Chronic undernutrition accentuates insulin deficiency in rats with mild streptozocin-induced diabetes.

The effect of chronic undernutrition on coexisting diabetes mellitus was studied in pair-fed littermate rats with mild streptozocin-induced diabetes. They were either fed ad libitum or 50% food restricted for 9 wk. Undernourished diabetic rats, in which weight gain was reduced by 40%, had significantly higher glucose intolerance (mean +/- SE, fractional rate of glucose disappearance during glucose tolerance test [Kgtt] 1.77 +/- 0.16) than diabetic littermates fed ad libitum (2.33 +/- 0.21, P less than 0.05) or nondiabetic controls (3.8 +/- 0.7, P less than 0.01). The total area under the insulin response curve was significantly reduced in both groups of diabetic rats, but the undernourished group showed a 45% greater reduction in overall secretion than normally nourished diabetic littermates (21.3 +/- 2.7 vs. 39.4 +/- 6.3 nM.min in the diabetic group, P less than 0.01, and 65.7 +/- 6.1 nM.min in controls). There was also a marked reduction in first-phase insulin secretion in the undernourished rats (4.75 +/- 0.24 vs. 9.84 +/- 1.36 nM, P less than 0.05, and 14.3 +/- 1.8 nM, P less than 0.01, respectively, in normally nourished diabetic littermates and controls). After refeeding, a significant improvement occurred in Kgtt (to 2.67 +/- 0.24, P less than 0.01) and first-phase insulin secretion (to 9.69 +/- 1.65 nM, P less than 0.05). The postrefeeding values were not different from those in the normally nourished diabetic littermates, indicating that the effect was fully reversible and solely attributable to undernutrition rather than to enhanced beta-cell cytotoxicity from streptozocin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of streptozocin diabetes and diabetes treatment by islet transplantation on in vivo insulin signaling in rat heart.

The insulin signaling cascade was investigated in rat myocardium in vivo in the presence of streptozocin (STZ)-induced diabetes and after diabetes treatment by islet transplantation under the kidney capsule. The levels of insulin-stimulated tyrosine phosphorylation of the insulin receptor beta-subunit, insulin receptor substrate (IRS)-2, and p52(Shc) were increased in diabetic compared with control heart, whereas tyrosine phosphorylation of IRS-1 was unchanged. The amount of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) and the level of PI 3-kinase activity associated with IRS-2 were also elevated in diabetes, whereas no changes in IRS-1-associated PI 3-kinase were observed. Insulin-induced phosphorylation of Akt on Thr-308 was increased fivefold in diabetic heart, whereas Akt phosphorylation on Ser-473 was normal. In contrast with Akt phosphorylation, insulin-induced phosphorylation of glycogen synthase kinase (GSK)-3, a major cellular substrate of Akt, was markedly reduced in diabetes. In islet-transplanted rats, the majority of the alterations in insulin-signaling proteins found in diabetic rats were normalized, but insulin stimulation of IRS-2 tyrosine phosphorylation and association with PI 3-kinase was blunted. In conclusion, in the diabetic heart, 1) IRS-1, IRS-2, and p52(Shc) are differently altered, 2) the levels of Akt phosphorylation on Ser-473 and Thr-308, respectively, are not coordinately regulated, and 3) the increased activity of proximal-signaling proteins (i.e., IRS-2 and PI 3-kinase) is not propagated distally to GSK-3. Islet transplantation under the kidney capsule is a potentially effective therapy to correct several diabetes-induced abnormalities of insulin signaling in cardiac muscle but does not restore the responsiveness of all signaling reactions to insulin.     

Composition changes in hepatic microsomal cytochrome P-450 during onset of streptozocin-induced diabetes and during insulin treatment

Monospecific polyclonal antibodies to five constitutive hepatic microsomal cytochromes P-450 were prepared. These antibodies were used to monitor alterations in the content of the enzymes in livers of diabetic male rats. Within 3 wk of onset of streptozocin-induced diabetes, immunodetectable levels of RLM3 and RLM5 were decreased by 85 and greater than 95%, respectively. Insulin treatment for 1 wk reversed the decline in these isozymes and restored RLM3 to 60% and RLM5 to 53% of levels found in the untreated rat. After a 2nd wk of therapy, these levels were returned to 86 and 92%, respectively. In contrast, the levels of RLM5b and RLM6 were elevated in diabetes 1.7- and 8-fold, respectively. Insulin treatment for 1 wk only slightly decreased the levels of RLM5b but completely reduced RLM6 levels to those seen in age-matched untreated rats. After the 2nd wk of insulin treatment, the level of RLM5b was almost completely restored to normal, with no additional change in the RLM6 level. The level of a fifth enzyme, RLM5a, was not markedly altered by diabetes or by insulin treatment. The results suggest there are at least three types of responses by constituents of the cytochrome P-450 population to diabetes: no change in the microsomal content, a rapid increase when insulin level declines and restoration when insulin is supplied, and a rapid decline when insulin level declines and a restoration by insulin treatment.     

Abnormal insulin secretion in a streptozocin model of diabetes. Effects of insulin treatment

We have proposed that chronic hyperglycemia causes the abnormal glucose influence on arginine-stimulated insulin secretion in the neonatal streptozocin (STZ) rat model of NIDDM and therefore studied the effect of 24 h of mild insulin-induced hypoglycemia on this defect. Ultralente insulin, 20 U/kg, was given at 9 a.m. and 10 U/kg at 5 p.m., and insulin and glucagon secretion were then studied the next morning using the in vitro isolated, perfused pancreas. The fed plasma glucose concentrations decreased in the STZ rats from 191 +/- 13 to 101 +/- 9 mg/dl and from 133 +/- 4 to 99 +/- 8 mg/dl in the controls. As expected, 10 mM arginine caused a trivial insulin response at 2.8 mM glucose in the treated and untreated control groups compared with the marked one at 16.7 mM. The response to arginine at 2.8 mM glucose in the untreated STZ rats, however, was strikingly elevated (7.65 +/- 2.29 versus 0.41 +/- 0.16 ng/ml in the untreated controls) and it was not potentiated by the high glucose background, but the result at 2.8 mM glucose in the treated STZ rats was similar to that of the treated controls (0.46 +/- 0.12 versus 0.16 +/- 0.03 ng/ml). A return of glucose influence on IBMX-stimulated insulin secretion was also noted. Glucose-induced insulin release, however, was not restored in the treated STZ rats, but it was markedly suppressed in the controls by the insulin treatment. Glucose influence on the glucagon response to arginine was maintained in the STZ model even though the glucagon release to a lowered glucose concentration was lost.(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-term effects of vanadyl treatment on streptozocin-induced diabetes in rats

The vanadate and vanadyl forms of vanadium have been shown by many investigators to have insulinlike effects on glucose metabolism. Many investigators have shown that vanadium, or its salts, counteracts the hyperglycemia associated with streptozocin-induced diabetes (STZ-D) in the rat, although insulin secretion remains depressed. Studies of the action of vanadate on insulin secretion and glucose metabolism have not addressed the question of possible long-term effects of this compound on glucose metabolism extending beyond the period of oral administration. This study was undertaken to assess the effects of treatment (3 wk) and withdrawal of vanadyl sulfate (13 wk) on glucose metabolism, insulin secretion, and islet insulin content of STZ-D rats. Our results indicate that STZ-D rats that have had blood glucose levels normalized by 3 wk of vanadyl treatment remain normoglycemic after 13 wk of withdrawal from treatment. Normal glucose tolerance was observed in vanadyl-treated diabetic animals despite depressed fasting and glucose-stimulated plasma insulin levels. Insulin secretion from the isolated perfused pancreas was greater after vanadyl treatment than in untreated diabetic rats, although it was only 12% of values from controls. Three weeks of vanadyl treatment of STZ-D rats, followed by 13 wk of withdrawal, yielded islets close in size and insulin content of control islets, even though in vivo and in vitro insulin secretion was impaired. This study has shown that short-term vanadyl treatment of STZ-D rats yields normalization of glucose tolerance and protection of islets from destruction by STZ.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Effects of streptozocin-induced diabetes and food restriction on quantities and source of T4 and T3 in rat tissues.

Diabetes mellitus and fasting are both associated with low plasma thyroid hormone concentrations and loss of body weight. To discriminate between the separate effects of energy shortage and insulin, we studied control rats, diabetic rats (DM), DM rats treated with insulin (DMI), and rats after modified fasting (MF1 and MF2; 70 and 30% of normal daily food intake, respectively). In double-isotopic equilibrium experiments, we determined the tissue thyroxine (T4) and triiodothyronine (T3) concentrations and the contribution of local T4-to-T3 conversion to total T3 in rat tissues; thyroidal T4 and T3 secretion and extrathyroidal T3 production were calculated. In DM and DMI rats, plasma T4 and T3 decreased; in MF1 and MF2 rats, only plasma T4 decreased. Thyroidal T4 secretion decreased, whereas that of T3 remained normal. The decrease in tissue T4 in MF and DM rats paralleled the decrease in plasma T4. Although plasma T3 did not differ in DM and DMI rats, total T3 concentrations in all tissues were not the same due to changed uptake of T3 from plasma and local T4-to-T3 conversion; these changes were not found in several tissues of MF1 and MF2 rats. Our results suggest that the decrease in tissue T4 during diabetes mellitus is due to the decrease in plasma T4 caused by the decreased thyroidal secretion, possibly due to intracellular energy shortage. The changes in tissue T3 during diabetes mellitus are only partly attributable to the same phenomenon; in several tissues, the decrease in T3 seems more related to the lack of insulin.     

Deficient axonal transport of substance P in streptozocin-induced diabetic rats. Effects of sorbinil and insulin

This study measured the accumulation of substance P-like immunoreactivity (SPLI) proximal and distal to 12-h constricting ligatures applied to rat sciatic nerves. There were three separate experiments, and the baseline for each consisted of control and age-matched rats with 3 wk of untreated streptozocin-induced diabetes. We compared the effects of twice-daily insulin treatment, daily sorbinil (25 mg.kg-1.day-1 p.o.), and a combination of both treatments. In untreated diabetic rats the anterograde accumulation of SPLI was reduced by 30-40%. This deficit was unaffected by sorbinil alone but was attenuated by insulin and prevented completely by insulin and sorbinil combined. There were also indications that diabetes caused reductions in retrograde accumulation of SPLI and its content in unconstricted nerve and the L4 dorsal root ganglion. The fraction of SPLI undergoing net anterograde or retrograde movement and the velocities of accumulation were unaffected by diabetes or the treatment regimens. These findings indicate a reduction in the amount of substance P moved by axonal transport in diabetic rats that is related partly to aldose reductase activity and partly to some other insulin-correctable consequence of experimental diabetes.     

Marked depletion of plasma 1,5-anhydroglucitol, a major polyol, in streptozocin-induced diabetes in rats and the effect of insulin treatment

Plasma levels of 1,5-anhydroglucitol (1,5AG), a major polyol resembling glucose in structure, fell rapidly and dramatically in streptozocin (STZ)-treated rats. 1,5AG fell immediately after STZ injection, reaching a plasma level 6 h after administration of the drug that was one-third that in the plasma of control rats. Reduction of 1,5AG was independent of the profile of blood glucose induced by STZ. After intravenous injection of [14C]-1,5AG, its plasma half-life was determined to be between 120 and 180 min. After a phase of acute decrease, the reduction of 1,5AG became gradual, stopping within 6 days after treatment. However, in some cases, the drop in 1,5AG was partially reversible by insulin treatment. The extent to which 1,5AG fell did not strictly correspond to the dose of STZ. The particular organ(s) consuming or accumulating 1,5AG was not identified. However, aside from the large amount of 1,5AG in plasma and the small amount of 1,5AG in the urine, the liver appears to be a significant organ for metabolism of 1,5AG.     

Oxygen free radical regulation of rat splanchnic blood flow.

BACKGROUND. The role of oxygen-derived free radicals on splanchnic prostaglandin (PGI2) synthesis and superior mesenteric artery (SMA) blood flow was examined during acute hemorrhage. METHODS. Sprague-Dawley rats were hemorrhaged to 30 mm Hg for 5, 30, or 45 minutes without (SK5, SK30, and SK45) or with (SK5 + R, SK30 + R, and SK45 + R) blood reperfusion. The SK30 + R and SK45 + R groups were treated with or without superoxide dismutase (10,000 units/kg intravenously). In vivo SMA blood flow was measured continuously for 100 minutes by a transonic flow probe, or in other groups the in vitro-perfused SMA and its end-organ intestine (SV+SI) were assayed for release of PGI2 by radioimmunoassay after 15 and 30 minutes of perfusion. RESULTS. Acute hemorrhage at all time periods increased SV+SI PGI2 release twofold to threefold compared with sham animals (p less than 0.01), which was abolished after blood reperfusion. SMA blood flow was decreased by 79.6% +/- 3.3% and 88.2% +/- 1.4% in the SK30 + R and SK45 + R groups compared with the sham animals (p less than 0.01). Superoxide dismutase treatment restored both SV+SI release of PGI2 after SK and SMA blood flow to control levels. CONCLUSION. Oxygen-derived free radicals locally produced during SK30 + R and SK45 + R inhibited splanchnic PGI2 synthesis, which contributed to decreased splanchnic blood flow.     

Low-dose streptozocin-induced diabetes in mice. Electron microscopy reveals single-cell insulitis before diabetes onset

We investigated the morphology of mouse islets 5 days after completion of low-dose streptozocin treatment of C57BL/6 mice by electron microscopy. At this stage, mice were still normoglycemic and light microscopy did not reveal massive islet infiltration. The electron-microscopic investigation revealed two characteristics indicative of ongoing islet cell destruction. In all islets investigated, lysed islet beta-cells were recognized by disrupted plasma membranes and concomitantly decreased plasma contrast. Many of the lysed islet beta-cells still contained numerous insulin granules. We also found immunocytes scattered throughout the islets, most of which could be identified as macrophages. Some were found engaged in phagocytosis of islet beta-cell debris. This early stage of islet lesion termed single-cell insulitis is followed by the well-known later stage of massive infiltration easily recognized in light microscopy. Administration of silica particles to mice treated with low-dose streptozocin inhibited macrophage infiltration of islets as shown by immunocytochemistry with macrophage-specific monoclonal antibody F4/80. In parallel, the development of hyperglycemia was suppressed. The observations favor a pathogenic role of macrophages in islet destruction.     

Starvation-related alterations in free radical tissue defense mechanisms in rats

Alterations in endogenous free radical-scavenging defense mechanisms of rat tissues after body weight loss (induced by starvation for 72 h) associated with hypoinsulinemia were investigated. The activities of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and glutathione (GSSG) reductase as well as levels of reduced glutathione (GSH) were examined in several tissues and in erythrocytes. A complex pattern of changes was observed. CAT activities were increased in the heart and pancreas and decreased in the liver. SOD levels were decreased in the heart and increased in the kidney and pancreas. GSH-PX activities were increased only in the kidney, and levels of GSH were decreased only in the liver of starved animals. Erythrocytes from starved animals showed no alterations in the levels of major free radical-scavenging enzymes. However, GSSG reductase levels were lower in erythrocytes from starved animals, and this was associated with an increased susceptibility to H2O2-induced GSH depletion. Paradoxically, H2O2-induced malondialdehyde (MDA) production in erythrocytes from starved animals was lower than that in control erythrocytes. Our results suggest that, in studies of experimental diabetes, attention must be given to the influence of body weight loss per se on the biochemical alterations associated with this disease.     

Effects of insulin and myo-inositol on embryo growth and development during early organogenesis in streptozocin-induced diabetic rats.

We have previously shown that myo-inositol depletion in the embryonic tissue at a critical stage of organogenesis has a crucial role in hyperglycemia-induced embryopathy. This study tested whether myo-inositol depletion in early organogenesis contributes to the pathogenesis of streptozocin-induced diabetic embryopathy. Rats were made diabetic by streptozocin administration before conception, and the diabetic rats were treated with diet supplemented by 2% myo-inositol or insulin from 6 to 11 gestational days during the period of maximum teratological susceptibility. In each group on the 11th gestational day, growth retardation and incidence of malformations were recorded, and myo-inositol and sorbitol content in the embryonic and extraembryonic tissues were examined. In diabetic rats, the myo-inositol content of the embryos was decreased by 36% (P less than 0.01) compared with control rats, and there was growth retardation (crown-rump length 3.37 +/- 0.04 vs. 3.87 +/- 0.03 mm, P less than 0.01; somite no. 27.5 +/- 0.2 vs. 29.1 +/- 0.2, P less than 0.01) and a significantly increased incidence of the neural lesions (17.6 vs. 1.9%, P less than 0.01). Insulin treatment resulted in near normalization of maternal serum glucose and complete restoration of myo-inositol content in the embryos with significant improvement of the growth retardation (crown-rump length 3.55 +/- 0.06 vs. 3.37 +/- 0.04 mm, P less than 0.05; somite no. 28.2 +/- 0.13 vs. 27.5 +/- 0.2, P less than 0.05) and a significantly lowered incidence of neural lesions (2.5 vs. 17.6%, P less than 0.01) compared with those of the untreated diabetic rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cerebral taurine transport is increased during streptozocin-induced diabetes in rats.

Taurine is a cerebral osmolyte whose intracellular content changes in parallel with plasma osmolality. We conducted experiments to assess whether cerebral taurine transport is modified during chronic hyperglycemia. Rats with STZ-induced diabetes were studied after 1 wk of sustained hyperglycemia. Cerebral taurine uptake in synaptosomes (metabolically active nerve terminal vesicles) was measured using a rapid filtration technique. The synaptosomes were isolated by homogenization of the brain and purification on discontinuous Ficoll gradients (n = 8 synaptosome preparations). Diabetic rats (n = 13) displayed a 15-25% increase in synaptosomal taurine uptake compared with normoglycemic control animals (n = 12) at all time points assayed between 5 and 120 min. Thus, after a 30-min incubation, cerebral taurine uptake increased from a control level of 3.53 +/- 0.23 to 4.10 +/- 0.24 mumol/mg protein (n = 10) in hyperglycemic rats, P less than 0.03. The magnitude of the plasma-to-brain cell taurine gradient was unchanged in diabetic animals. The intrasynaptosomal taurine concentration (approximately 2 microM) and taurine efflux from the synaptosomes were no different in hyperglycemic versus control rats; efflux amounted to less than 2.5% of the uptake value at corresponding time points. Maximal brain taurine uptake under both control and experimental hyperglycemic conditions required the presence of external Na+ and Cl-. Synaptosomal taurine transport was reduced by competing beta-amino acids such as beta-alanine, beta-aminoisobutyric acid, and hypotaurine (P less than 0.01). Addition of oubain and the anionic binding site inhibitors, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid and 4,4'diisothio-cyanatostilbene-2,2'-disulfonic acid, also decreased cerebral taurine uptake under normoglycemic and hyperglycemic conditions (P less than 0.01). The increased synaptosomal taurine uptake by diabetic rats was not a result of generalized membrane dysfunction because glycine transport was not elevated in hyperglycemic rats. The enhanced transport rate was attributable to a 35 and 81% increase in the Vmax of the high- and low-affinity taurine transporters, respectively (P less than 0.01), without significant change in the Km of the carrier systems. Treatment of hyperglycemic rats (n = 5) with ultra-long-acting insulin to normalize the serum glucose concentration restored synaptosomal taurine uptake to the level observed in normoglycemic controls. The effect of insulin was attributable to correction of hyperglycemia, because addition of insulin (500 mU/ml) to the in vitro assay system did not alter synaptosomal taurine uptake.(ABSTRACT TRUNCATED AT 400 WORDS)     

Influence of diabetes mellitus and insulin treatment on protein turnover in the rat

We studied the effects of diabetes mellitus and insulin treatment on total body protein synthesis and catabolism in the rat. Protein turnover was measured by infusing [¹⁵N]glycine intravenously and analyzing urine for [¹⁵N]urea enrichment. Diabetes mellitus was induced with streptozotocin (50 mg/kg), and diabetic animals were studied with and without insulin replacement. Normal animals, fed and starved, were studied as controls. Diabetes mellitus significantly decreased protein synthesis but did not change catabolism as compared with fed controls. Starvation of normal animals did not alter the protein synthesis rate but significantly increased the rate of protein catabolism. Insulin treatment restored the protein synthesis rate to normal while decreasing the rate of protein catabolism below normal. These findings for total body synthesis and catabolism are consistent with earlier studies of the effects of diabetes mellitus and insulin on protein metabolism in specific organs. It appears that the major effect of the diabetic state on total body protein turnover is to decrease synthesis without significantly changing catabolism.     

An insulin sensitizer improves the free radical defense system potential and insulin sensitivity in high fructose-fed rats.

Recently there has been growing interest in the effects of antioxidants on insulin activity. In the present study, we investigated the effect of metformin on free radical activity and insulin sensitivity in high fructose-fed rats, a diet that leads to insulin resistance. The animals were divided into four groups (n = 16 per group; experiment duration = 6 weeks): the control (C) group received a standard diet; the control metformin (CM) group was fed a control diet and received metformin (200 mg x kg(-1) x day(-1) in water); the fructose control (FT) group was fed a diet in which fructose composed 56.8% of the total carbohydrates; and the fructose metformin (FM) group received high-fructose diet and metformin (200 mg x kg(-1) x day(-1) in water). The glucose clamp technique was used to determine insulin sensitivity in eight animals per group. Metabolic and oxidative stress parameters were measured in the remaining rats. In the FT rats, insulin resistance, lower red cell CuZn superoxide dismutase activity and lower blood reduced glutathione were observed. Metformin treatment improved both the insulin activity and the antioxidant defense system. In the CM group, metformin had no effect on metabolic parameters, but improved red cell antioxidant enzyme activities and the blood GSH level, which suggests that it has an antioxidant activity independent of its effect on insulin activity.