The general significance of insulin induced ulcers remains not determined. We focused on hyperinsulinemia, deliberate injection of excessive insulin, and possibility that an anti-ulcer therapy with anti-ulcer peptide may be more successful counteracting therapy.

Insulin induced ulcers were long ago described, and related to gastric acid hypersecretion (1, 2). They were relatively little further investigated, and particularly not further explored in the broader context of hyperinsulinemia. Namely, accidental or deliberate injection of excessive insulin may derange insulin signaling and devastate many other organs and tissues producing various disturbances that may be concomitantly induced in addition to insulin-stomach ulcers. Thereby, the therapeutic possibilities rising from these disturbances associations still lack a comprehensive therapeutic demonstration (i.e., relevance of anti-ulcer peptide therapy for other organs and tissues).

Thus, to provide some novel intriguing insights, we focused on insulin induced gastric ulcers (1, 2) and then on other disturbances that may be concomitantly induced by insulin application (3-5). Hypoglycemic seizures induced by insulin injection in rats may closely represent seizures as a common and serious complication of hypoglycemia (4). Also, despite strong clinical evidence, we still lack a comprehensive explanation for the association between diabetes and vascular calcification (5). More recently, it was demonstrated that high insulin accelerated calcium deposition by human vascular smooth muscle cells (6).

We used a consistently higher insulin regimen to avoid known problems in patients with insulin application (considerable variations from patient to patient and from time to time in a given patient) and experimental inconsistency (for instance, different doses were used to induce stomach ulcer (1, 2) or hypoglycemic seizures (4)). This may invariably link insulin action with gastric ulcer, seizures, severely damaged neurons in cerebral cortex and hippocampus, hepatomegaly, fatty liver, breakdown of liver glycogen with profound hypoglycemia 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, no side effects in clinical trials (12, 13) stable in human gastric juice (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 than recombinant 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 by stable gastric pentadecapeptide BPC 157 (21).


MATERIALS AND METHODS
Animals

Male 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 by observers unaware of the given treatment.

Drugs

Medication, without carrier or peptidase inhibitor, includes pentadecapeptide BPC 157 (a partial sequence of human gastric juice 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 described before (7, 8) and insulin (Humulin R, Novo Nordisk).

Insulin (250 IU/kg) was given intraperitoneally. As an antidote after insulin, we applied the stable gastric pentadecapeptide 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 insulin over-dose application were further assessed.

Biochemical assay

To determine the serum values of glucose (mmol/L), aspartate transaminase (AST), alanine transaminase (ALT), amylase (IU/L) and total bilirubin (µmol/L) blood samples were centrifuged for 15 min at 3000 rpm, immediately after death. All tests were measured on an Olympus AU2700 analyzer with original test reagents (Olympus Diagnostica, Lismeehan, Ireland).

1. Gastric lesions. Injury severity was assessed immediately after sacrifice. The sum of the longest lesion diameters were assessed as described earlier and gastric tissue was processed for routine microscopy analysis as described before (7, 8).

2. Liver assessment. Animals were weighed before insulin protocol initiation and before sacrifice. The liver was removed and weighed. The absolute and relative liver weight was assessed. Part of the liver tissue was fresh frozen, immediately cut and stained with Sudan, in order to evaluate fatty change/lipid accumulation. The number of lipid-containing hepatocytes per high power visual field (HPF) was counted on three HPF and expressed as an average value per one HPF. Another part of the liver was placed in 10% neutral buffered formalin for 24 h and embedded in paraffin. Hematoxylin-eosin (HE) stained sections were analyzed on three HPF. Number of nuclei in each HPF was counted, and final values expressed as an average per HPF. In each of the three HPF diameters of six nuclei and area of six hepatocytes were measured using ISSA program (Vamstec, Zagreb, Croatia), and presented as average values. The number of binucleated cells was also counted. We used PAS stain to analyze glycogen content in hepatocytes using semiquantitative scoring system as follows: score 0-0% of cells with PAS positive content; score 1 up to 20% of hepatocytes with PAS positive content, score 2- between 20 and 50% of PAS positive hepatocytes, and score 3- more than 50% of PAS positive hepatocytes. For calcium deposits staining von Kossa method was applied and following scoring system: score 0- absence of positivity; score 1- dot-like positivity; score 2- granular staining; score 3- linear positivity.

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

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

5. Brain assessment. The brain was fixed in 10% formalin during two days and cut in coronal sections. Brain slabs were dehydrated in graded ethanol series and embedded in paraffin. Paraffin blocks were cut into 5 µm thin slices, deparaffinated in xylene, rehydrated in graded ethanol series and stained with haematoxylin and eosin. Distribution of brain lesions (dark neurons percentage) was evaluated.

Statistical analysis

Quantified data was performed by analysis of variance (ANOVA). Post hoc comparisons were appraised using the conservative Bonferroni/Dunn test. Data are presented as mean±standard deviation (SD). Non parametric statistic analysis was performed for categorical data using Kruskal-Wallis and post hoc Mann-Whitney U test. Values are expressed as min/med/max. Difference between proportions was evaluated by Fischer exact probability test. Test results with P<0.05 were considered statistically significant.


RESULTS
Generally, fatal chain induced by an application of insulin over-dose included gastric ulcer (Fig. 1, Table 1), seizures (Fig. 2, Table 1), 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 of liver glycogen with profound hypoglycemia (Fig. 6, Table 1), along with calcium deposition (Fig. 7, Table 1) unless BPC 157 was applied (Fig. 1, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Table 1).

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

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

Interestingly, pentadecapeptide BPC 157, as an antiulcer peptide, may besides stomach ulcer consistently counteract all insulin disturbances and fatal outcome.

Specifically, the threatening course of insulin-over dose had no an immediate presentation. However, all rats that received saline after insulin application were eventually presented with severe disturbances. Hypoglycemic seizures eventually leading to death, appeared already since 90 minutes (i.e., neck extension, vocalizations, tonic extension of the tail, digging or running limb movements with prolonged helpless inter-period when motionless animals lie down) and those animals that were convulsing and that survived till the 180 minutes after over-dose application were further assessed (Table 1). Severe damage of neurons appeared in the hippocampus and the cerebral cortex. Damaged, angulated, darkly stained neurons were present in the cerebral cortex and hippocampus (Fig. 3, Fig. 4) while Purkinje cells, 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 completely absent (Fig. 6, Table 1) and in the stomach multiple hemorrhagic lesions that were evenly distributed between the two sides of the stomach (Fig. 1, Table 1). Histologically, they appeared as mucosal defects ranging from one half of mucosal thickness to full-blown ulcers. The defect bed was debris-covered, partially with erythrocytes and surrounded by an edematous lamina propria with polymorphonuclear infiltration.

Fig. 3. Microscopic presentation of normal rat hippocampus (A) severe hippocampal hypoglycaemic injuries (arrow) in insulin treated rats (B) and mild hippocampal hypoglycaemic injuries in BPC 157 treated rats (C) (HE, objective x4).

Fig. 4. Microscopic presentation of normal rat cortex (A) severe cortical hypoglycaemic injuries in insulin treated rats (B) and mild hypoglycaemic cortical injuries in BPC 157 treated rats (C) (HE,objective x40).

In contrast, BPC 157 rats showed no fatal outcome within investigated experimental period, they were mostly without hypoglycemic seizures with apparently higher blood glucose levels (glycogen was still present in hepatocytes), less liver pathology (i.e., normal liver weight, less fatty liver), decreased ALT, AST and amylase serum values,, markedly less damaged neurons in brain and they only occasionally showed small gastric lesions (Fig. 1, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Table 1).

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 calcium deposit (healthy rats (A)); calcium deposits present in the blood vessel walls, hepatocytes sourrounding blood vessels and sometimes even in parenchyma of the liver, mainly as linear and only occasionally as granular accumulation (control insulin rats (B)); dot-like calcium presentation and in some samples granular positivity 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 in hepatocytes (BPC 157 rats (C)).

Interestingly, increased serum amylase levels and calcium deposits 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 other hand, more important seems to be calcium presentation in liver since markedly counteracted in BPC 157 rats (Fig. 7, Table 1). In insulin rats calcium deposits were present in the blood vessel walls, hepatocytes sourrounding blood vessels and sometimes even in parenchyma of the liver in control animals, mainly as linear and only occasionally as granular accumulation (Fig. 7, Table 1). BPC 157 rats exhibited dot-like calcium presentation and in some samples granular positivity was present in the blood vessels’ walls (Fig. 7, Table 1). Interestingly, no calcium deposit could be noticed in the brain.

Table 1. Gastric ulcer, seizures, damaged neurons in cerebral cortex and hippocampus, hepatomegaly, fatty liver, increased liver enzymes and amylase serum values, breakdown of liver glycogen with profound hypoglycemia, calcium deposition induced in insulin rats. Medication (BPC 157, or saline intraperitoneally or intragastrically. *P0.05, vs. control, at least.

DISCUSSION
We demonstrated that insulin gastric ulcer deserves to be evaluated along with various insulin disturbances. Rather, insulin gastric ulcer has a general significance and seizures, severely damaged neurons in cerebral cortex and hippocampus, hepatomegaly, fatty liver, increased AST, ALT and amylase serum values, breakdown of liver glycogen with profound hypoglycemia, along with calcium deposition may be the fatal chain induced by insulin application. Thereby, at the 180 minutes after over-dose application, all these disturbances together may comprehensively demonstrate endogenous coping breakdown. Moreover, seizures appeared already since 90 minutes (that correlated with supposed peak of insulin activity). Then, we could suggest development of definitive failure of endogenous controlling system with hyperinsulinemia involving decrease of endogenous insulin and likely also an (in) activity of glucagon and then epinephrine (which have to be secreted when blood glucose levels fall below the normal range)to prolong hypoglycemia).

Thus, when given to insulin-rats, BPC 157 would be confronted with the all processes simultaneously occurring that eventually lead to stomach ulcer (1, 2), hypoglycemia and all mentioned disturbances and death in insulin over-dose-rats (3-5). However, we shown that pentadecapeptide BPC 157, as an antiulcer peptide (7-11), may besides stomach ulcer consistently counteract all insulin disturbances and fatal outcome. This may also indicate that these disturbances are also interconnected throughout BPC 157 background. Moreover, considering the used insulin (250 IU/kg i.p.) /BPC 157 (10 µg, 10 ng/kg i.p. or i.g.) ratio (7, 8), it may be reasonably to assume that these therapy effects may indicate a likely role of BPC 157 in insulin controlling and influence on one or more causative process(es).

Unfortunately, the specific target(s) for these BPC 157 effects remained outside of the present investigation, but we should assume that in few hours period such a high insulin over-dose may be able also to reflect the disturbances that would otherwise require much longer period. Thus, the targets of BPC 157 counteraction may be at least partly approached within the frame of obtained insulin-damages. For instance, the most likely possibility may be the liver presenting its important role in glucose homeostasis, impaired peripheral glucose control (22) indicated by the minute blood glucose levels and absence of liver glycogen in insulin-rats, unless BPC 157 was applied. Hepatomegaly, fatty liver and pronounced elevation of liver enzymes (AST and ALT) (as in type 1 diabetic patients with poor metabolic control and treated with high daily doses of insulin (23, 24) or acute liver steatosis after insulin over-dose (25)) and obtained counteraction by BPC 157 may be also perceived in this context. Also, calcium deposition in the liver vessels appeared in these conditions. The demonstration that insulin given in such a high dose may induce prominent calcification in liver blood vessels in few hours period that was markedly attenuated by BPC 157 administration may be interesting presenting vascular calcification as a common finding in patients with long-lasting diabetes and hyperinsulinemia/vascular calcification relation shown in vitro (5). Like hepatic steatosis (26) calcium deposition is also suggestive for insulin resistance (5) (supportingly, at the cellular level, excessive circulating insulin appears to be a contributor to insulin resistance via down-regulation of insulin receptors and insulin itself can lead to insulin resistance; every time a cell is exposed to insulin, the production of GLUT4 (type four glucose receptors) on the cell’s membrane is decreased (27) and finally, fewer glucose receptors (28)). Both insulin pathways have to be inhibited for the calcification (interestingly, blocking both phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinases (MAPKs) pathways attenuated the inhibitory effect of insulin) (5).

Interestingly, since the increased amylase serum values that were attenuated were the only finding, less likely may be another option such an impaired pancreatic involvement because, during hypoglycemia, both insulin and glucagon release are under KATP channel control as well as under control of hypothalamus-brain stem 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, tonic extension of the tail, digging or running limb movements) always did occur presenting an absolute incidence and consistent latency to onset along with severely damaged neurons in cerebral cortex and hippocampus unless BPC 157 administration was given. A possible BPC 157 target for the less occurrence of these seizures (or even absence) may be the acute brain refueling from the peripheral source since without appropriate peripheral control the activity in previously hypoglycemia-silenced brain regions such as cortex, hippocampus, and amygdale may be recovered (4). Because the endogenous mechanisms involved in seizure control in substantia nigra have failed, the ensuing seizures rapidly evolve to bilaterally clonic and tonic-clonic and, eventually, to death (4). Finally, BPC 157 may also avoid the particular stomach vulnerability to hypoglycemic conditions that was long ago recognized (29).

Thus, we could argue that BPC 157 as antiulcer peptide (also effective against stomach ulcer in alloxan-animals (21) shown to maintain ATP content in stomach mucosa (30) may accordingly counteract also impaired peripheral glucose control providing at least some metabolic control, i.e., glycogen still preserved in the liver, normal liver weight, less fatty liver, less calcium deposition and no seizures despite excessive insulin consumption. Presenting that these may be end result of the complex but inevitable chain of events (i.e., arterial calcification), it seems that BPC 157 application may provide a positive shift (i.e., avoiding inhibition of either one or both insulin pathways) and an insight for novel therapeutic possibilities in insulin-disorders. Previously, BPC 157 was shown to particularly protect endothelium (9), angiogenesis (16) and modulate NO-system function (10, 31) (i.e., L-NAME-induced hypertension accompanied by an increase in insulin resistance in rats (32) was prevented and reversed by BPC 157 application (10, 31)). In addition, in view to the pathways that maintain proper glucose homeostasis and atherosclerosis, BPC 157 stimulating both expression of egr-1 and its repressor NAB2 may be an important controlling regulator (18) presenting that NAB2 has been shown to be induced by the same stimuli as egr-1, thus regulating egr-1 activity in a negative feedback loop (33).

What’s more, the premise that when given peripherally, BPC 157 may have a particular beneficial effect on CNS (i.e., markedly less damaged neurons in most severely injuried areas) is in accord with: its neuroprotective properties (34, 35), consistent antagonization of different central disturbances (36-39), brain 5-HT synthesis and antagonization of serotonin-syndrome in rats based on region-specific influence on the brain given peripherally either 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 precise alpha-[14C]methyl-L-tryptophan (alpha-MTrp) autoradiographic method) (36, 40). Presenting the suggested significance of substantia nigra for controlling insulin seizures, it may be important that BPC 157/substantia nigra relation may be particularly substantiated: the increased 5-HT synthesis in substantia nigra was the most prominent one, counteracted 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) parkinsonian syndrome as well as the lethal outcome due to particular substantia nigra damage, maintained dopamine system function (BPC 157 may counteract akinesia, catalepsy induced by neuroleptics or reserpine 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 the substantia 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 beneficial effects at the general level may be obscured by an inability to more or less specifically identify the responsible target, we should emphasize a particular advantage. Whatever the mechanism of the maintenance of the glucose level, survival of these animals, and markedly less expressed disturbances, we suggest that a summation of the BPC 157-therapy and success against threatening insulin-course can be unmistakably attributed directly to this peptide itself (7, 8). Importantly, in all BPC 157 studies (7, 8), peptide was always given alone and carrier was not used. This may allow undisputed combining of the different BPC 157 effects, and substation of the mechanisms that were suggested for the obtained BPC 157 beneficial effect (7, 8). These may be additionally supported by the same beneficial effects obtained when this peptide was given intraperitoneally as well as intragastrically (7, 8). In contrast, with regular or occasional use of carriers, other peptides which use different carriers and peptide+carrier(s)-complexes bear considerable methodological/activity dilemmas (for review see (7, 8)) and thereby, less certain effects.

In conclusion, these findings demonstrate that when one application of very high dose of insulin may induce together stomach ulcer, seizures, severely damaged neurons in cerebral cortex and hippocampus, hepatomegaly, fatty liver, breakdown of liver glycogen with profound hypoglycemia, along with calcium deposition, and finally fatal outcome in rats, all damages were markedly attenuated when BPC 157 was applied. The same effectiveness when given intraperitoneally or intragastrically (i.e., stable in human gastric juice (14)) may be suggestive that BPC 157 may have also an incretin role in controlling insulin effects (7, 8, 11). Intriguingly, further studies may show whether standard anti-ulcer agents that prevent insulin-stomach ulcer (1, 2) may also have a broader beneficial effect on other disturbances presented in insulin-rats. Previously, BPC 157 was shown to counteract toxic effects of other insulin preparations (43). Also, additional studies may show whether standard peptides implicated in ulcer in diabetic rats may also have a broader beneficial effect on insulin-ulcer and other disturbances presented in insulin-rats (44-47).

Conflict of interests: None declared.


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