Low-dose Streptozocin Research Articles

Hypoglycemic Activity and Antioxidative Stress of Extracts and Corymbiferin fromSwertia bimaculata In VitroandIn Vivo

The present study was to investigate the anti-diabetic activities of Swertia bimaculata. Based on the glucose consumption of S. bimaculata extractsand different fractions (petroleum, dichloromethane, ethyl acetate, n-butanol and water extracts) in 3T3-L1 adipocyte assay, ethanol (ETH) and dichloromethane (DTH) extracts had the most effective potency. Furthermore, ETH, DTH and corymbiferin (the most abundant component of DTH) were evaluated for anti-diabetic effects in high fat and sucrose fed combined with low dose streptozocin induced diabetic rats. DTH and corymbiferin displayed remarkable anti-diabetic activities. The fasting blood glucose levels were significantly decreased, while the serum insulin levels were obviously increased. The oral glucose tolerance was also improved. The lowed serum total cholesterol, low density lipoprotein (LDL) and triglyceride levels and increased ratio of HDL (high density lipoprotein)/LDL were observed. The insulin sensitivity was improved on the basis of increased expressions of insulin-receptor substrate-2, phosphatidylinositol 3-kinase and Ser/Thr kinase AKT2. And also DTH and corymbiferin improved antioxidant capacity and carbohydrate metabolism in diabetic rats, along with the improvement of histopathology of livers and pancreatic β cells. Corymbiferin was one of active constituents, responsible for anti-diabetic properties. Therefore, S. bimaculata could be considered as an alternative agent against diabetes mellitus.

A site-specific PEGylated analog of exendin-4 with improved pharmacokinetics and pharmacodynamics in vivo

Our aim was to improve the in vivo pharmacokinetics and pharmacodynamics of exendin-4 by using site-specific PEGylation. We designed the PEGylated peptide based on its structure and activity relationship and prepared the conjugate by two steps of chromatographic purification. After obtained the conjugate we confirmed its glucose-lowering activity in normal mice and determined its half-life in SD rats. Then we evaluated its anti-diabetic activity in a multiple low-dose Streptozocin (STZ)-induced diabetic mice model. With the process established in this study the product conjugate was obtained with a yield of over 60% and purity of above 99%. The conjugate maintained its original conformation after modification. In SD rats its half-life was prolonged to 27.12 ± 5.75 h which was 17.61-fold longer than that of the natural exendin-4 for which the half-life was only 1.54 ± 0.47 h. Its anti-diabetic activity was significantly improved in the diabetic mice. Compare with native exendin-4, the C-terminal site-specific PEGylated analog of exendin-4 obtained in this study has an improved pharmacokinetics and pharmacodynamics in vivo and could be regarded as a potential candidate for the future development of anti-diabetic drugs.

Antidiabetic Effect of the Total Polyphenolic Acids Fraction from Salvia miltiorrhiza Bunge in Diabetic Rats

An investigation was made to evaluate the therapeutic potential of the total polyphenolic acids fraction (PAF) from Salvia miltiorrhiza Bunge in the type 2 diabetes mellitus rats model with an oral dose of 187 mg/kg for 28 days. The results showed that PAF induced a significant decrease in fasting blood glucose (FBG), fasting blood insulin (FINS), total cholesterol (TC), triglyceride (TG) and blood urea nitrogen (BUN), and an obvious increase in insulin sensitivity index (ISI) in diabetic rats induced by a high fat diet and a low dose of streptozocin (STZ). These results suggested that PAF has antidiabetic potential in vivo.

Metabolic effects of various antidiabetic and hypolipidaemic agents on a high-fat diet and multiple low-dose streptozocin (MLDS) mouse model of diabetes

Insulin resistance and subsequent insulin secretory defect are two main features of type 2 diabetes and associated metabolic disorders. The animal models of type 2 diabetes are very complex and are as heterogeneous as the disease. We have evaluated the effect of various antidiabetic and lipid lowering agents (fenofibrate, rosiglitazone, glimepiride, metformin and simvastatin) on the metabolic abnormalities induced by combining a high-fat diet and multiple low-dose streptozocin (MLDS) in mice. Male Swiss albino mice were orally treated with the above agents and fed with a diet containing high fat for 28 days. On day 15 the animals were injected intraperitoneally with low-dose streptozocin (40 mg kg(-1)), which was administered for five consecutive days. At the end of the 28-day treatment plasma metabolic parameters (glucose, triglyceride and immunoreactive insulin) were estimated. The antidiabetic and hypolipidaemic agents exhibited differential effects on these metabolic parameters. With the exception of fenofibrate all these agents reduced the plasma glucose levels, and the effects of metformin and rosiglitazone on glucose were found to be statistically significant. Although the effect of the test drugs on cholesterol was modest, a significant decrease in triglyceride levels was observed with sub-chronic treatment with these agents. Interestingly, glimepiride mildly elevated the insulin levels while the other antidiabetics and hypolipidaemics reduced the insulin levels, with metformin and rosiglitazone exhibiting statistically significant effects on insulin. To our knowledge this is the first report on the effect of various peroxisome proliferator-activated receptor modulators and newer antidiabetics on the metabolic effects induced by the combined high-fat diet and MLDS model of type 2 diabetes in Swiss albino mice. The results suggested the complexity of the hyperglycaemia, hyperinsulinaemia and hypertriglyceridaemia induced by the high-fat diet and MLDS mouse model, and their correction by various antidiabetics and antihyperlipidaemics may have involved diverse mechanisms.

Release of Glutamate Decarboxylase-65 into the Circulation by Injured Pancreatic Islet β-Cells

The enzyme glutamate decarboxylase-65 (GAD65) is a major autoantigen in autoimmune diabetes. The mechanism whereby autoreactivity to GAD65, an intracellular protein, is triggered is unknown, and it is possible that immunoreactive GAD65 is released by injured pancreatic islet beta-cells. There is a great need for methods by which to detect and monitor ongoing islet injury. If GAD65 were released and, furthermore, were able to reach the circulation, it could function as a marker of beta-cell injury. Here, a novel GAD65 plasma immunoassay is used to test the hypotheses that beta-cell injury induces GAD65 discharge in vivo and that discharged GAD65 reaches the bloodstream. Plasma GAD65 levels were determined in rats treated with alloxan, and with diabetogenic and low, subdiabetogenic doses of streptozotocin. beta-Cell injury resulted in GAD65 release into the circulation in a dose-dependent manner, and low-dose streptozotocin resulted in a more gradual increase in plasma GAD65 levels than did diabetogenic doses. Plasma GAD65 levels were reduced in rats that had undergone partial pancreatectomy and remained undetectable in mice. Together, these data demonstrate that GAD65 can be released into the circulation by injured beta-cells. Autoantigen shedding may contribute to the pathogenesis of islet autoimmunity in the multiple low-dose streptozocin model and perhaps, more generally, in other forms of autoimmune diabetes. These results demonstrate that, as is true with other tissues, islet injury, at least in some circumstances, can be monitored by use of discharged, circulating proteins. GAD65 is the first such confirmed protein marker of islet injury.

Inhibition of Autoimmune Diabetes by Oral Administration of Anti-CD3 Monoclonal Antibody

Anti-CD3 monoclonal antibody (mAb) has been shown to induce tolerance and to be an effective treatment for diabetes both in animal models and in human trials. We have shown that anti-CD3 mAb given orally is biologically active in the gut and suppresses experimental autoimmune encephalitis by the induction of a regulatory T-cell that expresses latency-associated peptide (LAP) on its surface. In the present study, we investigated the effect of oral anti-CD3 mAb on the prevention of autoimmune diabetes in AKR mice in which the low-dose streptozocin (STZ) model induces autoimmunity to the beta-cells of the islets. We found that oral anti-CD3 mAb given at doses of 50 and 250 microg/feeding suppressed the incidence of diabetes in this model with the best effects seen at the 50 microg/dose. Associated with suppression, we observed decreased cell proliferation in the spleen and conversion of T-helper (Th)1 responses into Th2/Th3 responses in the periphery, including the pancreatic lymph nodes. Oral anti-CD3 mAb increased the expression of LAP on CD4(+) T-cells, and these cells could adoptively transfer protection. Protection by oral anti-CD3 was transforming growth factor-beta dependent. Our results demonstrate that oral anti-CD3 is effective in the model of STZ-induced diabetes and may be a useful form of therapy for type 1 diabetes in humans.

Effects of High or Low Dose of Streptozocin on Pancreatic Islets in C57BL/6 and C.B17-SCID Mice

Effects of High or Low Dose of Streptozocin on Pancreatic Islets in C57BL/6 and C.B17-SCID Mice

The immunosuppressant FK506 inhibits the damage to mouse pancreatic islets induced by low dose streptozocin.

Diabetes mellitus was induced in 40 male C57BL6 mice by injection of a low dose of streptozocin (45 mg/kg body weight) on 5 consecutive days. Twenty four of the mice were immunosuppressed by administration of 1.5 mg FK506/kg body weight daily for 10, 15, 18 and 24 days. Administration of FK506 almost completely inhibited the streptozocin-induced islet damage, and consequently glycaemia remained normal. In FK506-treated animals any inflammatory infiltrate was very sparse and was limited to the vascular pole of the islets. Immunocytochemical results demonstrated that infiltrating cells were Ia-immunoreactive, but were not activated. Ultrastructural observations confirmed the absence of B cell necrosis and degranulation in FK506-treated mice; the few infiltrating elements encountered did not contain phagocytic vesicles or show other signs of activation.

The immunosuppressant FK506 inhibits the damage to mouse pancreatic islets induced by low dose streptozocin

The immunosuppressant FK506 inhibits the damage to mouse pancreatic islets induced by low dose streptozocin

An increase in superoxide dismutase counteracts islet vascular alterations in low-dose streptozocin-treated mice.

A decrease in superoxide dismutase (SOD), the first cellular defence against free radicals, occurs at about the same time as the activation of macrophages within the islets of low-dose streptozocin (LDS)-treated mice. Furthermore, a decrease in the total islet capillary area also has been shown to occur by 10 days after the first streptozocin (STZ) injection and this decline in capillary area is concomitant with the activation of macrophages as is the fall in SOD. Intracellular levels of SOD have been shown to increase after administration of acetyl-homocysteine-thiolactone (citiolone); therefore, the aim of the present study was to observe any relationship between the citiolone-induced increase in SOD levels and islet microvasculature area during LDS-induced diabetes. C57BL6/J male mice were pretreated with daily intramuscular injections of 50 mg citiolone/kg body wt. for 30 days and were then rendered diabetic with 45 mg STZ/kg body wt. given for 5 days; citiolone was given until the animals were killed (days 6, 11 and 18 after the first STZ injection). Further animals were used as non-diabetic and diabetic (STZ-only) controls. The results show that LDS-treated animals when given citiolone: (1) were generally normoglycaemic; (2) had SOD levels that were higher than those of STZ-only control animals; (3) had an islet capillary area that was larger than that of LDS-treated mice. Therefore, the administration of a free radical scavenger, namely citiolone, is able partly to counteract and delay the reduction of islet vascular area and oedema formation in LDS-treated mice.

Multiple low-dose streptozocin-induced diabetes in NOD-scid/scid mice in the absence of functional lymphocytes.

The murine severe combined immunodeficiency (scid) mutation was used to assess whether the diabetogenic effects of multiple low-dose streptozocin (MD-STZ) administration required the presence of functional T-cells. An STZ dose as low as 30 mg/kg body wt for 5 days induced hyperglycemia in young NOD/Lt-+/+ male mice, whereas a dose of 50 mg/kg for 5 days was required to elicit comparable hyperglycemia in C.B.-17-+/+ male mice. The greater NOD strain sensitivity was not a function of preexisting insulitis, because insulitis- and diabetes-free NOD male mice congenic for a diabetes-resistant major histocompatibility complex haplotype were equally susceptible to MD-STZ. This was confirmed in NOD-scid/scid and C.B.-17-scid/scid males. Both were completely insulitis-free, and despite the absence of functional T- cells and B-cells, both congenic stocks were as sensitive to MD-STZ as congenic +/+ controls. Indeed, MD-STZ-induced hyperglycemia in NOD-scid/scid male mice was significantly higher than in NOD/Lt-+/+ male mice. The NOD-scid/scid mouse as a recipient of adoptively transferred splenocytes clearly delineated a distinct pathogenesis of spontaneous insulin-dependent diabetes mellitus (IDDM) versus MD-STZ-induced hyperglycemia. Splenocytes from spontaneously diabetic NOD/Lt males, but not those from donors given MD-STZ, readily transferred IDDM, even when host beta-cells were sensitized by a single injection of STZ before adoptive transfer. We conclude that IDDM induced by MD-STZ is not mediated by T-cell- or B-cell-dependent autoimmune mechanisms in a fashion analogous to the spontaneous IDDM characteristic of NOD mice.

Gangliosides prevent insulitis but not islet B cell destruction in low-dose streptozocin-treated mice

High doses of gangliosides were given to low-dose streptozocin(LDS)-treated mice, in order to observe their ability to prevent diabetes in this animal model and also to look for any modification in the early histopathological lesions occurring in pancreatic islets. Cronassial, 150 mg/kg body wt., (21% GM1, 40% GD1a, 16% GD1b, 19% GT1b and 5% other) was given to 20 C57BL6/J mice in which diabetes was induced 1 week after the start of the Cronassial treatment. Data obtained from animals killed 1 and 3 weeks from the end of the LDS treatment demonstrate that gangliosides stop infiltration of the islets but do not prevent destruction of islet B cells. Therefore, inflammation is not obligatory for the development of the disease in this model. Streptozocin exerts a direct cytotoxic action of considerable importance and is able to act prolongedly on target cells. The destruction achieved is similar to that produced by administration of a single high dose even though it takes place over a longer period. Our results underline the complexity of the LDS-induced diabetic disease.

Alterations of islet microvasculature in mice treated with low-dose streptozocin.

Islet capillary area was followed daily in mice after treatment with low-dose streptozocin (LDS), in order to elucidate the exact period during which the insular vascular bed undergoes a significant reduction. Forty C57BL6/J mice were diabetized with 5 x 40 mg streptozocin (STZ)/kg body wt and killed 6, 8, 9, 10, 11, 12, 15 or 18 days after the first STZ injection. Pancreases were sectioned and processed by staining for alkaline phosphatases using a method devised by Gomori. The percentage of the islet parenchymal area occupied by intra-islet capillaries was measured using a Videoplan videoanalyzer. LDS treatment did not significantly alter the islet capillary area up to day 8; the first signs of reduction were seen on days 9 and 10 (islet capillary area at days 9 and 10 respectively was 2.68% and 2.60% of controls). At day 11 a dramatic decrease in islet capillary area was seen (1.38%), which was not accompanied by a similar reduction of the islet parenchymal area. The reduction in islet capillary area continued to progress up to day 15 by which time it had achieved the lowest level (0.72%). On day 18, values remained practically unchanged.

Ultrastructural observations on cytotoxic effector cells infiltrating pancreatic islets of low-dose streptozocin treated mice.

The aim of this study was to observe the ultrastructural events, during the onset of diabetes mellitus in the low-dose streptozocin (LDS)-treated mouse model with emphasis on the infiltrating elements. Forty male C57 BL/6J mice were given 40 mg/streptozocin on 5 consecutive days and killed 5, 6, 7, 8, 9, 10, 15, and 18 days after the first injection. Results demonstrated that islet infiltration occurring in LDS-treated mice is characterized by a very early pre-infiltration state in which mononuclear phagocytes in islet capillary vessels were considerably increased in number. A new histopathological time sequence for the early insulitis is described, in which attraction of blood mononuclear phagocytes into the islet capillary lumen is the first step. During the successive stage, occurring on days 6-8 we observed that mononuclear phagocytes migrate through capillary and venule walls into the islet parenchyma, where they differentiate into tissue macrophages. It was only later (step 3) that these macrophages acquired novel properties, typical of their "activated state" and started to phagocytose islet beta-cell debris. These data suggest that during the pre-infiltration and early insulitis the mononuclear phagocyte system plays a key role in the onset of LDS diabetes.

Further Morphological and Biochemical Observations on Early Low Dose Streptozocin Diabetes in Mice

Conflicting published data regarding the role of macrophages and other cell types during the early stages of diabetes mellitus led us to further study this problem. To this end we diabetized mice, using low doses of streptozocin (STZ), 40 mg/kg body wt/day/5 days, and processed their pancreatic tissue for immunocytochemistry and ultrastructural observations; immunohistochemistry was performed on days 5 and 18 after the first STZ injection, and islets were observed ultrastructurally on days 5, 9, 10, and 18. Animals were tested for fasting serum glucose, and isolated islets were assayed for insulin secretion capacity. Immunohistology demonstrated that expression of major histocompatability complex class 2 antigens is strongly induced by multiple, low dose STZ treatments prior to impaired insulin release, and that different types of cells within the islet are capable of expressing Ia molecules. Ultrastructurally we found (a) a small number of macrophages (most probably resident monocytes/macrophages) containing B-cell debris, that were located close to either damaged or intact B cells; (b) a large number of recruited macrophages in a vascular or perivascular position; and (c) macrophages recognizable in the exocrine portion, close to the islets, occasionally containing exocrine cell debris. This led us to believe that recruited macrophages play an important role in the early islet-infiltrating stage.

Low-Dose Streptozocin-Induced Autoimmune Diabetes in Islet Transplantation Model

An islet transplant model was used to gain further insight into the immunologic mechanisms involved in low-dose streptozocin (STZ)-induced diabetes. As shown by others, male C57BL/KsJ mice developed diabetes and insulitis after five daily injections of STZ (40 mg.kg-1.day-1). Syngeneic islet transplants beneath the renal capsule developed insulitis when the islets were transplanted 10-14 days before the daily injections of STZ. In contrast, insulitis of the grafts did not occur when the syngeneic transplants were done 3 days after the five daily injections of STZ. If the donor islets were incubated in vitro with 0.5 mg/ml STZ for 1 h at 37 degrees C and then transplanted after the low-dose STZ administration of the recipients, then a definite insulitis was present in the syngeneic transplants. These findings indicated that this brief exposure to STZ in vitro was sufficient to permit immunologic recognition of the grafts. In vitro STZ-exposed islets transplanted into high-dose STZ-induced diabetic mice also developed insulitis, whereas STZ-exposed islets transplanted into alloxan-induced diabetic mice did not. Donor islets incubated in vitro with STZ and transplanted into unexposed mice did not develop insulitis in the grafts. Thus, preexposure of the recipient to STZ is essential to the development of insulitis within in vitro STZ-exposed islet grafts. This is also specific to islets because in vitro STZ-exposed thyroid tissue transplanted into low-dose STZ recipients failed to exhibit a thyroiditis. These data are consistent with the idea that STZ immunologically alters the beta-cell by inducing an antigenic change.

Exercise training normalizes glucose metabolism in a rat model of impaired glucose tolerance

The purpose of this study was to characterize an animal model of impaired glucose tolerance produced by streptozocin treatment of rats (45 mg/kg, intravenously [IV]) and selection of animals with plasma glucose concentrations less than 150 mg/dL. In addition, we determined the effects of physical training on glucose tolerance and metabolism in these animals. During 10 weeks of monitoring, it was determined that these animals have nearly normal plasma glucose concentrations; however, they have an impaired glucose tolerance when challenged with an oral glucose load. They also have normal fasting insulin, free fatty acid, and triglyceride concentrations, normal body weight and food consumption patterns, and normal rates of skeletal muscle glucose uptake, but impaired basal and insulin-stimulated glucose metabolism in isolated adipose cells. Ten weeks of exercise training normalized both the impaired glucose tolerance and adipose cell function present in the untrained streptozocin-treated rats. Low-dose streptozocin treatment of rats with appropriate selection of animals based on plasma glucose concentrations appears to be an excellent model of impaired glucose tolerance for studies of factors affecting insulin resistance and altered glucose metabolism.

Early macrophage infiltration in mice treated with low-dose streptozocin decreases islet superoxide dismutase levels: prevention by silica pretreatment.

It has been hypothesized that streptozocin (STZ) given in low doses for 5 consecutive days produces diabetes by induction of peroxidation phenomena similar to those induced by free radicals. Moreover, it has been demonstrated that macrophages are among the first to invade the pancreatic parenchyma and destroy islet B cells supposedly by the release of interleukin-1 that induces free radical formation. Superoxide dismutase (SOD) is a free radical scavenger present in cells, and islet B cells are known to have extremely low levels of this enzyme. Therefore, our aim was to observe SOD activity concomitantly with the appearance of intra-islet macrophages, in early diabetes induced by low-dose streptozocin (LDS). Silica-pretreated mice showed SOD values which were comparable to those found in control animals. In LDS-only-treated mice we found that SOD levels were decreased even after only 4 days from the last STZ injection and that it is at this time that the first 'recruited' macrophages appear in the islets. Moreover, the SOD levels found at this early stage (animals were still normoglycaemic and therefore not as yet diabetic) were similar to levels found by us in a previous work, in prediabetic Bio Breeding rats, thereby ascribing a crucial factor to the lowering of SOD levels even in LDS-induced diabetes.

Capillary area in early low-dose streptozocin-treated mice

It has been reported that vasoconstriction of intra-islet capillaries plays an important role in the initiation of the insulitis seen in the islets of Langerhans of diabetic animals. Nevertheless, only a few studies have concentrated on islet vessels. This led us to perform an experiment with the aim to compare the islet capillary area of normal untreated and multiple low-dose streptozocin (LDS) (40 mg/kg b.wt. i.p./5 days)-treated mice. In order to identify endothelial cells a method devised by Gomori, based on the fact that these cells present alkaline phosphatases on their surface, was used. Results revealed that in LDS-treated animals the capillary area per islet is significantly reduced when compared to the vascular area of controls (p less than 0.05). This could be due to a vasoconstriction phenomenon that occurs in the islet capillaries after the streptozocin administration and before the appearance of any inflammation. Our findings could demonstrate that vasoconstriction events are involved in initiation of the diabetic disease.

Fish Oil–Enriched Diet and Reduction of Low-Dose Streptozocin–Induced Hyperglycemia: Inhibition of Macrophage Activation

Repeated low doses of streptozocin (STZ; 40 mg/kg, 5 injections/day) induce hyperglycemia in certain strains of mice after a latency of 1 wk. Omega-3 polyunsaturated fatty acids (omega 3FA) have been reported to suppress immune processes by blockade of the cyclooxygenase pathway of arachidonic acid metabolism. We investigated the effects of diets high in omega 3FA on the development of diabetes in the low-dose STZ-induced diabetes (LDSTZ-D) model. Male C57BL/6J mice were on a fish oil diet (FOD) as a source of omega 3FA 8 wk before STZ injection. Controls received laboratory chow only or a coconut oil diet (COD). Blood glucose levels in FOD mice were reduced (12.5 vs. 28 mM for COD mice, P less than .001) 60 days after STZ injection with a diet in which 20% of the calories were from fish oil. In FOD mice, immunohistology showed reduced numbers of class II antigen-expressing cells in pancreatic islets followed by a decreased extent of insulitis. FOD significantly decreased the number of Fc receptor-negative dendritic cells in cytospin preparations of islets isolated from diabetic mice. Interleukin 1-like activity of peritoneal exudate cell supernatants isolated from mice on FOD was reduced. FOD did not improve insulin secretion of isolated islets from LDSTZ-D mice. These data indicate a beneficial effect of FOD on the immune component of the mouse LDSTZ-D model.