Hanoch Bar-On

Biochemical and morpho-cytochemical evidence for the intestinal absorption of insulin in control and diabetic rats. Comparison between the effectiveness of duodenal and colon mucosa

SummaryA combined biochemical and morpho-cytochemical investigation was carried out in order to assess insulin absorption by the duodenal and colon epithelium. Insulin was introduced in the lumen of the rat duodenum or colon in combination with sodium cholate and aprotinin. Blood analysis made at several time points has demonstrated a rapid increase in circulating levels of insulin followed by significant and consistent decreases in blood glucose. This indicates that biologically active insulin is absorbed by the intestinal mucosa and transferred to the circulation. Because of the initial high blood glucose levels, the lowering of the glycaemic values was more significant in diabetic animals. Also, levels of circulating insulin remained higher for longer time when the administration was performed in the colon. The integrity of the intestinal wall after insulin administration, evaluated morphologically, was retained. Application of protein A-gold immunocytochemistry has established the pathway for insulin absorption. In both duodenal and colon epithelial cells the labelling for insulin was detected in the endosomal compartment, in the Golgi apparatus and in association with the baso-lateral plasma membrane interdigitations. Some labelling was also present in the interstitial space and in capillary endothelial plasmalemmal vesicles. Insulin introduced in the lumen of the rat duodenum and colon appears thus to be rapidly internalized by the epithelial cells and transferred through a transcytotic pathway to the interstitial space from which it reaches the blood circulation. This exogenous insulin then induces significant decreases in plasma glucose levels which lasts for several hours. The results obtained support the possibility for the clinical development of an oral preparation of insulin.

The absorption of insulin from various regions of the rat intestine

Abstract The absorption of intact, biologically active insulin from the ileum, or the ascending colon was measured by the resulting changes in blood glucose concentration. One hour after injection of the ascending colon with a 1 ml volume containing 12 u insulin and 2 mg DOC the blood glucose level was reduced to 50% of the initial value, i.e. 31±2.0 mg%. When insulin was injected directly into the lumen of the ileum, the addition of 3 mg soybean trypsin inhibitor boosted the insulin effect. Direct injection of the ileum with 12 u insulin and 3 mg soybean trypsin inhibitor resulted in a significant drop in blood glucose: 69±5.0 and 85±8.1% of the initial concentration, following 1 and 2 hours, respectively. In the presence of soybean trypsin inhibitor, it was found that the endogenous bile salts in the ileum aid in the absorption of biologically active insulin.

Insulin resistance in the NIDDM model Psammomys obesus in the normoglycaemic, normoinsulinaemic state

Summary The desert gerbil Psammomys obesus (“sand rat”), a model of nutritionally induced insulin resistance and non-insulin-dependent diabetes mellitus, was treated after weaning with exogenous insulin implants in the normoglycaemic, normoinsulinaemic state. Albino rats matched for weight and age served as high energy diet adjusted reference animals. Insulin administration, elevating the serum insulin to 6000 pmol/l resulted in only a mild reduction in blood glucose levels in Psammomys, but caused a severe, often fatal hypoglycaemia in the albino rats. The hepatic response to insulin-induced hypoglycaemia in rats involved a significant loss in glycogen and suppression of phosphoenolpyruvate carboxykinase (PEPCK) activity. In Psammomys under similar hyperinsulinaemia no appreciable changes in liver glycogen and PEPCK activity were evident, indicating that blood glucose was replenished by continuing gluconeogenesis. Euglycaemic, hyperinsulinaemic clamp caused a complete shut-down of hepatic glucose production in albino rats. However, in both diabetes-prone and diabetes-resistant Psammomys lines, mean hepatic glucose production was reduced by only 62 to 53 % respectively, despite longer lasting and higher levels of hyperinsulinaemia. These results indicate that Psammomys is characterized by muscle and liver insulin resistance prior to diet-induced hyperglycaemia and hyperinsulinaemia. This is assumed to be a species feature of Psammomys, exemplifying a metabolic adjustment to survival in conditions of food scarcity of both animal and human populations. It may reflect a propensity to insulin resistance and hyperglycaemia in population groups exposed to affluent nutrition. [Diabetologia (1996) 39: 1269–1275]

Removal defect of very-low-density lipoproteins from diabetic rats

The disappearance rate of triacyl[3H]glycerol carried on very-low-density lipoproteins (VLDL), isolated from diabetic rats and reinjected into normal recipient rats, was about twice as low as that of VLDL-triacyl[3H]glycerol from non-diabetic rats. The VLDL derived from diabetic rats was deficient in the apolipoprotein E component. These results indicate that the triacylglycerol removal defect in diabetes may be related to the quality of the protein carrier.

Chloroquine-induced interference with degradation of serum lipoproteins in rat liver, studied in vivo and in vitro☆

The effect of chloroquine, an inhibitor of certain lysosomal enzymes including cathepsin B (EC 3.4.22.1), on the degradation of serum lipoproteins in rat liver was studied in vivo and in liver homogenates. Chloroquine had no effect on the clearance from the circulation of 125I-labeled rat or human very low density lipoproteins or human low density lipoproteins. Pretreatment with chloroquine for 3 h, resulted in a 2-2.5 fold increase in 125i-labeled very low density lipoprotein recovered in the liver 45 min after injection of the homologous and heterologous lipoproteins. This effect was evident on both the 125I-labeled protein and 125I-labeled lipid moiety. 30 min after the injection of [3H]-cholesterol linoleate-labeled very low density lipoproteins, 70% of the injected label was recovered in the liver, both in control and chloroquine-treated rats. Since the perl and 20% in the experimental group, it was concluded that chloroquine interferes with the hydrolysis of [3H]cholesterol linoleate. Following injection of 125I-labeled human low density lipoproteins only 4% of the injected lipoprotein was recovered in the liver of control rats and not more than 10% after chloroquine treatment, when about 50% had been cleared from the circulation. Hence, while very low density lipoprotein protein and cholesterol ester are catabolized in the liver, the catabolism of low density lipoproteins occurs mainly in extra-hepatic tissues. Using post-nuclear liver suprnatant, optimal degradation of various serum lipoproteins was found at pH 4.4, and chloroquine inhibited their degradation. Degradation of very low density and low density lipoproteins was completely inhibited at 0.05 M chloroquine, while less pronounced inhibition was seen with high density lipoproteins, apolipoproteins and apolipoprotein AI. These results indicate that liver acid hydrolases in vivo participate in the degradation of serum lipoproteins. Cathepsin B is apparently responsible for the degradation of aplipoprotein B, while other cathepsins might also be active in the degradation of this and the other apolipoproteins.

Treatment of diabetes with vanadium salts: general overview and amelioration of nutritionally induced diabetes in the Psammomys obesus gerbil

Numerous investigations have demonstrated the beneficial effect of vanadium salts on diabetes in streptozotocin (STZ)‐diabetic rats, in rodents with genetically determined diabetes and in human subjects. The amelioration of diabetes included the abolition of hyperglycemia, preservation of insulin secretion, reduction in hepatic glucose production, enhanced glycolysis and lipogenesis and improved muscle glucose uptake through GLUT4 elevation and translocation. The molecular basis of vanadium salt action is not yet fully elucidated. Although evidence has been provided that the insulin receptor is activated, the possibility exists that cytosolic non‐receptor tyrosine kinase, direct phosphorylation of IRS‐1 and activation of PI3‐K, leading to GLUT4 translocation, are involved. The raised phosphorylation of proteins in the insulin signaling pathway appears to be related to the inhibition of protein tyrosine phosphatase (PTPase) activity by vanadium salts.

Morpho-cytochemical and biochemical evidence for insulin absorption by the rat ileal epithelium

SummaryIn order to investigate the mechanism through which insulin is absorbed by the intestinal epithelium and transferred to the circulation where it exercises its biological activity of lowering blood glucose levels, a combined biochemical morpho-cytochemical study was undertaken on rat ileal tissue, in vivo. Insulin was introduced into the lumen of the ileum in combination with sodium cholate and aprotinin and allowed to be absorbed for various periods of time. Analysis of blood samples from the inferior vena cava, at different time points has demonstrated an increase in plasma insulin followed by a decrease in blood glucose levels. The ileal tissues were studied at different time points after the introduction of the insulin, by applying the protein A-gold immunocytochemical technique. Insulin antigenic sites were detected with high resolution, at various levels of the enterocytes but were absent from goblet cells. At 2 to 5 min, the labelling was mainly associated with the microvilli and endocytotic vesicles in the apical portion of the epithelial cells. Some gold particles were in contact with the lateral membranes. At 10 min, the labelling was found at the level of the trans-side of the Golgi apparatus and mainly along the baso-lateral membranes of the epithelial cells. Labelling was also detected in the interstitial space. The control experiments have demonstrated the specificity of the labelling and confirmed the nature of the insulin molecules detected. Furthermore, the morphological study has confirmed that exposure of the tissue to the insulin-cholate-aprotinin solution does not affect the integrity of the epithelium while promoting insulin absorption. Thus, insulin introduced in the lumen of the rat ileum in conjunction with sodium cholate and aprotinin, appears to be rapidly absorbed by the epithelial cells and transferred to the circulation through a transcytotic pathway.

BILE SALTS PROMOTE THE ABSORPTION OF INSULIN FROM THE RAT COLON

Abstract The absorption of insulin mixed with sodium deoxycholate (DOC) or sodium cholate from the rectal mucosa of diabetic and non-diabetic rats was measured by the effect on blood glucose levels. Blood sugar was lowere by 50% one hour after administration of 12 u soluble insulin mixed with 1–10 mg/ml DOC, or 2–20 mg/ml sodium cholate. There was a linear correlation between the reduction in blood glucose and the amount of insulin administered (1–64 units) when mixed with 5 mg/ml DOC. Radioimmuno-assay of plasma insulin showed an increase from 16.2 μu/ml to 3335 muuu/ml after rectal administration of 12 u soluble insulin. We conclude that insulin when mixed with bile salts can be absorbed by the intestine to reach the circulation in a biologically active form.

Origin and pattern of glucocorticoid-induced hyperlipidemia in rats. Dose-dependent bimodal changes in serum lipids and lipoproteins in relation to hepatic lipogenesis and tissue lipoprotein lipase activity.

Rats maintained for five days on a low dose of triamcinolone (0.5 mg/kg) showed a 2-fold increase in serum triacylglycerol concentration, paralleled by a rise in all very low density lipoprotein (VLDL) components but no significant change in serum cholesterol or high density lipoproteins (HDL). In contrast, a high dose of triamcinolone (12.5 mg/kg) produced a fall in triacylglycerol and VLDL to the range of control levels coincident with doubling in serum cholesterol and HDL. The rise in VLDL was attributed in a large part to enhanced hepatic fatty acid synthesis as evident from the marked rises in activity of rate-limiting enzymes of lipogenesis and in 3H incorporation into liver and serum fatty acids from in vivo administered 3H2O. The induction of fatty acid synthesis was linked to pronounced hyperinsulinemia, elicited by the triamcinolone treatment, to which the liver remained selectively responsive, contrary to the general insulin antagonism in peripheral tissues. Triamcinolone treatment also resulted in small rises in serum glucagon but these changes did not appear to be of importance for the observed bimodal serum lipoprotein perturbations. Dexamethasone, prednisolone and cortisol, administered in doses equipotent to 0.5 mg/kg triamcinolone, produced similar changes in the levels of serum triacylglycerol and insulin and activities of hepatic enzymes of lipogenesis.

Irreversibility of Nutritionally Induced NIDDM in Psammomys obesus Is Related to β-Cell Apoptosis

Psammomys lapses into fully fledged diabetes when maintained on a high-energy diet. Progression to diabetes has been classified into stage A of normoglycemia and normoinsulinemia (<120 mg/ml and 100 mU/L, respectively); stage B of hyperinsulinemia (100-300 mU/L) with marked insulin resistance in the face of normoglycemia; stage C of pronounced hyperinsulinemia with hyperglycemia < or =500 mg/ml; stage D at 6-10 weeks after stage C, featuring further hyperglycemia and loss of insulin. Insulin resistance expressed in Psammomys at stages B and C was demonstrated by nonsuppression of the hepatic gluconeogenesis enzyme phosphoenolpyruvate carboxykinase by the endogenous hyperinsulinemia and by the reduced capacity of insulin to activate muscle and liver tyrosine kinase of the insulin receptor. Diabetes at stage C, but not at stage D, was fully reversed to stage A by restricting the food ration of animals by half (from 14 to 7 g/day) for 10-14 days. We examined islet beta cells of Psammomys in the four stages of progression to diabetes by staining for insulin as well as for apoptosis by the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) and visualizing the biotin-labeled cleavage sites. Psammomys in stage A had insulin-laden beta cells. In stage B, a hypertrophy and partial insulin depletion of beta cells was evident with negative TUNEL staining. In stage C, beta cells were markedly depleted of insulin, and their number within the islets decreased, but the TUNEL staining was virtually negative. In stage D, beta cells were markedly diminished within the islets, almost void of insulin, showing distinct TUNEL staining of beta cells. These results indicate that prolonged exposure of islets to in vivo hyperglycemia with beta-cell overtaxation induces nuclear disintegration with irreversible damage to the insulin-secretion apparatus. This precludes the return to normalcy by restricting the food intake of Psammomys. The appearance of cells with TUNEL-positive staining may serve as a marker of impending irreversibility of nutritionally induced diabetes.