Cells In Streptozotocin-induced Diabetic Rats Research Articles

Effects Of <italic>Annona Muricata</italic> (Linn) On The Morphology Of Pancreatic Islet Cells Of Experimentally-Induced Diabetic Wistar Rats

The study was designed to evaluate the effects of methanolic extract of Annona muricata leaves on the pancreatic islet cells of streptozotocin induceddiabetic rats. Thirty adult Wistar rats were randomly assigned into three groups (A, B and C) of ten rats each. Group A was the control, Group B was untreated diabetic group and group C was A. muricata-treated group. Diabetes mellitus was experimentally induced in groups B and C by a single intra-peritoneal injection of 80mg/kg streptozotocin dissolved in 0.1M citrate buffer. Group A rats were intraperitoneally injected with equivalent volume of citrate buffer. Daily intra peritoneal injection of 100mg/kg A. muricata was administered to group C rats for two weeks. The rats were sacrificed and the pancreas were removed and fixed in Bouins fluid. The tissues were processed for paraffin embedding and sections of 5μm thickness were produced and stained. Histopathological examination of the stained sections showed regeneration β-cells of islets of pancreas of A. muricata-treated rats when compared to untreated diabetic group of rats.

Streptozotocin-induced diabetes modulates GABA receptor activity of rat retinal neurons

Neural deficits suggestive of involvement of the GABA signaling pathway can often be detected early in the course of diabetic retinopathy, a leading cause of blindness in the United States. To examine in greater detail the nature of the neuronal changes associated with hyperglycemia, we investigated GABA receptor activity on retinal bipolar cells in streptozotocin-induced diabetic rats; cells from age-matched normal rats served as controls. Patch-clamp recordings from isolated rod-bipolar cells revealed that diabetes enhanced the whole cell currents elicited by GABA. Responses of the GABA C receptor, the predominant GABA receptor on rat rod bipolar cells, exhibited a greater sensitivity to GABA, larger maximum current responses, slower response kinetics, and a smaller single channel conductance among diabetic cells relative to those recorded from normal controls. Compared with the properties of homomeric ρ1 and heteromeric ρ1ρ2 receptors formed in a heterologous expression system, these results suggested that there was a greater contribution from the ρ1 subunit in the GABA C receptor-mediated response of diabetic cells. The levels of mRNA, measured with real-time RT-PCR, were consistent with this finding. There was a significant enhancement in the ratio of ρ1/ρ2 subunit expression in the retina of diabetic animals, although the levels of GABA ρ1 subunit expression were comparable in diabetic and normal retinas. Taken together, the results suggest that diabetes modifies the subunit composition of the GABA C receptor on retinal neurons, most likely through its effect on the efficacy of gene transcription.

Effects of Activin A on Pancreatic Ductal Cells in Streptozotocin-Induced Diabetic Rats

The shortage of islets for transplantation has led to find alternative insulin producing cells. Pancreatic progenitor cells in the duct have the potential to grow and differentiate into endocrine cells. In this study, we examined whether activin A can promote the expansion and/or differentiation of ductal cells into insulin-producing cells. Pancreatic ductal cells were treated with activin A for differentiation into endocrine cells, and transplanted into the renal subcapsular space of streptozotocin (STZ)-induced diabetic rats. The identity of the endocrine cells was confirmed by immunostaining and analysis of the expression of transcription factors and endocrine genes by reverse-transcriptase polymerase chain reaction. Activin A treatment significantly increased the DNA synthesis and the expression of insulin I, insulin II, PDX-1, Nkx 6.1, Glut-2, Pax-4, Pax-6, and Ngn-3. De novo synthesis of insulin in activin A-treated ductal cells was observed by the immunocytochemical detection of C-peptide and the differentiated ductal cells secreted significantly increased amount of insulin compared to nontreated ductal cells in response to glucose stimulation. When activin A-treated ductal cells were transplanted on STZ-induced diabetic rats, blood glucose levels were normalized and the removal of the transplanted kidney resulted in return to hyperglycemia. The pancreatic ductal cells could be efficiently differentiated into insulin secreting cells by activin A treatment in vitro and normalize hyperglycemia in vivo.

Antidiabetic effects of chitooligosaccharides on pancreatic islet cells in streptozotocin-induced diabetic rats

To investigate the effect of chitooligosaccharides on proliferation of pancreatic islet cells, release of insulin and 2 h plasma glucose in streptozotocin-induced diabetic rats. In vitro, the effect of chitooligosaccharides on proliferation of pancreatic islet cells and release of insulin was detected with optical microscopy, colorimetric assay, and radioimmunoassay respectively. In vivo, the general clinical symptoms, 2 h plasma glucose, urine glucose, oral glucose tolerance were examined after sixty days of feeding study to determine the effect of chitooligosaccharides in streptozotocin-induced diabetic rats. Chitooligosaccharides could effectively accelerate the proliferation of pancreatic islet cells. Chitooligosaccharides (100 mg/L) had direct and prominent effect on pancreastic beta cells and insulin release from islet cells. All concentrations of chitooligosaccharides could improve the general clinical symptoms of diabetic rats, decrease the 2 h plasma glucose and urine glucose, and normalize the disorders of glucose tolerance. Chitooligosaccharides possess various biological activities and can be used in the treatment of diabetes mellitus.

Amelioration of diabetic peripheral neuropathy by implantation of hematopoietic mononuclear cells in streptozotocin-induced diabetic rats

This study was performed in order to evaluate the angiogenic effect of implantation of either peripheral blood mononuclear cells (PBMNCs) or bone marrow mononuclear cells (BMMNCs) on diabetic peripheral neuropathy. Streptozotocin (50 mg/kg) was injected intravenously into 6-week-old male Lewis rats. Four weeks after the induction of diabetes, 6 x 10(7) of PBMNCs or 1 x 10(8) of BMMNCs were implanted into the left hindlimb muscle. Motor nerve conduction velocity (MNCV) was monitored before and after implantation. At the end of the experiment, bilateral nerve blood flow (NBF) was measured by laser Doppler and the number of vessels in the sciatic nerves quantified by Factor VIII staining of the sections. Diabetes resulted in an approximately 20% reduction (P < 0.01) in sciatic MNCV. Four weeks after implantation, MNCV was improved by 54% with PBMNCs and by 67% with BMMNCs (both P < 0.01). Moreover, the effects of implantation were almost abolished by administration of VEGF-neutralizing antibody. Sciatic NBF was reduced by approximately 50% by diabetes (P < 0.05). This reduction in perfusion was improved by 74% by implantation of PBMNCs and by 62% by implantation of BMMNCs (P < 0.05 and P < 0.01, respectively). These effects were observed only in the implanted limb. Immunohistochemical staining of sciatic nerve sections for Factor VIII showed no significant increase in the number of vessels in the sciatic nerve following implantation of either PBMNCs or BMMNCs. These data suggest that implantation of hematopoietic mononuclear cell fractions is associated with an improvement in MNCV as a result of arteriogenic effects in the sciatic nerve, and that VEGF may contribute to this effect. This improvement occurred in the absence of angiogenesis. Implantation of these cell fractions may therefore be a potential new therapeutic method for treating diabetic peripheral neuropathy.

The effects of acarbose and Rumex patientia L. on ultrastructural and biochemical changes of pancreatic B cells in streptozotocin-induced diabetic rats

This study was performed to observe the effects of acarbose and Rumex patientia on morphological change of pancreatic B cells in streptozotocin (STZ)-induced diabetic (type 2) rats. Two-day-old Wistar albino rats were intraperitoneally injected with 100mg/kg of STZ or vehicle alone for control. Vehicle and STZ given rats were divided into six groups (1st, 2nd and the 3rd groups are control; the 4th, 5th and 6th groups are STZ groups). The 1st and the 4th groups received water, the 2nd and the 5th groups received 40mg acarbose/100g feed, the 3rd and the 6th groups received 2% decoction of Rumex patientia grain. During experimentation period, blood glucose levels were checked periodically, and HbA1c level was measured from cardiac blood at the end of the experiment. Pancreas tissues were examined by electron microscope. Glucose and HbA1c levels increased by STZ were decreased by acarbose and Rumex patientia. Morphologically, we found a mitochondrial vacuolization and swelling as well as dilatation of the endoplasmic reticulum in the B cells of STZ-induced diabetic rats. Also, a decrease in the secretory granules of B cells was observed in the STZ-induced diabetic group. No pathological changes were observed in the STZ+acarbose group. In the STZ+Rumex patientia group, a weak swelling in the B cells was observed in the some of the mitochondria.

Histochemical assessment of nitric oxide synthase activity in aortic endothelial cells of streptozotocin-induced diabetic rats

Impaired endothelium-dependent relaxation of blood vessels is a common feature in diabetes, but the exact underlying mechanisms have not yet been clarified. In present study, endothelium-dependent vasorelaxation of aortic rings were evaluated in vitro in streptozotocin (STZ)-induced diabetic and age-matched control rats. Moreover, nitric oxide synthase (NOS) activity of aortic endothelial cells was assessed in both diabetic and healthy rats using histochemical staining for nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity. The results showed a significant decrease of endothelium-dependent relaxation in response to acetylcholine (ACh) in diabetic rings, compared with controls, that was accompanied by a remarkable attenuation of NOS activity in diabetic sections of rat aorta stained for NADPH-diaphorase. Furthermore, a membrane disruption of some endothelial cells was also observed in all diabetic sections. It can be concluded that a decrease in NOS activity together with a disruption of endothelial cell membrane play a major role in endothelial dysfunction observed in diabetes.

Increased spike broadening and slow afterhyperpolarization in CA1 pyramidal cells of streptozotocin-induced diabetic rats

Diabetes mellitus is associated with impairments of cognitive function both in humans and animal models. In diabetic rats cognitive deficits are related to alterations in activity-dependent synaptic plasticity in the hippocampus. Many similarities with the pathophysiology of normal brain aging have been noted, and the view emerges that the effects of diabetes on the brain are best described as “accelerated brain aging.” In the present study we examined whether CA1 pyramidal neurons from streptozotocin-induced diabetic rats display an increased slow afterhyperpolarization, often considered as a hallmark of neuronal aging. We found no differences in resting membrane potential, input resistance, membrane time-constant, and action potential amplitude and duration between CA1 pyramidal neurons from streptozotocin-induced diabetic and age-matched control rats. During a train of action potentials, however, there is an increased broadening of the action potentials in diabetic animals, so-called “spike broadening.” The amplitude of the slow afterhyperpolarization elicited by a train of action potentials is indeed increased in diabetic animals. Interestingly, when the slow afterhyperpolarization is elicited by a Ca 2+ spike, there is no difference between control and diabetic rats. This indicates that the increased slow afterhyperpolarization in diabetes is likely to be due to an increased Ca 2+ influx resulting from the increased spike broadening. These data underscore the notion that the diabetic brain at the neuronal level shares properties with brain aging.

Clusterin expression during regeneration of pancreatic islet cells in streptozotocin-induced diabetic rats.

Beta-cell regeneration has been reported after islet injury in an animal model for diabetes. Recently, we showed up-regulation of clusterin after islet injury and suggested that clusterin might be involved in cytoprotection and in the regeneration of islet cells. The aim of this study was to investigate the correlation of clusterin expression with islet regeneration and its effect on islet cell replication. Streptozotocin was administrated to rats to induce various types of diabetes. Islet regeneration and clusterin expression were examined after islet injuries. Clusterin cDNA was transfected to MIN6 cells and their proliferation activity was measured by a [3H]thymidine-incorporation assay. A diabetogenic dose of streptozotocin injected in rats provoked an immediate degeneration of beta cells. In this model, islets showed increased clusterin expression with extensive proliferation of alpha cells but showed poor beta-cell replication. A subdiabetogenic dose of streptozotocin, however, led to the proliferation of beta cells with clusterin up-regulation. In streptozotocin-treated neonatal rats, up-regulation of clusterin was noted during beta-cell proliferation. In all experimental models, clusterin was expressed in alpha cells in close correlation with islet cell proliferation, higher transcription of insulin mRNA and MAPKs activation. Cell replication was increased by 31 % in the MIN6 cells transfected by the clusterin cDNA. Up-regulation of clusterin in alpha cells might induce beta-cell proliferation and thus restore their population after islet injury. We suggest that clusterin could be considered as a growth factor-like molecule stimulating islet-cell proliferation by paracrine action.

Changes in adrenocorticotropic hormone (ACTH) release from the cultured anterior pituitary cells of streptozotocin-induced diabetic rats.

We examined ACTH release from cultured anterior pituitary cells of streptozotocin (STZ)-induced diabetic rats. Rats 1 week after the injection of STZ (55 mg/kg, i.v.) were used as a short-term diabetic model, while rats 8 weeks after the injection of STZ were used as a long-term diabetic model. ACTH release induced by corticotropin-releasing factors (CRF) was significantly increased in the short-term diabetic rats, whereas ACTH release decreased in the long-term diabetic rats. A stimulator of adenylate cyclase, forskolin, caused marked increases in ACTH release in short-term diabetic rats, whereas it did not affect the long-term diabetic rats. An L-type Ca2+ channel agonist, BAY K 8644, did not affect ACTH release in the short-term diabetic rats, although it decreased ACTH release in long-term diabetic rats. In addition, CRF-induced ACTH release also increased in normal anterior pituitary cells cultured under high glucose conditions (25 mmol/L) for 5 d. These results suggest that the increase in the CRF-induced ACTH release from cultured anterior pituitary cells of short-term diabetic rats appears to involve the cAMP system in part. In contrast, the decrease in the CRF-induced ACTH release from the cultured anterior pituitary cells of long-term diabetic rats may indicate a change in the properties of the L-type Ca2+ channel coupled with the CRF receptor, or in the CRF receptor itself.

Overexpression of pulmonary surfactant apoprotein A mRNA in alveolar type II cells and nonciliated bronchiolar (Clara) epithelial cells in streptozotocin-induced diabetic rats demonstrated by in situ hybridization.

Pulmonary surfactant is critical for gas exchange and is composed of both phospholipids and specific surfactant-associated proteins. The most abundant surfactant protein is termed surfactant apoprotein A (SP-A). This protein is thought to be important in the formation of tubular myelin, in absorption of surfactant to the air-liquid interface, in recycling of surfactant in alveolar type II cells, and in the regulation of secretion. We have examined the expression and localization of SP-A mRNA in streptozotocin-induced diabetic rats by in situ hybridization using a specific rat cDNA probe. Diabetes was induced by intraperitoneal injection of 60 mg/kg streptozotocin. After 10 wk, lungs were excised and examined by in situ hybridization and by light and electron microscopy. The ultrastructural examination demonstrated the marked changes of endoplasmic reticulum of alveolar type II cells, as reported previously. Immunohistostaining of SP-A in diabetic lungs was weak in alveolar type II cells. However, by autoradiographs of in situ hybridization, compared with the control lungs, a larger number of silver grains for the SP-A mRNA were shown in alveolar type II cells and also in some bronchiolar epithelial (Clara) cells from the diabetic lungs. Alveolar type II cells having high contents of silver grains were also increased in number. These results were confirmed by measurement of the SP-A content and by Northern blot analysis. The present study demonstrates an overexpression of SP-A mRNA despite the ultrastructural changes in the endoplasmic reticulum of alveolar type II cells in the diabetic lungs, which will provide new information on the regulatory mechanism of SP-A gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphology and functional responses of isolated zona glomerulosa cells of streptozotocin-induced diabetic rats

Streptozotocin-induced diabetes significantly decreased plasma aldosterone concentration in rats whose renin-angiotensin system had been pharmacologically interrupted. Isolated zona glomerulosa cells showed a marked atrophy, coupled with a reduced basal secretion of aldosterone and corticosterone. The secretory response to the three main physiological stimuli (ACTH, angiotensin II and potassium) was also notably impaired. The hypothesis is advanced that the chronic lack of insulin may directly impair the growth and steroidogenic capacity of rat adrenal zona glomerulosa.

Immunohisto- and Cytochemical Studies of Pancreatic Enzymes in Peri-Insular and Tele-Insular Acinar Cells of Streptozotocin-Induced Diabetic Rats

Previous studies have demonstrated that the exocrine pancreatic tissue is influenced by islet hormones and that under diabetic condition, amylase secretion is altered. On the other hand according to the topographical location of the acini with reference to the islet of Langerhans, the exocrine parenchyma has been partitioned into peri- and tele-insular regions. In the present study, we have analyzed by immunofluorescence and protein A-gold immunocytochemistry the distribution of two enzymes, amylase and chymotrypsinogen, on pancreatic tissues of short- and long-term diabetic rats. The alteration in amylase secretion occurring under diabetic condition was found to follow the topographical partition of the pancreas. Acinar cells, located in the peri-insular regions of the pancreas (close to the islets of Langerhans), appeared totally devoid of amylase immunolabeling while positive and negative cells were found in the tele-insular regions (at a distance from the islets). In addition, quantitation of amylase immunolabeling present in positive cells has demonstrated that although amylase is still secreted by these cells, its processing is altered. Since the partition of the exocrine parenchyma is due to the paracrine influence of islet hormones, the data obtained demonstrate and reinforce the fact that the endocrine and exocrine components of the pancreas are functionally interrelated in normal as well as in pathological conditions.

Ultrastructural lesions of retinal pericapillary Müller cells in streptozotocin-induced diabetic rats.

Seventeen Wistar inbred rats were made diabetic by a single injection of streptozotocin (50 mg/kg body weight) and were killed after periods of 3, 6, 9 or 12 months. Pathological changes in pericapillary Müller cells of the retina were studied using electron microscopy. Basement membrane-like material proliferated in the intercellular space of the Müller cell network and occasionally appeared to insinuate into the Müller cell cytoplasm far from capillary pericytes and endothelial cells. The part of the Müller cell that was enveloped by proliferating basement membrane-like material showed partial necrosis which was thought to contribute to the widening of the capillary wall. A breakdown of the retinal framework, which leads to capillary dilatation, was also thought to be associated with partial necrosis of Müller cells. Highly electron-dense bodies accumulated in the Müller cell cytoplasm which surrounded the retinal capillaries. Ultrastructurally, these dense bodies resembled lysosomes. Their increased number might reflect the altered metabolism of the diabetic retina.

Ultrastructural changes in Leydig cells of streptozotocininduced diabetic rats

Hyperglycemia (experimental diabetes) was induced in adult male rats by destruction of the pancreatic beta cells with a single intravenous injection of streptozotocin (STZ). Testes from diabetic, from insulin-treated diabetic, and from sham-injected normal rats were fxed by vascular perfusion. The fine structure of Leydig cells was examined at two, three, and four weeks after the STZ injection in the untreated diabetic animals, and at four weeks in the controls and insulin-treated diabetic rats. A number of morphological changes was observed in Leydig cells of untreated diabetic animals. Most obvious of these was an accumulation of lipid droplets, not normally present in Leydig cells in adults of this species. Smooth endoplasmic reticulum (SER) was markedly reduced in Leydig cells of the hyperglycemic rats. Several types of intracellular bodies were seen exclusively in Leydig cells of the untreated diabetic animals. Many resembled secondary lysosomes or dense bodies, while others appeared to be autophagic vacuoles. In addition, a small, granule-containing lamellar structure was seen either within a typical dense body or free in the cytoplasm. Myelin-like structures were commonly observed within the cytoplasm of the Leydig cell or within mitochondria. The appearance of the mitochondria in diabetic rats was otherwise normal. The extracellular spaces surrounding Leydig cells from untreated hyperglycemic rats also contained large accumulations of myelin-like material. These structural changes appear to be direct consequences of the diabetic state of the animals, since the ultrastructure of insulin-treated diabetic rats did not differ from that of the controls. These findings may reflect an alteration or breakdown of Leydig cell components normally involved in the synthesis of androgen, and correlate with previous reports of lowered circulating levels of testosterone in diabetic rats.