Lipid and exocrine pancreatic ultrastructural changes due to experimental diabetes.

The aim of this study was to establish if the changes in the ultrastructure of the exocrine part of the pancreas are correlated with changes in serum glucose, cholesterol and lipoprotein fractions during the progression of diabetes in rabbits. Diabetes mellitus was induced in male New Zealand rabbits by a single injection of alloxan into the auricular vein. On the day 7th the glucose level in the whole blood was measured and this day was designated as the first day of diabetes. Rabbits were divided into 5 groups: untreated control, 21-day diabetes, 42-day diabetes, 90-day diabetes and 180-day diabetes. The cholesterol, HDL (high-density lipoprotein) and LDL (low-density lipoprotein) levels were examined in the serum. The total pancreatic lipase activity was measured spectrophotometrically in the pancreatic homogenate. Histological specimens were examined under an electron microscopy. The glucose level increased significantly in all of the alloxan exposed animals. The significant elevation of cholesterol level was observed on day 21 and 180. The HDL level was increased (P<0.05) only on the day 21st. The LDL level and the total activity of pancreatic lysosomal lipase increased significantly on day 21, 42 and 90. Further dilation of granular endoplasmic reticular ducts and decrease in the number of zymogen granules were observed amongst exocrine cells. Fragmented mitochondrial and translucent matrix were also seen. Intensification of the pancreatic fibrosis was found on day 90. Microvascular changes were reported in exocrine cells after 180 days. Their nuclei were smaller with large bulges on the nuclear membrane, and the number of heterogeneous electron granules of zymogen further declined. We concluded that the intensification of ultrastructural changes of the exocrine part of the pancreas correlated with the changes of the pancreatic lipase activity, and glucose and lipoprotein levels.     

Developmental changes in the activities of prolinase and prolidase in rat salivary glands,and the effect of thyroxine administration

Summary As the salivary glands are interesting tissues to study proliferation, we studied the activities of prolinase and prolidase using Pro-Ala and Pro-Hyp as substrates, respectively, in developing rat salivary glands between day 1 and week 10 after birth. Developmental changes of prolinase activity in the submandibular and sublingual glands were similar to those in the parotid gland, which steadily increased and reached the adult level by 20–25 days after birth. However, the changes in the activity of prolidase in the submandibular and sublingual glands were different from those in the parotid gland: the activity in the parotid gland slowly increased with maturation and reached a maximum level on day 30, but the activity in the submandibular and sublingual glands continuously increased with maturation. When thyroxine was injected every two days from day 1 to day 19, both enzyme activities were induced precociously in the parotid gland but not in the submandibular and sublingual glands. On the study of regional distribution in rat tissues, the correlation coefficient between prolinase and prolidase activities was high in the peripheral but not high in the brain regions.These results indicate that the physiological roles of prolinase and prolidase are very similar but not the same.     

Hormonal regulation of lysosomal hydrolases in the reproductive tract of the rabbit

The total protein content and the activities of lysosomal hydrolases (arylsulphatase, alkaline and acid phosphatases, beta-glucuronidase, beta-N-acetylhexosaminidase, alpha-L-fucosidase and beta-galactosidase) in the uteri of ovariectomized rabbits treated with different concentrations of progesterone, oestradiol-17 beta and a combination of progesterone and oestradiol were determined. The enzyme activities were also measured in the reproductive organs of rabbits induced to superovulate by PMSG and hCG. In superovulated and steroid-treated rabbits, the changes in lysosomal hydrolases were more obvious in the endometrium than the myometrium. Except for the myometrial alkaline phosphatase and beta-galactosidase and the endometrial alkaline phosphatase, there were no significant changes in the solubilities of hydrolases after treatment with steroids. beta-Galactosidase levels were significantly higher in the ovariectomized rabbits treated with progesterone. An antagonistic effect of oestradiol and progesterone was observed with respect to uterine weight, protein content and enzyme activities in the ovariectomized rabbits treated simultaneously with oestradiol and progesterone.     

Comparative analysis of lysosomal hydrolases and natural glycoprotein substrates in the rat major salivary glands.

1. The activities of some lysosomal hydrolases and the concentrations of their natural substrates were studied in the submandibular and sublingual glands of male and female rats using biochemical procedures. 2. In sublingual gland enzyme activities and substrate concentrations show the highest values. 3. The enzyme activities appear, in general, lower and the natural substrate concentrations higher in the females with respect to males. 4. In both glands beta-galactosidase shows the highest activity and beta-glucosidase the lowest. 5. These findings suggest that metabolic turnover of glycoproteins is slower in females than in males, probably because the oestrogens control the activity of lysosomal hydrolases.     

Concentration and distribution of insulin and insulin-like protein in the organs of normal and diabetic mice

Presence of insulin or insulin-like protein has been studied in mouse liver, kidneys, lungs, duodenum, jejunum, submandibular and parotid salivary glands, in femoral, diaphragmal and abdominal wall muscles by means of the immunofluorescent method. In order to understand the role of the extrapancreatic insulin for compensation of the insular insufficiency, corresponding organs have been examined in mice with alloxan diabetes. The immunoreactive insulin is proved to be present only in cells of the granular parts of the salivary tubules of the submandibular and striated ducts of the parotid glands. As demonstrates microfluorometry, a relative amount of insulin in the submandibular gland cells is 1.5 times and in the parotid gland cells--2 times as small as in beta-cells of the pancreatic glands. Under alloxan diabetes insulin content in the salivary gland cells decreases by 1.3-1.9 times (in the beta-cells--by 2.7 times). This may designate that the extrapancreatic insulin (or insulin-like protein) participates in compensation of hypoinsulinemia. In mice with alloxan diabetes, immunoreactivity of insulin is also revealed in hepatocytes.     

Influence of experimental diabetes on the kinetic behaviour of renal cortex hexose monophosphate dehydrogenases

1. Short term (1-2 hr) and long-term (2 days) effects of experimental alloxan induced diabetes on the kinetics of the renal hexose monophosphate shunt dehydrogenases are reported. 2. Alloxan diabetes for 2 days significantly increased kidney weight (16%) adding about 80 mg/day per g of kidney. No significant changes were found in renal growth 1-2 hr after alloxan injection. 3. Under these experimental conditions, the activities of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase significantly increased (103 and 33% respectively) at all substrate concentrations, without affecting the KmS of either enzyme. 4. There was no effect of alloxan on the activity of these enzymes at 1-2 hr. Saturation curves show that all enzymes exhibited a M-M kinetic without evidence of sigmoidicity. 5. The results suggest that increased renal hexose monophosphate dehydrogenases activities are due to increased concentrations of the rate limiting proteins. 6. The relationship between these changes and renal hypertrophy is also discussed.     

Effect of fasting on islet lysosomal enzyme activities and the in vivo insulin response to different secretagogues

The effect of a 24 hr starvation period on islet lysosomal enzyme activities and the in vivo insulin response to glucose, glibenclamide and L-isopropyl-noradrenaline (L-IPNA) was studied in mice. It was observed that fasting induced a significant decrease of islet acid amyloglucosidase activity, whereas the activities of acid phosphatase, beta-N-acetyl-glucosaminidase, and beta-glucuronidase in islet tissue were unaffected by the fasting period studied. Starvation markedly reduced the acute insulin response to a maximal dose of glucose or glibenclamide. However, the insulin response to a maximal dose of L-IPNA was of similar magnitude in both fed and fasted animals. Pretreatment of fasted mice with purified fungal acid amyloglucosidase could restore the impaired insulin response to glucose to the normal level seen in fed mice. It is suggested that islet acid amyloglucosidase activity is of importance for glucose-stimulated insulin secretion, and that reduced levels of islet amyloglucosidase may contribute to the impairment of glucose-induced insulin release seen after fasting.     

Variations of susceptibility to alloxan induced diabetes in the rabbit

The variations of susceptibility to alloxan induced Diabetes in a total of seventeen rabbits was described. Our study was designed to explore dosage schedules which might improve rabbit responsiveness to and survival after alloxan treatment. A wide range of response to intravenously administered alloxan was observed. Permanent diabetes (blood glucose 350 mg/dl) was found in three rabbits after a single injection (60 mg/kg in one, 100 mg/kg in two). This effect has persisted for eight months. By contrast, two other rabbits injected with a single dose of alloxan (60 mg/kg) developed only transient hyperglycemia. Similarly, four other rabbits either did not respond or had an incomplete response after receiving a total dose of 120 mg/kg. These data suggest that there is extreme variability in individual rabbits susceptibility to the diabetogenic affects of alloxan.     

Effects of chemical sympathectomy by means of 6-hydroxydopamine on insulin secretion and islet morphology in alloxan-diabetic mice

Activation of sympathetic nerves increases circulating glucose and inhibits insulin release from the islet beta-cells, which might contribute to stress-related diabetes. Accordingly, we have shown previously that blockade of parasympathetic activity aggravates diabetes in alloxan-treated mice, suggesting that unopposed sympathetic activity impairs diabetes. In this study, we tested whether elimination of sympathetic nerve activity by chemical sympathectomy with 6-hydroxydopamine (6-OHDA; 60 mg/kg) ameliorates the diabetogenic effects of alloxan (50 mg/kg) in NMRI mice. Mice given alloxan alone developed manifest diabetes after 2 days, as indicated by hyperglycemia. The diabetes persisted throughout the 35-day study period. Pretreatment with 6-OHDA did not, however, affect the glucose levels or the low, 2-min in vivo insulin response to glucose (1 g/kg) after alloxan. In situ hybridization at day 35 revealed a significantly reduced grain area of insulin-mRNA in the alloxan-treated animals, which was not affected by 6-OHDA, and an altered islet architecture, with accumulation of glucagon cells in the central portion. Also 6-OHDA alone reduced the insulin mRNA area, but this was accompanied by an increase in the total islet area. We conclude that, in contrast to cholinergic inhibition, sympathectomy does not perturb the development of chemically induced diabetes in mice. Alone, however, sympathectomy reduces insulin gene expression and induces increased islet size, suggesting that sympathetic nerves are of importance for long-term islet function.     

Regeneration potential of the submandibular gland after transplantation: An immunohistological and scintigraphic study

The aim of this study was to evaluate the regeneration potential of the submandibular gland and the associated morphological and functional impairment after a transient warm ischemia as a result of submandibular gland transfer in humans. 42 rabbits were used for the study. After 1.5 h of transient ischemia, submandibular glands were studied histologically in the following 14 days, one month, two and four months in 32 rabbits. Additionally, the glands were functionally evaluated after one and four months in eleven of these rabbits. Ten rabbits were used to establish the scintigraphic method. In the evaluation of the results unpublished data derived from a previous study by our group has also been considered. The Ki-67 index showed a significant increase of the proliferating rate reaching a peak after 14 days (p = 0.006) and still evident after four months involving mostly the terminal ductal system. There was only a slight increase in the tracer uptake of ischemic glands after one month and a more pronounced one after four months.It was concluded that initial loss of acinar cells by 1.5 h of ischemia released a regeneration process which stabilised after 14 days but continued throughout the whole time of the study. These results correlate with clinical observation in patients with submandibular gland transfer for the treatment of keratoconjunctivitis sicca and explain the alternating secretory function observed within the four months following surgery. Scintigraphy has been found to play a limited role in the evaluation of the regeneration process.     

Cytological changes in the pars distalis of the female rat hypophysis grafted under the kidney capsule

Summary Intact female rats received transplants of two hypophysial partes distales under the kidney capsule. The plasma levels of prolactin were determined, and the ultrastructure of the grafted gland was studied 15, 45 and 90 days after the operation. Although prolactin levels in the three experimental groups were significantly higher than those in control rats, a decrease in prolactin level was detected in the 45-day samples. Parallel ultrastructural changes suggest that between the 45th and 90th postoperative day a process is initiated leading to hyperplasia and hypertrophy of prolactotrophs.Supported by Grants from PLAMIRH (93.173.2-77) and from the Dirección de Investigaciones, Universidad Austral (S-77-28)     

A Simple Bioassay for Chemicals Active to the Photosynthetic or Respiratory Systems of Plants

After male rats of the Sprague Dawley strain, 5 weeks old, were fed a 20% casein diet with or without 0.5% nicotinamide for 13 days, 180 mg/kg body weight of alloxan was injected in- traperitoneally into the rats. The rats were kept for 18 days with the same diet. The level of blood glucose was increased 6-fold in the group on a 20% casein diet by the injection of alloxan, while there was only a 2-foid increase in the group on a nicotinamide-containing diet and the decreased body weight was also lower in the group on the nicotinamide diet than the group on the casein diet. The body weight was indirectly related to the concentration of blood glucose. A marked increase was observed in the activities of tryptophan oxygenase, aminocarboxymuconate-semialdehyde decarboxylase, and nicotinamide methyltransferase upon the injection of alloxan with both diets; on the other hand, the activities of kynureninase and NAD⁺ synthetase were decreased by the injection of alloxan. The activity of kynurenine aminotransferase increased in the group on the 20% casein diet by the injection of alloxan, while in the group on the nicotinamide-containing diet its activity was not increased by the injection. These changes in the above enzyme activities mean that the conversion ratio from tryptophan to niacin is lower in the alloxan diabetic rat than normal rat. It was found that the activities of tryptophan oxygenase, aminocarboxymuconate-semialdehyde decarboxylase, and nicotinamide methyltransferase were directly related to the concentration of blood glucose, and that the activities of kynureninase and NAD⁺ synthetase were inversely related. There was no difference in the activities of 3-hydroxyanthranilic acid oxygenase and nicotinamide mononucleotide adenylyltransferase upon the injection of alloxan with both diets.     

C-Fos在db/db自发性糖尿病小鼠颌下腺的表达

目的观察转基因糖尿病小鼠下颌下腺形态学改变与原癌基因C-fos蛋白表达的关系,为糖尿病的临床及基础研究提供依据。方法引进日本C57BL/ksj-db/ m表型正常隐性基因小鼠,其近亲交配所得纯合子后代,即为db/db(单基因遗传自然发病型)糖尿病小鼠。取3、4、6、8、10月龄db/db糖尿病小鼠及相应月龄的db/ m正常小鼠下颌下腺,行HE染色及SP免疫组化染色后进行图象分析,统计各组下颌下腺C-fos阳性表达的细胞数,观察其形态学改变。结果糖尿病小鼠下颌下腺腺泡萎缩,细胞缩小,形态不规则,排列不整齐。各月龄糖尿病小鼠颌下腺C-Fos阳性细胞明显低于相应对照组(P<0.01),且逐渐减少,呈下降趋势。结论db/db糖尿病状态下颌下腺细胞表达C-Fos蛋白明显降低,c-fos低表达可能与下颌下腺实质细胞的增殖减弱性形态学变化密切相关。     

Insulin sensitivity of isolated muscle tissue of rats with manifest alloxan diabetes of different duration]

Insulin sensitivity of muscle tissue was assessed in 20 control rats and 27 rats with alloxan diabetes from the extent of its effect on C14-glucose incorporation by the diaphragmatic muscular glycogen. In diabetes of 5--8-day standing insulin sensitivity of muscle tissue remained unchanged. In alloxan diabetes of 22--24-day duration insulin sensitivity of muscle tissue diminished. With prolongation of decompensated alloxan diabetes up to 5--8 months a further decrease in incorporation of labelled glucose by the diaphragmatic glycogen was recorded. The data obtained indicate that metabolic disturbances inherent in diabetes mellitus lead to a decrease in insulin sensitivity of muscle tissue.     

High levels of GM1-ganglioside beta-galactosidase in the salivary glands and GM1-like-ganglioside storage in parotids of deficient mice.

We have previously demonstrated high levels of GM1-ganglioside beta-galactosidase (beta-gal) in the salivary glands of Swiss-Webster mice (Nowroozi et al., J Craniofac Genet Dev Biol 18:51, 1998), and suggested that this activity reflects an important role for the lysosome in catabolism of salivary glycoconjugates. Here, we characterized and compared activities of lysosomal glycosidases among the salivary glands, spleen, and muscle of C57BL/6 mice, beta-gal hexosaminidase, and beta-glucuronidase activities are high in all three glands relative to muscle. Enzyme activities in the sublingual gland were substantially higher than in the submandibular and parotid glands. Spleen displays levels of activity that are comparable or higher (for beta-glucuronidase) than those in the salivary glands, whereas muscle displays substantially lower levels of these lysosomal glycosidases. In order to investigate the role of beta-gal in the salivary glands, we further characterized the salivary phenotype of knock-out mice deficient in this enzyme, mimicking human GM1-gangliosidosis. In contrast with the relative levels of beta-gal specific-activity among the salivary glands, only the parotid developed severe, generalized, degenerative histopathological changes in beta-gal-deficient knock-out mice. GM1-like-ganglioside, typically found at high levels only in the nerve tissue, where its exact function is still not clear, was demonstrated in storage vacuoles of the parotid glands of the deficient mice by binding of cholera toxin subunit B. Thus, beta-gal activity observed in the parotid gland most likely reflects its role in GM1-ganglioside catabolism, and this ganglioside, never previously reported in the salivary glands, may have a role in parotid exocrine secretory functions. beta-gal may also serve in secretory glycoprotein catabolism in other salivary glands, but this function may be non-essential for these glands.     

Role of ubiquitin-proteasome-dependent proteolytic process in degradation of muscle protein from diabetic rabbits.

The activity of ATP, ubiquitin (Ub)-dependent proteases partially purified from skeletal muscle (psoas) from alloxan diabetic rabbits was determined at different periods of insulin deficiency. Two days after alloxan injection, no change was observed in the activity of ATP, Ub-dependent proteases, but this activity increased 3 and 5 days after diabetes induction, attaining 181% of control values on the 5th day. However, after this early rise, the activity of muscle ATP, Ub-dependent proteases decreased, returning to values that did not differ significantly from controls 7 and 10 days after alloxan injection. After 15 days, the activity of these proteases was 57% lower than in muscle from control rabbits. Both the initial increase and the subsequent fall in the activity of the enzymes were prevented by insulin treatment of alloxan diabetic rabbits. The data suggest that Ub-proteasome-dependent proteolysis have an important role in the control of muscle protein degradation and may be regulated by insulin.     

Changes in the function and ultrastructure of vessels in the rat model of multiple low dose streptozotocin-induced diabetes

In this study we investigated functional changes in the femoral artery and ultrastructural alterations in mesenteric vessels and capillaries in the rat model of multiple low dose streptozotocin (STZ)-induced diabetes. Participation of oxidative stress in this model of diabetes was established by studying the effect of the pyridoindole antioxidant stobadine (STB) on diabetes-induced impairment. Experimental diabetes was induced by i.v. bolus of STZ (20 mg/kg) given for three consecutive days to male rats. At the 12(th) week following STZ administration, the animals revealed typical signs of diabetes, such as polyphagia, polydypsia and polyuria. There was no weight gain in the diabetic groups throughout the experiment. No exitus occurred in any group. Diabetes was characterised with high levels of plasma glucose, no significant changes in lipid metabolism, decreased serum levels of glutathione, increased serum levels of the lysosomal enzyme N-acetyl-beta-D-glucosaminidase (NAGA), injured endothelial relaxant capacity of the femoral artery and alterations in ultrastructure of mesenteric arteries and capillaries. Antioxidant STB in the dose of 25 mg/kg body weight i.p. (5 times per week) did not influence glucose levels, however, it mitigated biochemical, functional and ultrastructural changes induced by diabetes, suggesting a role of reactive oxygen species in diabetes-induced tissue damage.     

Dynamic aspects of amino acid metabolism in alloxan-induced diabetes and insulin-treated rabbits: in vivo studies with 15N and gas chromatography-mass spectrometry

The present study was designed to determine the effect of alloxan-induced diabetes in rabbits on L-[15N]alanine and [15N]glycine kinetic parameters. This process was measured by single-dose administration of 15N-labeled amino acids to postabsorptive control rabbits and alloxan-induced diabetics and insulin-treated diabetic rabbits. Gas chromatography-mass spectrometry was used to determine the 15N enrichment of plasma glycine and alanine. Glycine and alanine pools and turnover rate constants were estimated from isotope enrichment time decay curves. The data from the present study indicate that plasma glycine and alanine turnover rate constants increased by 25-50% after alloxan administration but pool sizes showed only little changes, resulting in highly significant increases in fluxes and metabolic clearance rates of both alanine and glycine following alloxan administration; single-dose crystalline insulin or protamine zinc insulin treatment failed to restore the turnover rate constants of glycine or alanine toward control values and caused a depletion of 50% in glycine pool size; 7 days prolonged treatment with protamine zinc insulin restored alanine and glycine fluxes and metabolic clearance rates towards control postabsorptive values; and the reduction in flux values following insulin treatment is consistent with the reduction in the plasma glucose levels in rabbits. The data suggest that the regulatory mechanisms for uptake and metabolism of circulating glycogenic amino acids no longer are operative as a consequence of insulin deficiency following alloxan administration. Exogenous insulin restored the activity of the regulatory mechanism toward the postabsorptive control state.     

Acute and acclimated digestive responses of the cockle Cerastoderma edule (L.) to changes in the food quality and quantity. II. Enzymatic, cellular and tissular responses of the digestive gland

Cockles Cerastoderma edule were fed two different concentrations of two diets with different qualities which were achieved by mixing different proportions of ashed silt particles with cells of the microalgae Tetraselmis suecica. After 3 days (acute response) and 11 days (acclimated response) of exposure to the diets, we analysed the digestive activity of the digestive gland using cyto-histological and enzymatic techniques. We measured (i) the volumetric fraction of digestive and basophilic cells in digestive tubules, (ii) the diverticular radius and the thickness of digestive epithelia, (iii) the stereological parameters characterizing the lysosomal system and, (iv) dry weight, soluble protein content and specific and total amylase, cellulase, laminarinase, and protease activities of the digestive gland. In the conditions of the present study, specific cellulase and laminarinase activities in the digestive gland of cockles were correlated with the volumetric fraction of basophilic cells (r=0.672 and 0.642, respectively), whereas the specific protease was highly correlated (r=0.866) with lysosomal volume density. The implications of these correlations are discussed in relation to the feeding and absorptive parameters reported in the preceding publication. In the acute response, adjustments of the synthesis of constituents of the lysosomal/proteolytic system of the digestive cells seemed to be the only mechanism operating at the digestive level to respond to the changes in food availability. Lysosomal volume density increased with rising ingestion rate of organic matter, however, the occurrence of a limit in this short-term tissular response would account for the recorded trade-off between absorption efficiency and ingestion rate with different food qualities. With regard to acclimation, food quality determined the nature of the response of the digestive gland. With low quality diets, a time-dependent capability of the digestive gland for intensifying lysosomal/proteolytic production explains the increase of food absorption rates that result from higher filtration and ingestion rates. With high quality food, digestive acclimation differed with food particle concentration: with low rations, in spite of constant morphometrical and stereological parameters, the significant changes in the absorptive balance of biochemical components suggests the existence of an increased production of lysosomes that promotes an accelerated turn-over rate of the digestive epithelia. With high food concentrations, this response was coupled with increased activities of cellulase and laminarinase enzymes, probably as a consequence of higher rates of enzyme secretions from basophilic cells.