Insulin: Almost a Century of Lifesaving.

The identification and purification of insulin in 1922 changed life for individuals with type 1 diabetes mellitus (T1DM). Its discovery was, to a certain extent, serendipitous. Although medical researchers suspected that some type of hormone was responsible for carbohydrate metabolism, by the end of the 19th century they had made little progress. When World War I broke out, efforts stalled. A somewhat cantankerous group of Canadian researchers led by Frederick Grant Banting, a surgeon, are credited with insulin's discovery. Their initial research was discredited and criticized for poor technique. Regardless, they persevered, and in January 1922 they successfully treated their first patient. A mere nine months later, collaboration between the University of Toronto and Eli Lilly Company made insulin available in North America. Derived from beef and pork pancreases, the 40 unit/mL product little resembled today's more refined human insulin. While insulin is indispensable to individuals with T1DM, it is also used or being studied for several different conditions. Some researchers have dubbed Alzheimer's disease "type 3 diabetes" because of similar aberrations in the blood-brain barrier and protein deposits.

Conditioned media trans-differentiate mature fibroblasts into pancreatic beta-like cells.

AIM: The study was carried out to evaluate the role of preconditioning strategies on the trans-differentiation of mature fibroblasts (NIH3T3 cells) into insulin producing ß-cells. METHODS: The NIH3T3 cells were treated with dexamethasone (5µM) and pancreatic extract (0.05 and 0.4mg/mL) separately or in combination. The treated cells were analyzed for the morphological changes, and expression of pancreatic genes and proteins by phase contrast microscopy, RT-PCR and flow cytometry/immunocytochemistry, respectively. RESULTS: Treatment of mature fibroblasts with different combinations of dexamethasone and pancreatic extract in the form of conditioned media resulted in comparable morphological changes and expression of certain pancreatic genes and proteins; however, their expression varied with each treatment. Most prominent effect was observed in case of combined treatment which resulted in significant increase (p<0.001) in gene expression levels of insulin, MafA, and Ngn3. Variable pattern was observed in insulin, MafA, Ngn3 and Sca1 expressions at the protein level. CONCLUSION: It is concluded from this study that preconditioning of NIH3T3 cells with conditioned media containing different combinations of dexamethasone and pancreatic extract can induce trans-differentiation of these cells into pancreatic ß-like cells. The conditioned media however, need to be optimized. The study may offer the possibility of improved regeneration of mature cell type that could serve as a future therapeutic option for diabetes.

[Differentiation of human amniotic mesenchymal stem cells into insulin-secreting cells induced by regenerating pancreatic extract].

In this study, the natural biological inducer, rat regenerating pancreatic extract (RPE), was used to induce human amniotic mesenchymal stem cells (hAMSCs) into insulin-secreting cells. We excised 60% of rat pancreas in order to stimulate pancreatic regeneration. RPE was extracted and used to induce hAMSCs at a final concentration of 20 microg/mL. The experiment methods used were as follows: morphological-identification, dithizone staining, immumofluorescence analysis, reverse transcription-PCR (RT-PCR) and insulin secretion stimulated by high glucose. The results show that the cell morphology of passge3 hAMSCs changed significantly after the induction of RPE, resulting in cluster shape after induction for 15 days. Dithizone staining showed that there were scarlet cell masses in RPE-treated culture. Immumofluorescence analysis indicated that induced cells were insulin-positive expression. RT-PCR showed the positive expression of human islet-related genes Pdx1 and insulin in the induced cells. The result of insulin secretion stimulated by high glucose indicated that insulin increasingly secreted and then kept stable with prolongation of high glucose stimulation. In conclusion, hAMSCs had the potential to differentiate into insulin-secreting cells induced by RPE in vitro.

Comparison of a recombinant trypsin with the porcine pancreatic extract on sperm used for the in vitro production of bovine embryos.

The objective was to compare the effects of treating bovine semen with two trypsin products (the porcine pancreas extract and a recombinant) and a control (no trypsin) on in vitro embryo production. Our hypothesis was that the trypsin treatments would not cause any significant difference in fertilization and embryo development as compared to the control. Semen was washed through a gradient system containing a porcine-origin trypsin, a recombinant bovine-sequence trypsin, or the control (no trypsin). Oocytes (n=3036) were collected from abbatoir-derived ovaries, matured for 24h, and allocated into three groups: porcine trypsin (n=1040), recombinant trypsin (n=972), and control (n=1024). Ova were inseminated with 1 x 10(6) motile sperm/mL and cultured for 18-24h. Thereafter, presumptive zygotes were cultured for 7 days in 50 microL G1/G2 micro-droplets under mineral oil. Overall, sperm motility was lower before than after each treatment (mean of 51.4% versus 70.2%, respectively; P<0.001); however, motility was not significantly different among the three groups (porcine-origin trypsin=68.8%, recombinant trypsin=69.0%, and control=72.6%). Similarly, there was no significant difference among these groups for cleavage rates (70.1, 70.9, and 73.9%), or the number of morula/blastocyst stage embryos (53.4, 53.3, and 48.7%). In conclusion, treatment of bovine sperm with either porcine-origin trypsin or recombinant trypsin prior to insemination had no detrimental effects on in vitro embryo development.

Characterization of liposomes coated with S-layer proteins from lactobacilli.

The stability of liposomes coated with S-layer proteins from Lactobacillus brevis and Lactobacillus kefir was analyzed as a previous stage to the development of a vaccine vehicle for oral administration. The interactions of the different S-layer proteins with positively charged liposomes prepared with soybean lecithin or dipalmitoylphosphatidylcholine were studied by means of the variation of the Z potential at different protein-lipid ratios, showing that both proteins were able to attach in a greater extent to the surface of soybean lecithin liposomes. The capacity of these particles to retain carboxyfluorescein or calcein by exposure to bile salts, pancreatic extract, pH change and after a thermal shock showed that both S-layer proteins increased the stability of the liposomes in the same magnitude. The non-glycosylated protein from L. brevis protects more efficiently the liposomes at pH 7 than those from L. kefir even without treatment with glutaraldehyde.

Mechanisms for cell activation and its consequences for biorheology and microcirculation: Multi-organ failure in shock.

Activation of cells in the vascular compartment causes profound alteration of cell rheological properties with impairment of the microcirculation and initiation of inflammatory reactions. Many cardiovascular diseases have been shown to be associated with cell activation and inflammation. While this situation offers the opportunity for new interventions against the deleterious effects of cell activation, there is the need for a better understanding of the mechanisms that lead to cell activation in the first place. We review here several mechanisms for cell activation in the circulation. We show that in shock, a condition associated with severe forms of cell activation, humoral cell activation factors can be detected in plasma. Further analysis indicates that the source of these humoral activators may be due to the action of pancreatic digestive enzymes in the intestine. Ischemia may serve to open the intestinal brush border and permit entry of pancreatic enzymes into the wall of the intestine to initiate self digestion. In this process low molecular weight but potent cell activators are produced which may escape via the intestinal circulation and the lymphatics into the general circulation. Inhibition of pancreatic enzymes in the lumen of the intestine leads to complete attenuation of humoral activator production as well as many of the deleterious sequelae that accompany shock, such as inflammation and multi-organ failure. We outline a method to carry out biochemical isolation of the cell activators derived from pancreatic enzymes. This analysis shows that there are multiple species of cell activators above and beyond currently known species, many of which have molecular weights below 3000 Da. Identification of the mechanisms that lead to cell activation is an important part to understand the mechanisms that lead to alterations of rheological properties of blood cells in disease and dysfunction of the endothelium and parenchymal cells. Our current evidence suggests that pancreatic digestive enzymes and tissue enzymes may play a central role in humoral activator production.

[The effect of the preparation Wobe-Mugos E on the functional and biochemical status of the kidneys in the polyuric stage of sublimate-induced nephropathy]. / Vplyv preparatu Wobe-Mugos E na funktsional'nyi ta biokhimichnyi stan nyrok u poliurychnu stadiiu sulemovoï nefropatiï.

The protective effect of Wobe-Mugos appliance on the kidney function and biochemical state in polyuric stage of sublimate nephropathia at the moment of tubulointerstitial component formation was revealed in experiments on 40 white male rats. It appeared in the increase of hydrogenous ion excretion, titred acids, renal tissue fibrinolytic and proteolytic activity. The succinatdehydrogenase activation in renal cortex matter pointed out on the improvement of energy balance.

Pancreatic supplements reduce symptomatic response of healthy subjects to a high fat meal.

In a double-blind, crossover study, we determined whether microencapusulated pancreatic enzymes reduce postprandial symptoms experienced by healthy volunteers after ingestion of a high calorie, high fat meal. At 7 AM, 18 subjects ingested 185 g of cookies (1196 calories and 72 g of fat) with three pancrelipase capsules or a placebo. The severity of gastrointestinal symptoms and flatus passages were recorded for 15-17 hr, and end-alveolar samples were obtained hourly for 10 hr. Ingestion of pancreatic supplements was associated with a significant (P = 0.049) reduction in bloating over the entire recording period, and with significant reductions in bloating, gas, and fullness during the dinner to bedtime period. Pancreatic supplements had no significant effect on breath H2 or CH4 concentration. The finding that pancreatic supplements reduce postprandial symptoms in healthy subjects suggests that these supplements also might be beneficial in irritable bowel syndrome.

Proteinases reduce metastatic dissemination and increase survival time in C57Bl6 mice with the Lewis lung carcinoma.

The effect of combined proteolytic enzymes, administered by the rectal route, on the metastatic process and the time of survival in C57Bl6 mice with the Lewis lung carcinoma inoculated subcutaneously was investigated. In the control group, which received no enzyme treatment, 90% of animals died of the metastatic spread of cancer by day 18 after primary tumor extirpation. In Group A, which received the multi-enzyme solution from the time of primary tumor extirpation, 30% of mice died of disseminated cancer by day 25. In Group B, which was treated with the enzymes from 6 days before primary tumor extirpation, only 10% of animals showed the metastatic process by day 15. In Group C, which received the enzymes from 24 hours after intracutaneous tumor inoculation, no metastatic dissemination was discernible. In these three groups, the enzyme treatment was carried out throughout the study. None of the control animals survived for 100 days when the study was ended. The treated groups A, B and C showed survival rate 60%, 90% and 100% of animals, respectively, by 100 days.

Effects of pancreatic enzyme preparations on erythrocyte glutathione peroxidase activities and plasma selenium concentrations in cystic fibrosis.

To substitute for exocrine pancreatic insufficiency, patients with cystic fibrosis (CF) take pancreatic enzymes (PE) originating from porcine pancreas. Five different pancreatic enzyme preparations used by our patients contained 0.5-1.4 microg selenium per g tablet. In patients taking PE in doses that were gradually increased to improve fat absorption during a 48-month period, the effects of PE dose on erythrocyte selenium-dependent glutathione peroxidase (SeGSH-Px) activities and plasma selenium concentrations were studied. At baseline, erythrocyte SeGSH-Px activities were significantly lower in patients (p=.01), while plasma selenium concentrations did not differ between patients and healthy subjects. When PE dose and, consequently, selenium intake from PE was increased, erythrocyte SeGSH-Px activities (p < .001) and plasma selenium concentrations (p=.02) increased. Changes in SeGSH-Px activities during the initial 8 months correlated with those in selenium intake from PE (r=0.67, p < .001). Plasma selenium concentrations plateaued at 12 months and erythrocyte SeGSH-Px activities did so at 36 months, when patients had reached SeGSH-Px activities similar to those of healthy subjects. At 48 months, patients took an average lipase dose of 17400 U x kg(-1) x d(-1) and selenium dose from PE of 0.53 microg x kg(-1) x d(-1). We conclude that selenium content of PE preparations has a significant effect on SeGSH-Px activity in patients with CF. This form of selenium supply needs to be taken into account when selenium supplements are given to patients with CF.

Polyenzyme preparation Wobe-Mugos inhibits growth of solid tumors and development of experimental metastases in mice.

Long-term rectal administration of enzyme mixture containing papain, trypsin and chymotrypsin in the same ratio as the preparation Wobe-Mugos E (Mucos Pharma, Germany) was evaluated for their antitumor effects in C57Bl6 inbred mice inoculated with Bl6 melanoma cells. 30% of animals in the test group (3 pcs) have been cured of cancer. In the rest of animals (70%) the survival time was prolonged by 58.3% compared to the control group (from average survival time of 24 days in control group to 38 days in the test group). Based on histological and immunohistochemical evaluation a faster process of metastasizing was found in control group than in the group treated with the polyenzyme preparation. In the case of melanoma Bl6 an antimetastatic effect of the preparation was thus proved.

[An experimental model of depletion of the blood kallikrein-kinin system]. / Eksperimental'naia model' istoshcheniia kallikrein-kininovoi sistemy krovi.

A new way of kallikrein-kinin system exhaustion in experiments on animals is suggested. This approach consists in a simultaneous rise in the activity of kinin formation and destruction. It is shown that insignificant activation of the kallikrein system and kininase activity leads to activation of the kinin system and rise in active kinin levels. A 1.5-3-fold increase in the total activity of kallikrein and a simultaneous 3-6-fold increase in kininase activity cause exhaustion of the blood kallikrein-kinin system in 2 hours and makes active kinin formation impossible by the body's requirements when the synthesis of kinin system components is maintained.

In vitro stimulation of hamster pancreatic duct growth by an extract derived from the "wrapped" pancreas.

Partial obstruction of the hamster pancreas in the cellophane wrap model leads to the induction of duct and ductular proliferation followed by endocrine cell differentiation. This effect appears to be mediated by the local action of a growth factor. The purpose of the present study was to determine if cytosolic extract prepared from the wrapped pancreas had trophic activity on purified hamster pancreatic ductal epithelium in tissue culture. Cultures of purified pancreatic ducts were prepared by digestion of the hamster pancreas using a solution of collagenase type XI and chymotrypsin infused directly into the pancreatic duct. The ducts were separated and purified by a series of steel mesh filtrations. Ducts were embedded in 1.5% Seaplaque agarose and fed a liquid medium containing serum-free Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 Ham (DME/F-12), 12.5% cytosol extract+DME/F-12, or 25% cytosol extract+DME/F-12. The trophic effect of the extract on the tissue in culture was evaluated by the incorporation of tritiated thymidine ([3H]TdR) into DNA. Duct fragments cultured in medium supplemented with 12.5% cytosol showed no difference in their [3H]TdR uptake compared with control ducts (908 +/- 147 vs. 913 +/- 151 dpm/micrograms DNA). The incorporation of [3H]TdR by ducts maintained in medium supplemented with 25% cytosol extract was increased 78% over serum-free controls (1,632 +/- 386 vs. 913 +/- 147 dpm/micrograms DNA; p < 0.025). We conclude that a cytosol extract prepared from the partially obstructed cellophane-wrapped pancreas contains a factor(s) trophic for pancreatic ductal cells.

Influence of exogenous application of pancreatic extracts on endogenous pancreatic enzyme secretion.

The existence of negative feedback inhibition of human pancreatic enzyme secretion by proteases is controversially discussed. We have recently demonstrated that jejunal application of porcine pancreatic extracts, in a dose commonly used to treat digestive insufficiency, stimulated rather than inhibited, human pancreatic enzyme secretion. We have now studied the influence of duodenal application of high concentrations of either pure trypsin or porcine pancreatic extracts with trypsin-equivalent activity, on human pancreatic enzyme secretion. Twenty-three male volunteers were intubated with a gastric tube and a two-lumen jejunal tube to collect secretions separately via the first and third tubes and to perfuse either pure trypsin or porcine pancreatic extracts distal to the pylorus via the second tube. PEG-4.000 was continuously perfused via the second tube to correct for losses of volume. Volunteers received PEG alone during the first hour, phenylalanine during the second, PEG alone again during the third, and either phenylalanine together with trypsin or porcine pancreatic extracts during the fourth h. Activities of lipase, amylase, and chymotrypsin were measured in 15-min fractions. In addition, human lipase secretion was measured with an enzyme immunoassay, which does not crossreact with porcine lipase. Plasma cholecystokinin (CCK) was measured using a sensitive bioassay, which utilizes amylase release by isolated rat pancreatic acini. Perfusion of the duodenum with phenylalanine caused a statistically significant stimulation of enzyme secretion. This stimulation could be inhibited by high concentrations of pure trypsin. In contrast, application of porcine pancreatic extracts, which contained the equivalent activity of trypsin, caused further increases of lipase secretion when compared to phenylalanine alone.(ABSTRACT TRUNCATED AT 250 WORDS)

[Effect of inflammatory mediators on respiration in rat liver mitochondria]. / Vliianie mediatorov vospaleniia na dykhanie mitokhondrii pecheni krys.

Some inflammatory mediators have been studied for their influence on the energy reactions of the liver mitochondria. Mediators were injected intraperitoneally to rats 15 min before decapitation in the following doses (per 100 g of the body) weight: histamine--0.5 mg, serotonin--0.5 mg, bradykinin--0.2 mg, andekalin--0.5 units. Histamine action in the body is connected with modification of the respiratory mitochondria chain and, like the oligomycin action, is directed to attended oxidation and phosphorylation points. Serotonin increases the mitochondria sensitivity to separating agents in succinate oxidation. It is supposed that serotonin-induced inhibition of oxidation of NAD-dependent substances is connected with NADH2 dehydrogenase inhibition or transhydrogenase reaction activation. Bradykinin has activated NAD-dependent substance oxidation and increased respiratory chain sensitivity on the SoQ link to 2,4-dinitrophenol action. Andekalin exerts an analogous effect intensifying ADP-, DNP- and Ca-stimulated respiration of mitochondria during succinate oxidation. Mechanism of the inflammatory mediators influence on the energy metabolism is discussed.

Influence of treatment with pancreatic extracts on pancreatic enzyme secretion.

We have evaluated the effects of porcine pancreatic extracts on human pancreatic secretion. Ten male volunteers were intubated with a 4-lumen jejunal tube to collect gastric and duodenal secretions separately via the first and third tube, to infuse PEG 4000 distal the pylorus via the second tube and to apply porcine pancreatic extracts via the fourth tube distal the ligament of Treitz. Pancreatic extracts were given four times at 40 minute intervals; the first two as active enzymes and subsequently as heat denatured proportions. Secretin was continuously infused intravenously (0.5 E/kg bw/h) to achieve minimal pancreatic flow. Lipase, amylase, trypsin, chymotrypsin, volume, and bicarbonate were measured in duodenal contents in eight pooled 15 minute fractions. Three subjects who received HEPES-Ringer buffer instead of pancreatic enzymes served as controls. Plasma cholecystokinin (CCK) was measured using a sensitive bioassay. Both active and heat denatured pancreatic extracts caused a small but significant increase in amylase and chymotrypsin secretion. Basal plasma CCK values were 0.85 (0.05) pM. After intrajejunal instillation of either active or heat denatured pancreatic extracts plasma CCK rose to 3.25 (0.30) pM and to 3.28 (0.36) pM respectively. In a second group of five volunteers, plasma CCK concentrations were measured after a test meal. On day 1, volunteers received a liquid fat and protein rich meal and on day 2, the same test meal containing porcine pancreatic extracts. In both cases, a similar increase in plasma CCK was observed. We conclude that therapy with pancreatic extracts stimulate pancreatic enzyme secretion. This may be mediated through release of CCK.