tetrascaaharide composed of h-D-glucose and h-D-mannose by h-glycoside bond, with reductive terminal of a-D-mannose. Its molecular weight (Mw) is 666 Da. Other study has shown Konjac prevented the senescence of cranial nerve cells and cardiac muscle cells in mice [15,16], and inhibited tumor genesis and metastasis [17]. It is well known that cell senescence and tumor genesis are intimately related with free radical damages. Then does it mean there is something in Konjac with radical scavenging function? The result in this work provides some hints. Because the pathogenesis of T2DM has been proved related with free radicals damages on islets [25–27], according to the study, it provides the possibility that the clinical hypoglycemic function of Konjac [13,14] is related with free radical attenuation mechanism. KOS, mainly referred to KOS-A, scavenges environmental nitric oxide level in certain dosage range, thus lowers the risks of radical attack on islets. Attenuated free radicals environment helps islets recover normal insulin secretion and keep plasma glucose balance, consequently. For all these results, a special phenomenon should not be ignored, that is at high concentration of 15 mM, KOS-A alters NO pathway and increases free radical release. Related work for the further study of this mechanism is being undertaken in our lab.
Conclusions All above research of Amorphophallus Konjac oligosaccharides on NO release and insulin secretion demonstrates that KOS-A is a potential free radical scavenger for isolated islets. It attenuates environmental NO level, but does not affect islets normal NO release and insulin secretion pathway at low
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