Insulin-treated Diabetic Animals Research Articles

Troxerutin affects nephropathy signaling events in the kidney of type-1 diabetic male rats.

Objective:Nephropathy is known to be the leading cause of kidney failure in diabetic patients. Troxerutin, as a flavonoid component, could provide a novel protective strategy in the prevention of diabetic nephropathy. A large number of reports on the salutary effects of troxerutin inspired us to investigate its effect on the nephropathy signaling events (i.e., expression of TGF-β, miRNA192, and SIP1) in type-1 induced diabetic rats.Materials and Methods:50 male Wistar rats were divided into 5 groups including control group, sham group treated with troxerutin for 4 weeks, diabetic group induced by streptozotocin (STZ) injection, DI group including insulin-treated diabetic animals and DT group treated with troxerutin. Ultimately, rat kidneys were extracted, and the level of miR-192 (using qPCR), transforming growth factor-beta (TGF-β), and smad interacting protein 1 (SIP1) using an ELISA kit, was measured.Results:The level of TGF-β and miRNA192 significantly increased in the diabetic group. However, their expression levels decreased following the administration of troxerutin and insulin (p<0.05) compared to control group. SIP1 was down-regulated in the diabetic group, whereas a spike in the expression levels was observed after troxerutin administration compared to control and troxerutin groups (p<0.05). However, no significant difference was found in the effects of insulin and troxerutin on the level of miR-192, SIP1, and TGF- β. Conclusion:According to the previous literatures, during the progression of nephropathy, TGF-β represses SIP1 (the repression region in the collagen gene) by increasing the expression of miR-192. Ultimately, in this study, diabetes led to up-regulation of TGF-β while troxerutin proved to have a protective effect on the kidney by increasing SIP and lowering miR-192 levels.

Insulin Treatment Reduces Susceptibility to Atrial Fibrillation in Type 1 Diabetic Mice.

Diabetes has been identified as an independent risk factor for atrial fibrillation (AF), the most common chronic cardiac arrhythmia. Whether or not glucose and insulin disturbances observed during diabetes enhance arrhythmogenicity of the atria, potentially leading to AF, is not well-known. We hypothesized that insulin deficiency and impaired glucose transport provide a metabolic substrate for the development and maintenance of AF during diabetes. Transesophageal atrial pacing was used to induce AF in healthy, streptozotocin-induced insulin-deficient type 1 diabetic, and insulin-treated diabetic mice. Translocation of insulin-sensitive glucose transporters (GLUTs) to the atrial cell surface was measured using a biotinylated photolabeling assay in the perfused heart. Fibrosis and glycogen accumulation in the atrium were measured using histological analysis. Diabetic mice displayed mild hyperglycemia, increased duration and frequency of AF episodes vs. age-matched controls (e.g., AF duration: 19.7 ± 6.8 s vs. 1.8 ± 1.1 s, respectively, p = 0.032), whereas insulin-treated diabetic animals did not. The translocation of insulin-sensitive GLUT-4 and -8 to the atrial cell surface was significantly downregulated in the diabetic mice (by 67 and 79%, respectively; p ≤ 0.001), and rescued by insulin treatment. We did not observe fibrosis or glycogen accumulation in the atria of diabetic mice. Therefore, these data suggest that insulin and glucose disturbances were sufficient to induce AF susceptibility during mild diabetes.

Volumetric investigation of the hippocampus in rat offspring due to diabetes in pregnancy–A stereological study

BackgroundThe brain development during the prenatal period is affected by various factors, including the mother's metabolic condition. It has been revealed that diabetes in pregnancy is associated with structural and functional alterations in offspring’s hippocampus. Hippocampus, as a critical region with well-known roles in learning and memory consolidation, is vulnerable to changes in glucose level. This study was designed to investigate the effects of maternal diabetes during the pregnancy period and insulin therapy on the neuronal density and the volume of different subfields of the hippocampus in rat offspring at postnatal day 14 (P14). MethodsWistar female rats were randomly divided into diabetics (STZ-D), diabetes treated with insulin (STZ-INS) group, and controls (CON). The animals in all groups were mated by non-diabetic male rats. Two weeks after birth, male pups from each group were sacrificed. The Cavalieri method was carried out to estimate the total volume, and the numerical density of the neurons in the hippocampus and its sub regions was measured by the optical dissector technique. ResultsBilateral hippocampal volume decreased in the diabetic group, mainly in the CA1, dentate gyrus (DG) and subiculum areas (P ≤ 0.05), when compared to control and insulin-treated diabetic animals. In all hippocampus sub-regions, maternal diabetes resulted in a significant decrease in the number of cells in comparison with two other groups (P ≤ 0.05 each). ConclusionThese data indicate that diabetes during pregnancy has a negative impact on the development of the hippocampus in the rats. These changes in the volume of hippocampal CA1, DG, and subiculum areas might be at the core of underlying neurocognitive and neurobehavioral impairments observed in the children of diabetic mothers.

Insulin Deficiency Is a Major Contributor to Atrial Fibrillation

Diabetes has been identified as a major independent risk factor for atrial fibrillation (AF), the most common sustained cardiac arrhythmia. However, it is unknown whether glucose and insulin disturbances observed during diabetes directly affect the function of the atria, leading to AF. Thus, we hypothesized that insulin deficiency and impaired glucose transport provide a metabolic substrate for the development and maintenance of AF during diabetes. Transesophageal atrial pacing was used to induce AF in healthy, streptozotocin-induced type 1 diabetic and insulin-treated (via a subcutaneous pump) diabetic mice (n=4-11/group). Translocation of insulin-sensitive glucose transporters (GLUTs) was measured using a photolabeled biotinylation assay in the perfused heart. Atrial fibrosis and glycogen accumulation were measured using histological analyses. As expected, insulin-deficient diabetic mice displayed hyperglycemia (434 ± 50.9 mg/dl) while insulin-treated diabetic mice remained euglycemic (118.8 ± 3.3 mg/dl, p&amp;lt;0.05). At 8 weeks, diabetic mice displayed increased duration and frequency of AF episodes vs. age-matched controls (e.g., AF duration: 18.7 ± 6.2 s vs. 1.8 ± 1.1 s, respectively, p=0.032), whereas insulin-treated diabetic animals did not. In addition, the frequency of AF episodes was significantly correlated to blood glucose level (p=0.01). GLUT-4 and -8 translocation to the atrial cell surface was significantly downregulated in diabetic mice (by 67% and 79%, respectively; p≤0.001), and rescued by insulin treatment. We did not observe any fibrosis or glycogen accumulation in the atria of (treated and untreated) diabetic mice. We conclude that, in the absence of structural remodeling and atrial fibrosis, insulin and glucose disturbances were sufficient to induce AF during diabetes. Therefore, these data suggest that insulin deficiency during diabetes leads to alterations in glucose transport and energy production in the atria, which enhances its vulnerability to atrial fibrillation. Disclosure Z. Maria: None. A. Campolo: None. B.J. Scherlag: None. J.W. Ritchey: None. V. Lacombe: None.

Automatic Diabetes Detection from Histological Images of Rats Phrenic Nerve Using Two-Dimensional Sample Entropy

In microscopy, morphological characteristic of the axon are the most common features assessed in histological images of nerves. Although morphometric indexes are widely used to describe histological data, the calculation of those indexes is a highly time-consuming task that demands great manual effort from the specialist. Recently, two-dimensional sample entropy (SampEn2D) was proposed to quantify the degree of irregularity present in an image, based on the spatial patterns of pixels. Here, we propose the use of SampEn2D as a suitable metric for classifying diabetic status of rats from histological images of the phrenic nerve. Microscopy images of three different Wistar rats groups (untreated diabetic (N = 24), insulin-treated diabetic (N = 9), and non-diabetic control (N = 11)) were analysed. The results show that for the optimal SampEn2D parameters (m = 1, r = 0.1), control rats have significantly (p < 0.01) lower entropy (3.76 ± 0.26) as compared to both insulin-treated (5.09 ± 0.20) and untreated (5.30 ± 0.13) diabetic animals. Performance of SampEn2D for image classification between untreated and control groups was assessed by ROC analysis and area under the ROC curves (AUROC = 0.96). SampEn2D reaches a sensitivity of 87% and specificity of 82% when a threshold of SampEn2D = 4.73 is taken to separate the two assessed groups. In conclusion, SampEn2D method arises as a useful tool in the screening of diabetic rats. The method may be useful to pre-select animals for further morphometric evaluation, reducing the burden of manual processing.

Insulin prevents aberrant mitochondrial phenotype in sensory neurons of type 1 diabetic rats

Diabetic neuropathy affects approximately 50% of diabetic patients. Down-regulation of mitochondrial gene expression and function has been reported in both human tissues and in dorsal root ganglia (DRG) from animal models of type 1 and type 2 diabetes. We hypothesized that loss of direct insulin signaling in diabetes contributes to loss of mitochondrial function in DRG neurons and to development of neuropathy. Sensory neurons obtained from age-matched adult control or streptozotocin (STZ)-induced type 1 diabetic rats were cultured with or without insulin before determining mitochondrial respiration and expression of mitochondrial respiratory chain and insulin signaling-linked proteins. For in vivo studies age-matched control rats and diabetic rats with or without trace insulin supplementation were maintained for 5months before DRG were analyzed for respiratory chain gene expression and cytochrome c oxidase activity. Insulin (10nM) significantly (P<0.05) increased phosphorylation of Akt and P70S6K by 4-fold and neurite outgrowth by 2-fold in DRG cultures derived from adult control rats. Insulin also augmented the levels of selective mitochondrial respiratory chain proteins and mitochondrial bioenergetics parameters in DRG cultures from control and diabetic rats, with spare respiratory capacity increased by up to 3-fold (P<0.05). Insulin-treated diabetic animals exhibited improved thermal sensitivity in the hind paw and had increased dermal nerve density compared to untreated diabetic rats, despite no effect on blood glucose levels. In DRG of diabetic rats there was suppressed expression of mitochondrial respiratory chain proteins and cytochrome c oxidase activity that was corrected by insulin therapy. Insulin elevates mitochondrial respiratory chain protein expression and function in sensory neurons and this is associated with enhanced neurite outgrowth and protection against indices of neuropathy.

Tissue specific oxidative stress profile in relation to glycaemic regulation in mice

Diabetes mellitus is a metabolic disorder characterized by hyperglycaemia resulting from uncontrolled glucose regulation. Reactive oxygen species are recognized as one link between hyperglycaemia and diabetic complications. Studies have shown that diabetes mellitus is associated with decreases in antioxidant potential and increased formation of free radicals leading to oxidative stress. The present study was undertaken because an unequivocal demonstration that control of hyperglycaemia can reduce oxidative stress is still lacking. In the present study, we investigated oxidative stress profile of normal, streptozotocin-induced diabetic, insulin-treated and untreated diabetic animals. On the one hand, oxidative damage caused to lipids, proteins and DNA was measured. On other hand, antioxidant defense was measured in terms of specific activities of antioxidant enzymes (AOEs) and antioxidant molecules. It was observed that the damage to lipids, proteins and DNA caused by free radicals increased in diabetic animals compared with that in controls. In diabetic animals not treated with insulin, damage to all biological molecules increased further significantly (p ≤ 0.005). Changes in AOEs from different tissues were complex depicting a varied AOE level in different tissues. Insulin treatment significantly improved the oxidative stress profile in all tissues studies. The control of hyperglycaemia improves oxidative stress profile, that is, the ability of cells to cope up with oxidative stress.

Early-Stage Retinal Melatonin Synthesis Impairment in Streptozotocin-Induced Diabetic Wistar Rats

Retinal melatonin synthesis occurs in the photoreceptor layer in a circadian manner, controlling several physiologic rhythmic phenomena, besides being the most powerful natural free radical scavenger. The purpose of the present work was to evaluate the diurnal profile of retinal melatonin content and the regulation of its synthesis in the retina of streptozotocin-induced diabetic rats. Diabetes was induced in male Wistar rats (12 hour-12 hour light/dark cycle) with streptozotocin. Control, diabetic, and insulin-treated diabetic animals were killed every 3 hours throughout the light-dark cycle. Retinal melatonin content was measured by high-performance liquid chromatography, arylalkylamine N-acetyltransferase (AANAT) activity was analyzed by radiometric assay, Bmal1 gene expression was determined by qPCR, and cyclic adenosine monophosphate (cAMP) content was assessed by ELISA. Control animals showed a clear retinal melatonin and AANAT activity daily rhythm, with high levels in the dark. Diabetic rats had both parameters reduced, and the impairment was prevented by immediate insulin treatment. In addition, the Bmal1 expression profile was lost in the diabetic group, and the retinal cAMP level was reduced 6 hours after lights on and 3 hours after lights off. The present work shows a melatonin synthesis reduction in diabetic rats retinas associated with a reduction in AANAT activity that was prevented by insulin treatment. The Bmal1-flattened gene expression and the cAMP reduction seem to be responsible for the AANAT activity decrease in diabetic animals. The melatonin synthesis reduction observed in the pineal gland of diabetic rats is also observed in a local melatonin tissue synthesizer, the retina.

The effect of long-term insulin treatment with and without antecedent hypoglycemia on neuropeptide and corticosteroid receptor expression in the brains of diabetic rats

We previously demonstrated that while diabetic animals receiving long-term insulin treatment exhibited some impairment in their corticosterone response to hypoglycemia, the stress response to hypoglycemia was completely absent when these animals were subjected to recurrent hypoglycemia. In the current study, we examined potential mechanisms that may contribute to defects in the adrenocortical response to hypoglycemia in long-term insulin-treated diabetic animals exposed to antecedent hypoglycemia. Whereas insulin-treated diabetic animals exhibited a significant rise in corticotrophin-releasing hormone (CRH) mRNA levels during hypoglycemia, exposure to antecedent hypoglycemia completely abolished this response. Moreover, expression of hippocampal mineralocorticoid receptors (MR) mRNA, which normally act to suppress hypothalamo–pituitary–adrenal activity, decreased in the normal control and insulin-treated diabetic groups in response to hypoglycemia, whereas MR mRNA levels remained at baseline in animals subjected to antecedent hypoglycemia. Interestingly, hippocampal glucocorticoid receptor (GR) mRNA levels decreased in all three treatment groups following the hypoglycemic clamp. While GR mRNA levels in the paraventricular nucleus were lower in normal controls following hypoglycemia, this trend just failed to reach statistical significance in the two diabetic groups. These data suggest that (1) recurrent hypoglycemia, much like uncontrolled diabetes, has a pronounced effect on hippocampal mineralocorticoid receptor mRNA expression that may prevent it, and presumably also the stress axis, from responding properly to a subsequent bout of hypoglycemia, and (2) while long-term insulin treatment was sufficient to restore some of these responses in diabetic animals, tighter glycemic control may be necessary to see full restoration of the stress response.

Influence of diabetes on liver injury induced by antitubercular drugs and on silymarin hepatoprotection in rats

Isoniazid, rifampicin and pyrazinamide during short-course chemotherapy for tuberculosis can result in liver injury. The coexistence of tuberculosis and diabetes is common in patients who receive inadequate treatment. The risk of hepatotoxicity from many toxicants is increased in diabetic rats. Silymarin provides protection against liver injury caused by many hepatotoxicants, including antitubercular drugs (ATDs). In the wake of increased severity of ATD-induced hepatotoxicity in diabetes we report here the results of a study on the influence of diabetes on silymarin hepatoprotection in rats. Rats with diabetes induced via intraperitoneally injected streptozotocin (50 mg/kg), nondiabetic rats and insulin-treated diabetic rats received isoniazid (7.5 mg/kg/day), rifampicin (10 mg/kg/day) and pyrazinamide (35 mg/kg/day) orally (p.o.) with or without silymarin (100 mg/kg/day p.o.) treatment for 45 days. Compared to nondiabetic rats, liver function tests and histological changes of liver revealed exaggerated liver injury in diabetic rats caused by ATDs which was evident by 5- to 8-fold increases in serum levels of marker enzymes (aspartate and alanine aminotransferase, alkaline phosphatase and gamma-glutamyltranspeptidase) and 1- to 2-fold increases in bilirubin accompanied by a 2-fold decrease in total serum proteins, intense fatty and inflammatory infiltrations, necrosis and fibrosis. Coadministration of silymarin provided protection against ATD hepatotoxicity in all animals. However, insulin-treated diabetic animals showed greater silymarin-induced hepatoprotection against ATD-induced liver injury, which was characterized by near normal levels of marker enzymes, an increase in total proteins and normal hepatic structure. These results thus indicate that diabetes exaggerates ATD-induced liver injury and attenuates silymarin-induced hepatoprotection. However, insulin treatment for diabetes offers greater silymarin-induced hepatoprotection against ATD-induced liver injury.

Runt-related transcription factor 2 (RUNX2) and RUNX2-related osteogenic genes are down-regulated throughout osteogenesis in type 1 diabetes mellitus.

Type 1 diabetes mellitus is associated with a number of disorders of skeletal health, conditions that rely, in part, on dynamic bone formation. A mouse model of distraction osteogenesis was used to study the consequences of streptozotocin-induced diabetes and insulin treatment on bone formation and osteoblastogenesis. In diabetic mice compared with control mice, new bone formation was decreased, and adipogenesis was increased in and around, respectively, the distraction gaps. Although insulin treatment restored bone formation to levels observed in nondiabetic control mice, it failed to significantly decrease adipogenesis. Molecular events altered during de novo bone formation in untreated type 1 diabetes mellitus, yet restored with insulin treatment were examined so as to clarify specific osteogenic genes that may contribute to diabetic bone disease. RNA from distraction gaps was analyzed by gene microarray and quantitative RT-PCR for osteogenic genes of interest. Runt-related transcription factor 2 (RUNX2), and several RUNX2 target genes, including matrix metalloproteinase-9, Akp2, integrin binding sialoprotein, Dmp1, Col1a2, Phex, Vdr, osteocalcin, and osterix, were all significantly down-regulated in the insulin-deficient, hyperglycemic diabetic animals; however, insulin treatment of diabetic animals significantly restored their expression. Expression of bone morphogenic protein-2, transcriptional coactivator with PDZ-binding motif, and TWIST2, all important regulators of RUNX2, were not impacted by the diabetic condition, suggesting that the defect in osteogenesis resides at the level of RUNX2 expression and its activity. Together, these data demonstrate that insulin and/or glycemic status can regulate osteogenesis in vivo, and systemic insulin therapy can, in large part, rescue the diabetic bone phenotype at the tissue and molecular level.

Loss of capsaicin-induced meningeal neurogenic sensory vasodilatation in diabetic rats

Neuropathic alterations of sensory nerves involved in the mediation of neurogenic inflammation of the meninges may contribute to the increased incidence of headaches in diabetics. In the rat, activation of capsaicin-sensitive nociceptors, which express the transient receptor potential vanilloid type 1 (TRPV1) receptor, induces meningeal vasodilatation, a significant component of neurogenic inflammation, through the release of calcitonin gene-related peptide (CGRP). This study examines the effects of streptozotocin-induced diabetes on TRPV1 receptor-mediated neurogenic sensory vasodilatation, CGRP release and nerve fiber density in the rat dura mater. In a cranial window preparation, epidural application of capsaicin (10 −7 M) produced distinct vasodilatory responses in control animals as measured by laser Doppler flowmetry. In diabetic rats, capsaicin-induced vasodilatation was reduced or even abolished 6, but not 2 or 4 weeks after diabetes induction. In contrast, vasoconstriction, a non-neurogenic response to capsaicin at a higher concentration (10 −5 M), was not altered in diabetic rats. The vasodilatory effects of histamine (10 −5 M), acetylcholine (10 −4 M) and CGRP (10 −5 M) were similar in control, diabetic and insulin-treated diabetic animals. In diabetic rats, a significant decrease in the capsaicin-evoked release of CGRP and reduction in the density of TRPV1-immunoreactive (IR) nerves were demonstrated. Treatment of the diabetic rats with insulin restored both the vasodilatory response and the capsaicin-induced CGRP release toward control values. In conclusion, this study revealed a marked impairment of meningeal TRPV1-IR nerves in streptozotocin diabetic rats by showing reduced neurogenic sensory vasodilatation, decreased capsaicin-evoked CGRP release and reduction in the number of TRPV1-IR nerve fibers of the dura mater. The findings suggest that capsaicin-sensitive afferents may play an important role in meningeal nociceptor function and their dysfunction, e.g. due to a limited removal of inflammatory mediators and/or tissue metabolites from the meningeal tissue, may contribute to the enhanced incidence of headaches in diabetics.

Nitric oxide restores impaired healing in normoglycaemic diabetic rats

Hyperglycaemia impairs wound healing. However, little is known about the underlying cellular mechanisms that lead to diminished wound repair in insulin-controlled and non-insulin-controlled diabetes. This study investigated the role of endogenous and exogenous nitric oxide on incisional wound healing in diabetic rats. Groups of 10 wild-typeWistar control rats - 10 genetically diabetic BioBreeding rats and 10 genetically diabetic BioBreeding rats treated with subcutaneous insulin implants to render them normoglycaemic - underwent dorsal skin incision followed by subcutaneous insertion of polyvinyl alcohol sponges. The rats were sacrificed 10 days later to determine the wound-breaking strength and reparative collagen deposition. Nitric oxide, an important mediator in diabetic wound healing and collagen synthesis, was measured in wound fluid. Wound-derived fibroblasts were tested for ex vivo synthesis of nitric oxide and collagen. Exogenous nitric oxide was used for the therapeutic interventions. Wound-breaking strength and wound collagen deposition were significantly impaired in the hyperglycaemic diabetic animals (p<0.01). Wound nitric-oxide synthesis and ex vivo wound fibroblast nitric-oxide production were reduced in the hyperglycaemic rats (p<0.01). Insulin treatment partially reversed some of the effects of hyperglycaemia on wound repair (p<0.05). Exogenous nitric oxide further restored wound mechanical strength, collagen deposition and fibroblast collagen synthesis (p<0.01) in insulin-treated (normoglycaemic) diabetic animals. Wound healing is impaired in hyperglycaemic and normoglycaemic diabetic rats. This is reflected in impaired wound fibroblast nitric-oxide synthesis. Used in combination with insulin, exogenous nitric oxide further improves healing outcomes, making it a potential target for therapeutic intervention in insulin-treated normoglycaemic diabetes.

Insulin-status-dependent modulation of FoF1 ATPase activity in rat kidney mitochondria

The early and late effects of alloxan-diabetes and insulin treatment on kinetic properties of mitochondrial FoF1 ATPase were examined. Diabetic state resulted in significant decrease in the activity while insulin treatment caused hyper-stimulation. In control animals the enzyme activity resolved in three kinetic components. In diabetic condition only component I and II were present. With insulin treatment component III was restored but component II was abolished. Diabetic state and insulin treatment had varied effects on Km values of the three components, whereas the Vmax values were generally on the higher side. Evaluation of the AppKcat/Km values revealed that diabetic state resulted in increased catalytic efficiency; insulin treatment brought back these values to normality. Temperature kinetics studies indicated that the phase transition temperature decreased significantly in the diabetic and insulin-treated diabetic animals. The energy of activation in low temperature range increased in the diabetic animals. Insulin treatment corrected the Arrhenius pattern at early stage of diabetes; at late stage the pattern was reversed. The results are suggestive of subtle insulin-status-dependent alterations in membrane structure – function relationships.

Effect of streptozotocin‐induced diabetes on oxidative energy metabolism in rat kidney mitochondria. A comparative study of early and late effects

The effects of streptozotocin (STZ)-induced diabetes on oxidative energy metabolism of rat kidney mitochondria were examined at the end of 1 week and 1 month of STZ treatment. At the end of 1 week of induction of diabetes, respiration rates with pyruvate + malate and succinate as the substrates increased while those with beta-hydroxybutyrate and ascorbate + TMPD decreased. Respiration with glutamate was not affected. Insulin treatment had no alleviating effect. The changes persisted through 1 month of induction of diabetes and were not corrected by insulin treatment even at this stage. beta-hydroxybutyrate dehydrogenase activity registered significant decrease while the succinate dehydrogenase activity increased in diabetic and insulin-treated diabetic animals whereas only marginal changes were evident in the composition of the cytochromes. The ATPase activity tended to be high in the diabetic groups and was restored by insulin treatment. At both the stages, i.e. early and late stages of diabetes the mitochondria were tightly coupled and the ADP/O ratios were in normal expected ranges. Taken together, the results suggest that kidney is the major target tissue to suffer impairment of mitochondrial function with the onset of the disease which persists throughout and that insulin treatment is ineffective in restoring the normal state.

Insulin-dependent diabetes impairs the inflammatory response and delays angiogenesis following Achilles tendon injury.

Although impaired wound healing associated with type 1 diabetes mellitus has been well studied in skin tissue, the influence of this metabolic disorder on tendon healing and recovery has not been extensively investigated. Because tendons are known to have limited repair potential, we studied the tendon-healing process by using a diabetic rat tendonitis model. We tested the hypothesis that diabetes influences the inflammatory response, cell proliferation, and angiogenesis in injured Achilles tendons. Diabetes was induced by injecting streptozotocin at 45 mg/kg body wt. Non-diabetic rats as well as diabetic and insulin-treated diabetic animals were then injected with collagenase. The accumulation of inflammatory cells was quantified in transversal sections of Achilles tendon by using immunohistochemical staining at days 0, 1, 3, 7, 14, and 28 posttrauma. The number of proliferative cells and the extent of neovascularization was also quantified in the paratenon and the core of the tendon at days 0, 3, 7, 14, and 28 posttrauma. Relative to nondiabetic and insulin-treated diabetic animals, the numbers of accumulated neutrophils and ED1(+) and ED2(+) macrophages in diabetic rats decreased by 46, 43, and 52%, respectively, in the first 3 days after injury compared with levels in nondiabetic and insulin-treated diabetic animals. The density of newly formed blood vessels decreased by 35 and 29% in the paratenon and the core of tendon, respectively, at days 3 and 7 after injury. Lastly, the concentration of proliferative cells decreased by 34% in the paratenon at day 7 posttrauma in injured tendons from diabetic rats relative to nondiabetic rats. These results indicate that alterations in inflammatory, angiogenic, and proliferative processes occurred in the diabetic state that might eventually perturb tendon healing and remodeling.

Hyperactivation of the hypothalamo-pituitary-adrenocortical axis in streptozotocin-diabetes is associated with reduced stress responsiveness and decreased pituitary and adrenal sensitivity.

Although increased hypothalamo-pituitary-adrenocortical (HPA) activity has been reported in diabetic patients, the mechanisms underlying hyperactivation are still unclear. We investigated whether alterations in pituitary, adrenal and/or glucocorticoid negative feedback sensitivity in diabetes are responsible for 1) the impaired HPA response to stress and 2) basal hyperactivation of the HPA axis. Normal control, untreated streptozotocin-diabetic and insulin-treated diabetic rats were chronically catheterized. Eight days following surgery, pituitary-adrenal function was monitored throughout the day. Stress responsiveness was evaluated using 20 min of restraint on d 10. Thereafter, the rats were treated with CRH (0.5 microg/kg), ACTH(1-24) (75ng/kg) or dexamethasone (25 microg/kg) iv on d 12, 14, and 16 to evaluate pituitary, adrenal and glucocorticoid feedback sensitivity, respectively. Plasma ACTH and corticosterone (B) concentrations in untreated diabetic rats were significantly higher at 0800 h, but no different at 1300 h or 1800 h. Insulin treatment of diabetic rats normalized ACTH and B concentrations at 0800 h. The pituitary-adrenal response to restraint was greatly diminished in untreated diabetic rats, whereas insulin treatment partially restored this response in diabetic rats. Administration of CRH and ACTH revealed reduced pituitary and adrenal sensitivity in untreated diabetic animals compared with both control and insulin-treated diabetic animals. The dexamethasone suppression test indicated decreased glucocorticoid negative feedback sensitivity in diabetic rats, which was restored with insulin treatment. In conclusion, these studies demonstrate that: 1) impaired stress responsiveness of the diabetic HPA axis involves both decreased pituitary and adrenal sensitivity; and 2) basal hyperactivation of the diabetic HPA axis in the morning is due, in part, to decreased glucocorticoid negative feedback sensitivity.

Oxidant stress is increased within the glomerulus in experimental diabetic nephropathy

SUMMARY: The mechanisms by which the metabolic consequences of hyperglycaemia induce diabetic renal injury remain ill‐defined. We hypothesized that oxidant stress, a consequence of hyperglycaemia, is increased in glomeruli from Lewis rats with streptozotocin (STZ)‐induced diabetes prior to major structural and functional glomerular damage. After 12 weeks of diabetes, Lewis rats had not developed proteinuria and their glomeruli appeared normal by light microscopy. However, kidneys of diabetic animals had higher levels of lipid peroxides and malondialdehyde (MDA) than control rats. Immunohistochemistry demonstrated MDA–lysine adducts in glomeruli of diabetic rats, and that lipid peroxides and MDA were increased in glomerular lysates of diabetic rats. A possible mechanism for this finding was suggested by the observation that freshly isolated whole glomeruli from STZ rats showed a greater capacity than glomeruli from control rats to produce H2O2. Activity of the inducible form of superoxide dismutase (SOD) Mn‐SOD was increased in glomerular lysates from STZ rats, consistent with its induction by oxidant stress. Immunostaining for Cu,Zn SOD showed increased protein in glomeruli, although compared with the increase in Mn‐SOD activity, Cu,Zn‐SOD activity was not substantially increased, potentially as a result of partial inactivation of this enzyme by glycation. The increased oxidant stress in untreated diabetic rats was a consequence of hyperglycaemia and not due to a direct nephrotoxic effect of STZ, as at least some of these changes were attenuated by insulin treatment of diabetic animals. Collectively, these results demonstrate that experimental diabetes mellitus is accompanied by increased oxidant stress within glomeruli.

Induction of Ca-independent PLA(2) and conservation of plasmalogen polyunsaturated fatty acids in diabetic heart.

Diabetes-induced changes in phospholipase A(2) (PLA(2)) activity have been measured in several tissues but are undefined in diabetic myocardium. We measured ventricular PLA(2) activity in control, streptozotocin-induced diabetic, and insulin-treated diabetic rats and characterized myocardial phospholipids to determine whether diabetes altered myocardial phospholipid metabolism. Increased membrane-associated Ca(2+)-independent PLA(2) (iPLA(2)) activity was observed in diabetes that was selective for arachidonylated phospholipids. Increased iPLA(2) activity was accompanied by an increase in choline lysophospholipids. Diabetes was associated with marked alterations in the phospholipid composition of the myocardium, characterized by decreases in esterified arachidonic and docosahexaenoic acids and increases in linoleic acid. The decrease in polyunsaturated fatty acids was confined to diacylphospholipids, whereas the relative amount of these fatty acids in plasmalogens was increased. Diabetes-induced changes in PLA(2) activity, lysophospholipid production, and alterations in phospholipid composition were all reversed by insulin treatment of diabetic animals. Diabetes-induced changes in membrane phospholipid content and phospholipid hydrolysis may contribute to some of the alterations in myocardial function that are observed in diabetic patients.

Insulin regulation of leptin expression in streptozotocin diabetic pigs.

The relationship between leptin mRNA and insulin status was explored using streptozotocin diabetic pigs. Twelve male Yorkshire x Landrace crossbred swine (approximately 40 kg BW) were divided into three groups. Two groups were rendered diabetic with the use of streptozotocin (75 mg/kg BW). Diabetes was confirmed 24 h after streptozotocin treatment by the presence of hyperglycemia. One group of diabetic animals received daily injections of insulin (.5 U/(kg x d)(-1)) for 7 d, whereas the other group of diabetic animals received saline injections. The nondiabetic group also received saline injections (controls). Tissue and blood were collected after 7 d of treatment. Leptin mRNA concentrations in dorsal s.c. adipose tissue were measured by Northern analysis and standardized against 28S rRNA expression. Diabetes reduced leptin mRNA concentration by 67% in s.c. adipose tissue (P < .05). Serum insulin concentrations in the diabetic animals were reduced by 69% (P < .05). Insulin treatment of diabetic animals resulted in an increase in leptin mRNA concentration to levels in controls. Primary cell culture of porcine adipose tissue was used to assess whether these actions were the direct or indirect action of insulin. Acute exposure (1 to 24 h) of primary cultures of porcine adipocytes to insulin did not result in a change in leptin expression. However, chronic (7-d) exposure to insulin elevated leptin mRNA levels by 73%. These data suggest that insulin mediates changes in porcine leptin mRNA levels in vivo or in vitro, most likely by an indirect action.