Ehud Ziv

Intestinal absorption of peptides through the enterocytes.

Transport of intact peptides and proteins from the intestinal lumen into the blood is a unique phenomenon, which differs from the regular process of food digestion and absorption. Intestinal absorption of minute amounts of proteins is, however, being considered as a normal physiological process. It is thus important to define and understand the routes for protein transfer from the intestinal lumen to the blood and the mechanisms by which the macromolecules overcome the sieving barrier of the intestinal wall. The study on insulin has demonstrated that, upon proper introduction into the intestinal lumen, insulin is absorbed by the epithelial cells and transferred to the circulation. The peptides absorbed and transferred to the blood retained their biological activity and induced significant lowering of blood glucose levels. The efficiency of the absorption does not differ among the ileum, duodenum, and colon. Morphological examination demonstrated no alteration of the structural integrity of the epithelia, the enterocytes stay intact with well‐developed microvilli, and the cells remain joined by tightly closed junctions. Application of immunocytochemistry on thin tissue sections revealed insulin antigenic sites at different locations depending on the time point. Insulin detected in the lumen of the intestinal tract is absorbed through the endosomal compartment of the epithelial cells rather than passing between cells. Internalization occurs through invaginations of the luminal plasma membrane and vesicular structures of the endosomal compartment. In 5–10 minutes, insulin is transferred to the basolateral membrane and released into the interstitial space to reach the circulation. Definition of the transcytotic pathway will contribute to a better understanding of drug delivery for potential therapeutic applications. Microsc. Res. Tech. 49:346–352, 2000.

Nutritionally induced insulin resistance and receptor defect leading to beta-cell failure in animal models

ABSTRACT: Animals with genetically or nutritionally induced insulin resistance and Type 2 diabetes comprise two groups: those with resilient β‐cells, e.g., ob/ob mice or fa/fa rats, capable of longstanding compensatory insulin hypersecretion and those with labile β‐cells in which the secretion pressure leads to β‐cell degranulation and apoptosis, e.g., db/db mice and Psammomys gerbils (sand rats). Psammomys features low insulin receptor density; on a relatively high energy diet it becomes hyperinsulinemic and hyperglycemic. In hyperinsulinemic clamp the hepatic glucose production is only partially suppressed by insulin, even in the normoglycemic state. The capacity of insulin to activate muscle and liver receptor tyrosine kinase is nearly abolished. GLUT4 content and mRNA are markedly reduced. Hyperinsulinemia was also demonstrated to inhibit insulin signaling and glucose transport in several other species. Among the factors affecting the insulin signaling pathway, phosphorylation of serine/threonine appears to be the prominent cause of receptor malfunction as inferred from the finding of overexpression of PKCɛ isoforms in the muscle and liver of Psammomys. The insulin resistance syndrome progressing in animals with labile β‐cells to overt diabetes and β‐cell failure is a “thrifty gene” characteristic. This is probably also true for human populations emerging from food scarcity into nutritional affluence, inappropriate for their metabolic capacity. Thus, the nutritionally induced hyperinsulinemia, associated with PKCɛ activation may be looked upon from the molecular point of view as “PKCɛ overexpression syndrome.”

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.

The efficiency of sand rat metabolism is responsible for development of obesity and diabetes.

Two separate lines--diabetic and partially diabetes-resistant--have been isolated from the sand rat (Psammomys obesus), each with different growth characteristics in response to diets of varying digestible caloric densities (high energy, HE, 2.93 kcal/g, or low energy, LE, 2.38 kcal/g). Over a two week period all animals consumed similar quantities (c. 125 g) irrespective of the diet consumed. Weight gains were as follows: diabetic line on HE diet - 59.7 g, on LE - 46.2 g; non-diabetic animals from the diabetes-resistant line on HE - 44 g. Only animals from the diabetic line, fed the HE diet, developed hyperinsulinemia, obesity and diabetes. The energy cost of weight gain for the diabetic line fed either HE or LE diets was 6.0 - 6.3 kcal/g whereas for the diabetes-resistant line on the HE diet, the cost of growth was 50% higher at 9.3 kcal/g. These differences could be due either to alterations in the content of tissue laid down or to differences in energy expenditure. It has already been established that diet-induced obesity and diabetes develop in the diabetic line with features typical of insulin resistance in the metabolism of the pancreas, liver and peripheral tissues. Some of the animals of the diabetes-resistant line may also develop diabetes over a long time period and go through a phase of transient hyperinsulinemia-normoglycemia. This may represent an intermediate stage in the development of the diabetic syndrome and serve as a model of type 2 diabetes in man.

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.

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.

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.