INTRODUCTIONThe general significance of insulin induced ulcers remains

not determined. We focused on hyperinsulinemia, deliberateinjection of excessive insulin, and possibility that an anti-ulcertherapy with anti-ulcer peptide may be more successfulcounteracting therapy.

Insulin induced ulcers were long ago described, and relatedto gastric acid hypersecretion (1, 2). They were relatively littlefurther investigated, and particularly not further explored in thebroader context of hyperinsulinemia. Namely, accidental ordeliberate injection of excessive insulin may derange insulinsignaling and devastate many other organs and tissues producingvarious disturbances that may be concomitantly induced inaddition to insulin-stomach ulcers. Thereby, the therapeuticpossibilities rising from these disturbances associations still lacka comprehensive therapeutic demonstration (i.e., relevance ofanti-ulcer peptide therapy for other organs and tissues).

Thus, to provide some novel intriguing insights, we focusedon insulin induced gastric ulcers (1, 2) and then on otherdisturbances that may be concomitantly induced by insulin

application (3-5). Hypoglycemic seizures induced by insulininjection in rats may closely represent seizures as a common andserious complication of hypoglycemia (4). Also, despite strongclinical evidence, we still lack a comprehensive explanation forthe association between diabetes and vascular calcification (5).More recently, it was demonstrated that high insulin acceleratedcalcium deposition by human vascular smooth muscle cells (6).

We used a consistently higher insulin regimen to avoidknown problems in patients with insulin application(considerable variations from patient to patient and from time totime in a given patient) and experimental inconsistency (forinstance, different doses were used to induce stomach ulcer (1,2) or hypoglycemic seizures (4)). This may invariably linkinsulin action with gastric ulcer, seizures, severely damagedneurons in cerebral cortex and hippocampus, hepatomegaly,fatty liver, breakdown of liver glycogen with profoundhypoglycemia and calcification development.

We focused on a peptide therapy, using a small, orally active,anti-ulcer peptide (7-11) stable gastric pentadecapeptide BPC 157(MW 1419) with very safe profile (LD1 could be not achieved, noside effects in clinical trials (12, 13) stable in human gastric juice

JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2009, 60, Suppl 7, 107-114www.jpp.krakow.pl

S. ILIC, I. BRCIC, M. MESTER, M. FILIPOVIC, M. SEVER, R. KLICEK, I. BARISIC, B. RADIC, Z. ZORICIC, V. BILIC, L. BERKOPIC, L. BRCIC, D. KOLENC, Z. ROMIC, L. PAZANIN, S. SEIWERTH, P. SIKIRIC

OVER-DOSE INSULIN AND STABLE GASTRIC PENTADECAPEPTIDE BPC 157. ATTENUATED GASTRIC ULCERS, SEIZURES, BRAIN LESIONS,HEPATOMEGALY, FATTY LIVER, BREAKDOWN OF LIVER GLYCOGEN,

PROFOUND HYPOGLYCEMIA AND CALCIFICATION IN RATS

Department of Pharmacology and Pathology Medical Faculty University of Zagreb, Zagreb, Croatia

We focused on over-dose insulin (250 IU/kg i.p.) induced gastric ulcers and then on other disturbances that wereconcomitantly induced in rats, seizures (eventually fatal), severely damaged neurons in cerebral cortex andhippocampus, hepatomegaly, fatty liver, increased AST, ALT and amylase serum values, breakdown of liver glycogenwith profound hypoglycemia and calcification development. Calcium deposits were present in the blood vessel walls,hepatocytes sourrounding blood vessels and sometimes even in parenchyma of the liver mainly as linear and onlyoccasionally as granular accumulation. As an antidote after insulin, we applied the stable gastric pentadecapeptide BPC157 (10 µg/kg) given (i) intraperitoneally or (ii) intragastrically immediately after insulin. Controls receivedsimultaneously an equivolume of saline (5 ml/kg). Those rats that survived till the 180 minutes after over-doseapplication were further assessed. Interestingly, pentadecapeptide BPC 157, as an antiulcer peptide, may besidesstomach ulcer consistently counteract all insulin disturbances and fatal outcome. BPC 157 rats showed no fatal outcome,they were mostly without hypoglycemic seizures with apparently higher blood glucose levels (glycogen was still presentin hepatocytes), less liver pathology (i.e., normal liver weight, less fatty liver), decreased ALT, AST and amylase serumvalues, markedly less damaged neurons in brain and they only occasionally had small gastric lesions. BPC 157 ratsexhibited mostly only dot-like calcium presentation. In conclusion, the success of BPC 157 therapy may indicate a likelyrole of BPC 157 in insulin controlling and BPC 157 may influence one or more causative process(es) after excessiveinsulin application.

K e y w o r d s : stable gastric pentadecapeptide BPC 157, insulin, calcium deposits, glycogen, hepatomegaly

(14), effective in trials for inflammatory bowel disease therapy(12, 13) and wound healing (15-18) also in diabetic rats (18, 19).Importantly, pentadecapeptide BPC 157 was more active thanrecombinant human platelet-derived growth factor (PDGF-BB),stimulating both expression of early growth response 1 (egr-1)gene and its repressor nerve growth factor 1- A binding protein-2(NAB2) (18). Thereby, it may be important for cytokine induction,growth factor generation, early extracellular matrix (collagen)formation (18), but also for gluconeogenesis regulation (20).Finally, alloxan-gastric ulcers in rats and mice may be inhibited bystable gastric pentadecapeptide BPC 157 (21).

MATERIALS AND METHODS

AnimalsMale Albino Wistar (200 g) rats, were used in all of the

experiments (10 rats at least per each experimental group)approved by the Local Ethic Committee and assessed byobservers unaware of the given treatment.

DrugsMedication, without carrier or peptidase inhibitor, includes

pentadecapeptide BPC 157 (a partial sequence of human gastricjuice protein BPC, freely soluble in water at pH 7.0 and in saline;peptide with 99% (HPLC) purity (1-des-Gly peptide as impurity,manufactured by Diagen, Ljubljana, Slovenia,GEPPPGKPADDAGLV, M.W. 1419 prepared as describedbefore (7, 8) and insulin (Humulin R, Novo Nordisk).

Insulin (250 IU/kg) was given intraperitoneally. As anantidote after insulin, we applied the stable gastricpentadecapeptide BPC 157 (10 µg/kg, 10 ng/kg) given (i)intraperitoneally or (ii) intragastrically immediately after insulin.Controls received simultaneously an equivolume of saline(5 ml/kg). Those that survived till the 180 minutes after insulinover-dose application were further assessed.

Biochemical assayTo determine the serum values of glucose (mmol/L),

aspartate transaminase (AST), alanine transaminase (ALT),amylase (IU/L) and total bilirubin (µmol/L) blood samples werecentrifuged for 15 min at 3000 rpm, immediately after death. Alltests were measured on an Olympus AU2700 analyzer withoriginal test reagents (Olympus Diagnostica, Lismeehan,Ireland).

1. Gastric lesions. Injury severity was assessed immediatelyafter sacrifice. The sum of the longest lesion diameters wereassessed as described earlier and gastric tissue was processed forroutine microscopy analysis as described before (7, 8).

2. Liver assessment. Animals were weighed before insulinprotocol initiation and before sacrifice. The liver was removed andweighed. The absolute and relative liver weight was assessed. Partof the liver tissue was fresh frozen, immediately cut and stainedwith Sudan, in order to evaluate fatty change/lipid accumulation.The number of lipid-containing hepatocytes per high power visualfield (HPF) was counted on three HPF and expressed as anaverage value per one HPF. Another part of the liver was placed in10% neutral buffered formalin for 24 h and embedded in paraffin.Hematoxylin-eosin (HE) stained sections were analyzed on threeHPF. Number of nuclei in each HPF was counted, and final valuesexpressed as an average per HPF. In each of the three HPFdiameters of six nuclei and area of six hepatocytes were measuredusing ISSA program (Vamstec, Zagreb, Croatia), and presented asaverage values. The number of binucleated cells was also counted.

We used PAS stain to analyze glycogen content in hepatocytesusing semiquantitative scoring system as follows: score 0-0% ofcells with PAS positive content; score 1 up to 20% of hepatocyteswith PAS positive content, score 2- between 20 and 50% of PASpositive hepatocytes, and score 3- more than 50% of PAS positivehepatocytes. For calcium deposits staining von Kossa method wasapplied and following scoring system: score 0- absence ofpositivity; score 1- dot-like positivity; score 2- granular staining;score 3- linear positivity.

3. Pancreas assessment was as described before (11) on HEslides and using von Kossa method.

4. Behavioral assessment. Intensity of presentation ofbehavioral disturbances was assessed as incidence and latency toonset seizure-like behaviour (neck extension, vocalizations, tonicextension of the tail, digging or running limb movements) (4).

5. Brain assessment. The brain was fixed in 10% formalinduring two days and cut in coronal sections. Brain slabs weredehydrated in graded ethanol series and embedded in paraffin.Paraffin blocks were cut into 5 µm thin slices, deparaffinated inxylene, rehydrated in graded ethanol series and stained withhaematoxylin and eosin. Distribution of brain lesions (darkneurons percentage) was evaluated.

Statistical analysisQuantified data was performed by analysis of variance

(ANOVA). Post hoc comparisons were appraised using theconservative Bonferroni/Dunn test. Data are presented asmean±standard deviation (SD). Non parametric statistic analysiswas performed for categorical data using Kruskal-Wallis andpost hoc Mann-Whitney U test. Values are expressed asmin/med/max. Difference between proportions was evaluated byFischer exact probability test. Test results with P<0.05 wereconsidered statistically significant.

RESULTSGenerally, fatal chain induced by an application of insulin over-

dose included gastric ulcer (Fig. 1, Table 1), seizures (Fig. 2, Table1), severely damaged neurons in cerebral cortex (Fig. 3, Table 1)and hippocampus (Fig. 4, Table 1), hepatomegaly (Fig. 5, Table 1),fatty liver, increased liver enzymes and amylase serum values(while total bilirubin was not increased) (Table 1), breakdown ofliver glycogen with profound hypoglycemia (Fig. 6, Table 1), alongwith calcium deposition (Fig. 7, Table 1) unless BPC 157 wasapplied (Fig. 1, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Table 1).

Interestingly, pentadecapeptide BPC 157, as an antiulcerpeptide, may besides stomach ulcer consistently counteract allinsulin disturbances and fatal outcome.

Specifically, the threatening course of insulin-over dose hadno an immediate presentation. However, all rats that receivedsaline after insulin application were eventually presented withsevere disturbances. Hypoglycemic seizures eventually leadingto death, appeared already since 90 minutes (i.e., neck extension,vocalizations, tonic extension of the tail, digging or running limbmovements with prolonged helpless inter-period whenmotionless animals lie down) and those animals that wereconvulsing and that survived till the 180 minutes after over-doseapplication were further assessed (Table 1). Severe damage ofneurons appeared in the hippocampus and the cerebral cortex.Damaged, angulated, darkly stained neurons were present in thecerebral cortex and hippocampus (Fig. 3, Fig. 4) while Purkinjecells, cerebellar and brain stem nuclei were universally spared.No inflammatory reaction and interstitial edema were found.Those rats exhibited only minute blood glucose levels(glycogen, usually present in normal hepatocytes, was

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completely absent (Fig. 6, Table 1) and in the stomach multiplehemorrhagic lesions that were evenly distributed between thetwo sides of the stomach (Fig. 1, Table 1). Histologically, theyappeared as mucosal defects ranging from one half of mucosalthickness to full-blown ulcers. The defect bed was debris-covered, partially with erythrocytes and surrounded by anedematous lamina propria with polymorphonuclear infiltration.

In contrast, BPC 157 rats showed no fatal outcome withininvestigated experimental period, they were mostly withouthypoglycemic seizures with apparently higher blood glucoselevels (glycogen was still present in hepatocytes), less liverpathology (i.e., normal liver weight, less fatty liver), decreasedALT, AST and amylase serum values,, markedly less damagedneurons in brain and they only occasionally showed small gastriclesions (Fig. 1, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Table 1).

Interestingly, increased serum amylase levels and calciumdeposits were present in pancreas without noticeable pathology(Table 1). Only serum amylase levels were attenuated by the

BPC 157 administration (but not calcium deposits). On the otherhand, more important seems to be calcium presentation in liversince markedly counteracted in BPC 157 rats (Fig. 7, Table 1).In insulin rats calcium deposits were present in the blood vesselwalls, hepatocytes sourrounding blood vessels and sometimeseven in parenchyma of the liver in control animals, mainly aslinear and only occasionally as granular accumulation (Fig. 7,Table 1). BPC 157 rats exhibited dot-like calcium presentationand in some samples granular positivity was present in the bloodvessels’ walls (Fig. 7, Table 1). Interestingly, no calcium depositcould be noticed in the brain.

DISCUSSIONWe demonstrated that insulin gastric ulcer deserves to be

evaluated along with various insulin disturbances. Rather,insulin gastric ulcer has a general significance and seizures,

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Fig. 1. Characteristic stomach presentation in insulin rats.Multiple hemorrhagic lesions (control (C)), presentation close tonormal (BPC 157 (B)).

Fig. 2 Characteristic seizure-like behaviour (neck extension,vocalizations, tonic extension of the tail, digging or running limbmovements) starting 90 min after insulin over-dose application,unless BPC 157 was applied.

Fig. 3. Microscopicpresentation of normal rathippocampus (A) severehippocampal hypoglycaemicinjuries (arrow) in insulintreated rats (B) and mildhippocampal hypoglycaemicinjuries in BPC 157 treatedrats (C) (HE, objective x4).

Fig. 4. Microscopicpresentation of normal ratcortex (A) severe corticalhypoglycaemic injuries ininsulin treated rats (B) andmild hypoglycaemic corticalinjuries in BPC 157 treatedrats (C) (HE,objective x40)

severely damaged neurons in cerebral cortex and hippocampus,hepatomegaly, fatty liver, increased AST, ALT and amylaseserum values, breakdown of liver glycogen with profoundhypoglycemia, along with calcium deposition may be the fatalchain induced by insulin application. Thereby, at the 180minutes after over-dose application, all these disturbancestogether may comprehensively demonstrate endogenous copingbreakdown. Moreover, seizures appeared already since 90minutes (that correlated with supposed peak of insulin activity).Then, we could suggest development of definitive failure ofendogenous controlling system with hyperinsulinemia involvingdecrease of endogenous insulin and likely also an (in) activity ofglucagon and then epinephrine (which have to be secreted whenblood glucose levels fall below the normal range)to prolonghypoglycemia).

Thus, when given to insulin-rats, BPC 157 would beconfronted with the all processes simultaneously occurring thateventually lead to stomach ulcer (1, 2), hypoglycemia and allmentioned disturbances and death in insulin over-dose-rats (3-5). However, we shown that pentadecapeptide BPC 157, as anantiulcer peptide (7-11), may besides stomach ulcer consistentlycounteract all insulin disturbances and fatal outcome. This mayalso indicate that these disturbances are also interconnectedthroughout BPC 157 background. Moreover, considering theused insulin (250 IU/kg i.p.) /BPC 157 (10 µg, 10 ng/kg i.p. ori.g.) ratio (7, 8), it may be reasonably to assume that thesetherapy effects may indicate a likely role of BPC 157 in insulincontrolling and influence on one or more causative process(es).

Unfortunately, the specific target(s) for these BPC 157effects remained outside of the present investigation, but weshould assume that in few hours period such a high insulin over-dose may be able also to reflect the disturbances that wouldotherwise require much longer period. Thus, the targets of BPC157 counteraction may be at least partly approached within theframe of obtained insulin-damages. For instance, the mostlikely possibility may be the liver presenting its important rolein glucose homeostasis, impaired peripheral glucose control(22) indicated by the minute blood glucose levels and absenceof liver glycogen in insulin-rats, unless BPC 157 was applied.Hepatomegaly, fatty liver and pronounced elevation of liverenzymes (AST and ALT) (as in type 1 diabetic patients withpoor metabolic control and treated with high daily doses ofinsulin (23, 24) or acute liver steatosis after insulin over-dose(25)) and obtained counteraction by BPC 157 may be alsoperceived in this context. Also, calcium deposition in the livervessels appeared in these conditions. The demonstration thatinsulin given in such a high dose may induce prominentcalcification in liver blood vessels in few hours period that wasmarkedly attenuated by BPC 157 administration may beinteresting presenting vascular calcification as a common

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Fig. 5. Characteristic liver presentation in insulin rats.Hepatomegaly (control (C)), liver presentation close to normal(BPC 157 (B)).

Fig. 6. Calcium deposits, von Kossa method x20. No calciumdeposit (healthy rats (A)); calcium deposits present in the bloodvessel walls, hepatocytes sourrounding blood vessels andsometimes even in parenchyma of the liver, mainly as linear andonly occasionally as granular accumulation (control insulin rats(B)); dot-like calcium presentation and in some samples granularpositivity in the blood vessels’ walls (BPC 157 insulin rats (C)).

Fig.7. Glycogen content in hepatocytes with PAS positive content x20. PAS positive hepatocytes glycogen present in hepatocytes(normal rats (A)); breakdown of liver glycogen with profound hypoglycemia (control insulin rats (B)); glycogen was still present inhepatocytes (BPC 157 rats (C)).

finding in patients with long-lasting diabetes andhyperinsulinemia/vascular calcification relation shown in vitro(5). Like hepatic steatosis (26) calcium deposition is alsosuggestive for insulin resistance (5) (supportingly, at thecellular level, excessive circulating insulin appears to be acontributor to insulin resistance via down-regulation of insulinreceptors and insulin itself can lead to insulin resistance; everytime a cell is exposed to insulin, the production of GLUT4 (typefour glucose receptors) on the cell’s membrane is decreased(27) and finally, fewer glucose receptors (28)). Both insulinpathways have to be inhibited for the calcification(interestingly, blocking both phosphoinositide 3-kinase (PI3K)and mitogen-activated protein kinases (MAPKs) pathwaysattenuated the inhibitory effect of insulin) (5).

Interestingly, since the increased amylase serum values thatwere attenuated were the only finding, less likely may be anotheroption such an impaired pancreatic involvement because, duringhypoglycemia, both insulin and glucagon release are under KATPchannel control as well as under control of hypothalamus-brainstem hypoglycemia-induced vagal signaling (4).

Whatever may be the final impairment in insulin-rats (4),behavioral manifestations associated with hypoglycemia,seizure-like behavior (i.e., neck extension, vocalizations, tonicextension of the tail, digging or running limb movements)always did occur presenting an absolute incidence and consistentlatency to onset along with severely damaged neurons incerebral cortex and hippocampus unless BPC 157 administrationwas given. A possible BPC 157 target for the less occurrence of

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Insulin (250 IU/kg) given intraperitoneally

Medication given

intraperitoneally immediately

after insulin

Medication given

intragastrically immediately

after insulin

Parameters assessed at

180 min after insulin application

saline

BPC 157

10µg/kg

saline

BPC 157

10µg/kg

Sum of longest lesions diameters,

means±SD, mm

15±2.7 1.5±0.6* 13±4.7 0.8±0.4*

Gastric

lesions Lesions incidence, rats with

lesions/ total rats number

10/10 2/10* 10/10 3/10*

Absolute weight, means±SD, g 17.3±2.4 12.7±2.2* 18.7±1.9 11.5±2.2*

Relative weight means±SD,

percentage

8.2±1.1 5.8±1.0* 9.1±0.9 5.4±1.0*

Number of hepatocytes,

means±SD

39.8±2.5 34.16±2.1* 41.6± 2.9 33.36±3.1*

Size of cytoplasmatic area,

means±SD,µm2

257.45±23.7 218.87±18.9* 263.95±31.1 211.32±22.2*

Size of nuclei, means±SD, µm 7.315±1.1 7.47±0.9 7.221±1.0 7.47±0.8

Number of hepatocytes with

binucleated nuclei, means±SD

0.61±0.31 1.33±0.42* 0.56±0.37 1.53±0.5*

Number of lipid-containing

hepatocytes, means±SD

16±4 8±3* 17±3 8±4*

Glycogen content in hepatocytes,

Min/Med/Max, score 0-3

0/0/1 1/2/3* 0/0/1 1/2/3*

Liver

lesions

Calcium deposits staining von

Kossa method, Min/Med/Max,

score 0-3

1/2/3 0/1/2* 2/2/3 0/1/2*

ALT, means±SD, IU/L 124±17 87±16* 138±22 77±13*

AST, means±SD, IU/L 412±22 260±31* 444±19 248±19*

Amylase, means±SD,IU/L 4423±301 2731±382* 4122±351 2604±249*

Serum values

ALT, AST,

amylase,

glucose Glucose, means±SD,mmol/L 0.8±0.1 3.5±0.5* 0.6±0.1 3.8±0.7*

Latency to onset after insulin

application means±SD,min

94±13 165±11* 97±20 170±24*

Hypoglycemic

convulsions Seizures incidence, rats with

seizure-like behavior/ total rats

number

10/10 2/10* 10/10 3/10*

Percentage of darkly stained

neurons in the cerebral cortex,

means±SD

79±9 31±9* 83±10 26±12*

Brain lesions

Percentage of darkly stained

neurons in the hypocampus,

means±SD

87±13 29±12* 85±9 25±10*

Table 1. Gastric ulcer, seizures, damaged neurons in cerebral cortex and hippocampus, hepatomegaly, fatty liver, increased liverenzymes and amylase serum values, breakdown of liver glycogen with profound hypoglycemia, calcium deposition induced in insulinrats. Medication (BPC 157, or saline intraperitoneally or intragastrically. *P≤0.05, vs. control, at least.

these seizures (or even absence) may be the acute brain refuelingfrom the peripheral source since without appropriate peripheralcontrol the activity in previously hypoglycemia-silenced brainregions such as cortex, hippocampus, and amygdale may berecovered (4). Because the endogenous mechanisms involved inseizure control in substantia nigra have failed, the ensuingseizures rapidly evolve to bilaterally clonic and tonic-clonic and,eventually, to death (4). Finally, BPC 157 may also avoid theparticular stomach vulnerability to hypoglycemic conditions thatwas long ago recognized (29).

Thus, we could argue that BPC 157 as antiulcer peptide (alsoeffective against stomach ulcer in alloxan-animals (21) shown tomaintain ATP content in stomach mucosa (30) may accordinglycounteract also impaired peripheral glucose control providing atleast some metabolic control, i.e., glycogen still preserved in theliver, normal liver weight, less fatty liver, less calcium depositionand no seizures despite excessive insulin consumption.Presenting that these may be end result of the complex butinevitable chain of events (i.e., arterial calcification), it seems thatBPC 157 application may provide a positive shift (i.e., avoidinginhibition of either one or both insulin pathways) and an insightfor novel therapeutic possibilities in insulin-disorders.Previously, BPC 157 was shown to particularly protectendothelium (9), angiogenesis (16) and modulate NO-systemfunction (10, 31) (i.e., L-NAME-induced hypertensionaccompanied by an increase in insulin resistance in rats (32) wasprevented and reversed by BPC 157 application (10, 31)). Inaddition, in view to the pathways that maintain proper glucosehomeostasis and atherosclerosis, BPC 157 stimulating bothexpression of egr-1 and its repressor NAB2 may be an importantcontrolling regulator (18) presenting that NAB2 has been shownto be induced by the same stimuli as egr-1, thus regulating egr-1activity in a negative feedback loop (33).

What’s more, the premise that when given peripherally, BPC157 may have a particular beneficial effect on CNS (i.e., markedlyless damaged neurons in most severely injuried areas) is in accordwith: its neuroprotective properties (34, 35), consistentantagonization of different central disturbances (36-39), brain 5-HT synthesis and antagonization of serotonin-syndrome in ratsbased on region-specific influence on the brain given peripherallyeither acutely or chronically (i.e., dorsal thalamus, hippocampus,lateral geniculate body, hypothalamus, dorsal raphe nucleus,substantia nigra, medial anterior olfactory nucleus, lateral caudate,accumbens nucleus, superior olive)) (shown by the very precisealpha-[14C]methyl-L-tryptophan (alpha-MTrp) autoradiographicmethod) (36, 40). Presenting the suggested significance ofsubstantia nigra for controlling insulin seizures, it may beimportant that BPC 157/substantia nigra relation may beparticularly substantiated: the increased 5-HT synthesis insubstantia nigra was the most prominent one, counteracted 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) parkinsoniansyndrome as well as the lethal outcome due to particular substantianigra damage, maintained dopamine system function (BPC 157may counteract akinesia, catalepsy induced by neuroleptics orreserpine as well as amphetamine-stereotypy behavior) (37-39).Also, BPC 157 was found to have anti-convulsive properties (37,41). Also very recently, with the respect of KATP channels in thesubstantia nigra as a predisposing factor for seizure development,BPC 157 inhibits K+ conductance in WT HEK293 cells (42). Thus,BPC 157 may markedly antagonize insulin-convulsions, or at least,it may significantly postpone their appearance.

Finally, although the evidence of the BPC 157 beneficialeffects at the general level may be obscured by an inability tomore or less specifically identify the responsible target, weshould emphasize a particular advantage. Whatever themechanism of the maintenance of the glucose level, survival ofthese animals, and markedly less expressed disturbances, we

suggest that a summation of the BPC 157-therapy and successagainst threatening insulin-course can be unmistakablyattributed directly to this peptide itself (7, 8). Importantly, in allBPC 157 studies (7, 8), peptide was always given alone andcarrier was not used. This may allow undisputed combining ofthe different BPC 157 effects, and substation of the mechanismsthat were suggested for the obtained BPC 157 beneficial effect(7, 8). These may be additionally supported by the samebeneficial effects obtained when this peptide was givenintraperitoneally as well as intragastrically (7, 8). In contrast,with regular or occasional use of carriers, other peptides whichuse different carriers and peptide+carrier(s)-complexes bearconsiderable methodological/activity dilemmas (for review see(7, 8)) and thereby, less certain effects.

In conclusion, these findings demonstrate that when oneapplication of very high dose of insulin may induce togetherstomach ulcer, seizures, severely damaged neurons in cerebralcortex and hippocampus, hepatomegaly, fatty liver, breakdownof liver glycogen with profound hypoglycemia, along withcalcium deposition, and finally fatal outcome in rats, all damageswere markedly attenuated when BPC 157 was applied. The sameeffectiveness when given intraperitoneally or intragastrically(i.e., stable in human gastric juice (14)) may be suggestive thatBPC 157 may have also an incretin role in controlling insulineffects (7, 8, 11). Intriguingly, further studies may show whetherstandard anti-ulcer agents that prevent insulin-stomach ulcer (1,2) may also have a broader beneficial effect on otherdisturbances presented in insulin-rats. Previously, BPC 157 wasshown to counteract toxic effects of other insulin preparations(43). Also, additional studies may show whether standardpeptides implicated in ulcer in diabetic rats may also have abroader beneficial effect on insulin-ulcer and other disturbancespresented in insulin-rats (44-47).

Conflict of interests: None declared.

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44. Ceranowicz P, Warzecha Z, Dembinski A, et al. Treatmentwith ghrelin accelerates the healing of acetic acid-inducedgastric and duodenal ulcers in rats. J Physiol Pharmacol2009; 60: 87-98.

45. Warzecha Z, Dembinski A, Ceranowicz P, et al. Influence ofghrelin on gastric and duodenal growth and expression ofdigestive enzymes in young mature rats. J PhysiolPharmacol 2006; 57: 425-437.

46. Kwiecien S, Pawlik MW, Sliwowski Z, et al. Involvement ofsensory afferent fibers and lipid peroxidation in thepathogenesis of stress-induced gastric mucosa damage. JPhysiol Pharmacol 2007; 58(Suppl 3): 149-162.

47. Kapica M, Puzio I, Kato I, Kuwahara A, Zabielski R. Roleof feed-regulating peptides on pancreatic exocrine secretion.J Physiol Pharmacol 2008; 59(Suppl 2):145-159.R e c e i v e d : October 25, 2009A c c e p t e d : December 11, 2009Author’s address: Predrag Sikiric, MD, PhD, Professor,

Department of Pharmacology, Medical Faculty, University ofZagreb, Salata 11, POB 916, 1000 Zagreb, Croatia; Phone: 385-1-4566; Fax: 385-1-492-00-50; E-mail: sikiric@mef.hr

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