Chronic Experimental Diabetes Research Articles

Morphological and morphometric study of the cervical vagus nerve myelinated fibers in acute and chronic experimental diabetes.

The literature is scarce on pathological studies of the vagus nerve in patients known to have chronic autonomic diabetic neuropathy. Most of the morphological studies of the vagus nerve in experimental diabetes involve the study of the abdominal segment of this nerve or its mesenteric branches. In these studies, the authors demonstrate a presence of diabetic neuropathy and characterize the dying back process of this neuropathy. However, the morphological lesions described by these authors are exclusive of the unmyelinated fibers, since in these segments, the vagus nerve no longer presents myelinated fibers. The objective of the present study was to investigate the existence of morphological and morphometric alterations of the cervical vagus nerve myelinated fibers in acute and chronic experimental diabetes. Male Wistar rats received a single injection of streptozotocin (40mg/kg) 15 days (n = 6) or 12 weeks (n = 6) prior to the experiments. Control rats (n = 6) received equal volume of citrate buffer solution. Proximal and distal segments of the cervical vagus nerves had their fascicle and myelinated fibers and respective axons measured with the aid of computer software. The morphometric data comparison was performed between segments in the same nerve, sides and groups and differences were considered significant when p <0.05. Our results show that there are alterations in the morphology and morphometry of myelinated fibers and axons of the vagus nerve in diabetic animals, especially in their distal segments in both experimental groups. However, in the chronic diabetic group the alterations were more evident and committed the proximal segments. Thus, the results suggest a presence of axonal neuropathy in the vagus nerve due to experimental diabetes, and that alterations were progressive in a time dependent manner.Support or Funding InformationFAEPA, CAPES and CNPqThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Фізико-хімічні властивості лігандних форм гемоглобіну за експериментального стрептозотоцинового цукрового діабету й за алкогольної інтоксикації

The study of hemoglobin affinity to oxygen, oxygen capacity and the content of ligand forms has been conducted in hemolyzates of rat peripheral blood at chronic alcohol intoxication models and experimental diabetes mellitus. The findings revealed that at alcohol intoxication both hemoglobin affinity to oxygen and hemoglobin oxygen capacity validly decrease, whereas at experimental streptozotocin-induced diabetes hemoglobin the affinity to oxygen increases and oxygen capacity is quite high. The results of study demonstrated that sulf- and methemoglobin levels rise under alcohol intoxication and sulf-, met- and carboxyhemoglobin levels rise under experimental diabetes mellitus. We have also undertaken study of hemoglobin affinity to oxygen, oxygen capacity and the ratio of hemoglobin ligand forms in alcoholics and healthy donors. A valid increase in sulf- and methemoglobin and a decrease in hemoglobin affinity to oxygen in alcoholics (32.50±0.45 versus 28.60±0.54 mm Hg in the control group) with insignificant changes in oxygen capacity were revealed. Since minor ligand forms contribute to the affinity of total hemoglobin to oxygen, their quantitative characteristics are essential. Thus, data on absorption spectra which characterize nitrosyl-hemoglobin ligand form and transition of deoxy- and methemoglobin into nitrosyl-hemoglobin have been collected. A combined absorption spectrum of nitrosyl-hemoglobin has been built based on the statistical data set deoxyhemoglobin and its full transition into nitrosyl-hemoglobin (60 analyses) in the examined wavelength range. The article provides the results of the comparative analysis of electronic absorption spectra characteristic peaks of six hemoglobin ligand forms taken from peripheral blood in healthy donors in 450–750 nm wavelength range. The studies can be a pre-requisite for the development of methods for determining six hemoglobin ligand forms in a single blood sample.

Anti-diabetic effect of a preparation of vitamins, minerals and trace elements in diabetic rats: a gender difference.

BackgroundAlthough multivitamin products are widely used as dietary supplements to maintain health or as special medical food in certain diseases, the effects of these products were not investigated in diabetes mellitus, a major cardiovascular risk factor. Therefore, here we investigated if a preparation of different minerals, vitamins, and trace elements (MVT) for human use affects the severity of experimental diabetes.MethodsTwo days old neonatal Wistar rats from both genders were injected with 100 mg/kg of streptozotocin or its vehicle to induce diabetes. At week 4, rats were fed with an MVT preparation or vehicle for 8 weeks. Well established diagnostic parameters of diabetes, i.e. fasting blood glucose and oral glucose tolerance test were performed at week 4, 8 and 12. Moreover, serum insulin and blood HbA1c were measured at week 12.ResultsAn impaired glucose tolerance has been found in streptozotocin-treated rats in both genders at week 4. In males, fasting blood glucose and HbA1c were significantly increased and glucose tolerance and serum insulin was decreased at week 12 in the vehicle-treated diabetic group as compared to the vehicle-treated non-diabetic group. All of the diagnostic parameters of diabetes were significantly improved by MVT treatment in male rats. In females, streptozotocin treatment resulted in a less severe prediabetic-like phenotype as only glucose tolerance and HbA1c were altered by the end of the study in the vehicle-treated diabetic group as compared to the vehicle-treated non-diabetic group. MVT treatment failed to improve the diagnostic parameters of diabetes in female streptozotocin-treated rats.ConclusionThis is the first demonstration that MVT significantly attenuates the progression of diabetes in male rats with chronic experimental diabetes. Moreover, we have confirmed that females are less sensitive to STZ-induced diabetes and MVT preparation did not show protection against prediabetic state. This may suggest a gender difference in the pathogenesis of diabetes.

Can metabolic impairments in experimental diabetes be cured with poly(amido)amine (PAMAM) G4 dendrimers? – In the search for minimizing of the adverse effects of PAMAM administration

Poly(amido)amine (PAMAM) G4 dendrimers, given intraperitoneally to diabetic rats, have been reported to scavenge excessive blood glucose and minimize the effects of hyperglycaemia, however, at the cost of reduced survival. This paper is the first to compare the effectiveness of three different routes of PAMAM G4 administration with regard to minimizing the adverse effects of hyperglycaemia in rats. Hence, the aim of the study is to identify the most effective and the least harmful method of dendrimer administration. Control and streptozotocin-diabetic Sprague-Dawley rats were exposed to PAMAM G4 (0.5 μmol/kg b.w.) for 60 days, administered intraperitoneally, intragastrically or subcutaneously. Intraperitoneal and subcutaneous administration of PAMAM G4 was found to be most effective in suppressing the long-term markers of hyperglycaemia, while the intragastric route appeared the least effective. Otherwise, the greatest incidence of adverse effects was associated with intraperitoneal and the lowest with subcutaneous delivery. Harmful effects of intragastrical administration were much lower compared to intraperitoneal route, but at the cost of reduced hypoglycaemizing potential. Otherwise, subcutaneous injection represents the best compromise of moderate PAMAM dendrimer toxicity and effective reduction in the markers of long-term severe hyperglycaemia in chronic experimental diabetes.

Experimental diabetes exacerbates skin transplant rejection in rats

To investigate the effect of chronic experimental diabetes on skin allografts in rats as a simple model that could clarify some basic aspects and mechanisms involved in transplant rejection in diabetes compared to normal animals. Skin grafting was performed with fragments of tail skin from sex matched non diabetic Wistar rats engrafted onto the thoracic area of diabetic and non diabetic recipients. Grafts were scored for rejection every other day and were removed on day 14. Skin grafts were graded according to the following itens: no rejection; or rejection including: acute, chronic and humoral and/or cellular rejection. Statistical analysis was performed using JMP 5.1 software with ANOVA test. Diabetes was induced with IV injection of alloxan 40 mg/kg. Inflammatory vascular infiltrate compromising the endothelium with areas of fibrinoid necrosis and thrombosis characteristics of acute humoral rejection and subendothelial lymphocyte infiltrate typical of acute cellular rejection were significantly (p<0.003) higher in diabetic than in non diabetic recipients as the inflammatory infiltrate in the epidermis (p<0.002). Skin transplant acute rejection from chronic alloxan diabetic rats to normal tissue was significantly more intense than the acute rejection between normal rats.

Is the phrenic nerve myelinated fiber distribution affected in short term experimental diabetes?

Previous reports from our laboratory showed that chronic experimental diabetes affects the shape of myelinated fiber normal distribution histogram in the phrenic nerve [1]. We aimed to investigate if this distribution is also affected in the short term experimental diabetes. Male Wistar rats (N=6) were rendered diabetic for 15 days, by a single injection of streptozotocin. Control rats received vehicle. Under pentobarbital anesthesia, rats were killed and phrenic nerves were prepared for semi‐thin transverse sections investigated by light microscopy and morphometry. Our results show that control nerves myelinated fiber distributions are bimodal, with peaks at 2.0 and 5.0 μm. Diabetic nerve distributions were skewed to the right, with peaks at 2.5 and 5.5 μm, showing reduction of the frequency of the large fibers. Control nerves myelinated axon distributions are unimodal, with peak at 3.0 μm and diabetic nerve distributions were skewed to the right, with peak at 3.5. G‐ratio distributions were unimodal in both groups, being the diabetic nerves distribution skewed to the left, suggesting the presence of axonal atrophy. Our results confirm previous reports [1] of axonal neuropathy in phrenic nerves of diabetic rats, adding to the literature that this neuropathy has a very early onset.

Chronic experimental diabetes accelerates urinary elimination of deprenyl and its metabolites.

Many diabetic patients take several medications to treat diabetes-associated complications and other ailments. The mode of elimination of these drugs and their metabolites are poorly understood. The elimination of deprenyl, a MAO-B inhibitor, used for the treatment of the early stage of Parkinson’s disease and senile dementia was investigated using thin layer chromatography.Male Wistar rats (180-200 g) were rendered diabetic by streptozotocin (STZ) treatment (60 mg/kg, i.v.). Rats having at least three times higher plasma glucose level than the normal were considered diabetic. Rats were treated with a single oral dose of 5 mg/kg 14C-(methyl)-labeled (-)-deprenyl, 98 µCi/mg. Diabetic rats excreted the majority of urinary radioactivity in 8 hours, while control rats did it in 16 hours. The approximate ratio of major metabolites as determined using thin-layer chromatography did not change. In conclusion, diabetic rats excreted radiolabelled-deprenyl more rapidly compared to control animals. Increased elimination of deprenyl should be taken into account in the management of patients suffering from diabetes.

Aortic Depressor Nerve Myelinated Fibers in Acute and Chronic Experimental Diabetes

Preliminary data from our laboratory have demonstrated baroreflex dysfunction as early as 5 days after the onset of experimental diabetes in rats. The aim of the present study was to determine the morphologic alterations of the aortic depressor nerve of rats submitted to acute (15 days) or chronic (12 weeks) experimental diabetes. Wistar rats received a single intravenous injection of streptozotocin (50 mg/kg) 15 days or 12 weeks before the experiments. Time control animals received vehicle. Under pentobarbital anesthesia the aortic depressor nerves were identified by means of electroneurographic recording simultaneously with the recording of arterial pressure. After identification, the aortic depressor nerves were prepared for light microscopy and our analysis only included the myelinated fibers. Five of 10 aortic depressor nerves from animals with acute diabetes showed clear signs of axonal atrophy with significantly smaller average diameter of myelinated fibers (1.9 +/- 0.1 microm) and their axons (1.0 +/- 0.1 microm) than the time control (2.1 +/- 0.1 microm and 1.2 +/- 0.1 microm, respectively). Histograms of the G-ratio (ratio between axon diameter and total fiber diameter--an index of axonal atrophy) distribution of acute diabetic animal nerves were shifted to the left. All aortic depressor nerves from animals with chronic diabetes showed signs of axonal atrophy with the average diameter of myelinated fibers (2.3 +/- 0.1 microm) and their axons (1.2 +/- 0.1 microm) also being significantly smaller than the time control (2.6 +/- 0.2 microm and 1.5 +/- 0.1 microm, respectively). These findings provide morphologic support for an impaired baroreflex during acute and chronic experimental diabetes.

Sensory neurons with activated caspase-3 survive long-term experimental diabetes.

Long-term experimental diabetes may best model the prominent and irreversible sensory deficits of chronic human diabetic polyneuropathy. Whereas irretrievable loss of sensory neurons, if present, would be an unfortunate feature of the disease, systematic unbiased counting has indicated that sensory neurons survive long-term experimental diabetes. In this study, we examined whether incipient cell loss from apoptosis in chronic experimental diabetes might nonetheless be in process, or whether neurons somehow adapt to their chronic insults. We examined sensory neurons in L4 and L5 dorsal root ganglia of long-term experimental streptozotocin-induced diabetic rats using transferase-mediated dUTP nick-end labeling (TUNEL), 4',6-diamidino-2-phenylindole (DAPI) staining of nuclear morphology, and electron microscopic appraisal of cell morphology. None provided any evidence for ongoing apoptosis. Despite this confirmation that sensory neurons survive, neurons had elevated expression of activated caspase-3 in unique patterns that included their nuclei, cytoplasm, and proximal axonal segments. Bcl-2 expression, a marker of antiapoptosis signaling, was observed in similar numbers of diabetic and nondiabetic neurons. In contrast, diabetic sensory neurons had elevated expression of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) in their nuclei, cytoplasm, and proximal axonal segments not overlapping with caspase-3 localization. Diabetic sensory neurons also had an apparent rise in cytoplasmic labeling of nitrotyrosine, a marker of peroxynitrite toxicity reported to activate PARP.

Arterial baroreceptors and experimental diabetes.

Alterations of the autonomic reflex control of the cardiovasclar system have been demonstrated in clinical and animal models of insulin-dependent diabetes mellitus. Established neuroaxonal dystrophy is considered the neuropathological hallmark of chronic experimental diabetes. However, the afferent arm of the arterial baroreflex, that is, the carotid sinus nerve and the aortic depressor nerve, has received much less attention in studies dealing with this physiopathological model. The attenuation of the pressure response to bilateral carotid occlusion in conscious rats indicates a derangement of the baroreflex, probably involving an alteration of the carotid sinus nerve. There is histological evidence obtained from adult spontaneous insulin-dependent diabetic rats (strain BB/S) of a carotid sinus nerve with signs of axonal swelling and intramyelinic edema, suggesting diabetic neuropathy. The study of aortic baroreceptor activity in anesthetized rats with short- and long-term streptozotocin diabetes by means of cross-spectral analysis of baroreceptor activity versus arterial pressure revealed a dysfunction in the afferent arm of the baroreflex even during a short period of diabetes. The morphology of the aortic depressor nerve of streptozotocin-diabetic rats indicated axonal atrophy by visual analysis remarkably at the distal segments of the nerves. This finding was confirmed by morphometric study of the myelinated fibers. In conclusion, although studies of the arterial baroreceptors related to experimental diabetes are scanty in the literature, there is electrophysiological and histological evidence demonstrating that the carotid sinus and the aortic depressor nerves are abnormal in this experimental model.

The renal expression of heat shock protein 47 and collagens in acute and chronic experimental diabetes in rats.

Glomerulosclerosis and tubulointerstitial fibrosis are the main structural changes found in the later stages of diabetic nephropathy, which is clinically characterized by proteinuria, and progressive renal insufficiency. Heat shock protein (HSP) 47, a collagen-binding stress protein, has a specific role in the intracellular processing of procollagen molecules during collagen synthesis. It is implicated in the pathogenesis of various fibrotic diseases. However, the expression and significance of HSP47 in acute and chronic phases of diabetic nephropathy is not yet known. In this study, we studied the expression of HSP47 in the kidneys obtained from streptozotocin-induced diabetic rats, in both short- and long-term diabetes. To determine the renal expression of HSP47, and collagens (type III and IV) in acute (days 1, 3 and 14) and chronic (weeks 4, 12 and 24) diabetes, we have performed a time-course study using streptozotocin-induced diabetic rats. The expression pattern of alpha-smooth muscle actin (to identify mesangial cell damage), vimentin (to identify tubular epithelial cell damage), and desmin (to identify glomerular epithelial cell damage) was also determined in kidneys of these diabetic rats. Antibodies specific for HSP47, type III and type IV collagens, alpha-smooth muscle actin, vimentin, and desmin were used to assess the relative expression of their proteins in paraffin-embedded kidney sections by immunohistochemistry. Compared to control rat kidneys, no significant changes in the expression of HSP47 was found in the kidneys of acute diabetic rats. However a significant increase in the expression of HSP47 was noted in the kidneys of chronic diabetic rats; increased expression of HSP47 correlated with an increased renal deposition of types III and IV collagens. Similarly, compared to kidneys of control and acute diabetic rats, an increased expression of alpha-smooth muscle actin (in mesangial cells), vimentin (in tubular epithelial cells), and desmin (in glomerular epithelial cells) was detected in the kidneys of chronic diabetic rats; by dual immunostaining, these phenotypically-altered renal cells in kidneys of chronic diabetic rats were found to be HSP47-producing cells. Importantly, HSP47 up-regulation coincided with the initiation and progression of renal fibrosis, as determined by the expression and deposition of collagens. Our results strongly support a pathological role for HSP47 in the later stages (sclerotic phase) of streptozotocin-induced diabetic nephropathy, which is associated with glomerulosclerosis and tubulointerstitial fibrosis.

The Renal Expression of Transforming Growth Factor- Isoforms and Their Receptors in Acute and Chronic Experimental Diabetes in Rats

Transforming growth factors-β (TGF-β) are fibrogenic factors that have been strongly implicated in the development of diabetic nephropathy. Our aim was to use two animal models [the streptozotocin (STZ)-induced diabetic rat and the genetically prone biobreeding (BB) rat] to fully characterize the responses of the renal TGF-β system in both short- and long-term diabetes. In this study changes in the entire renal TGF-β system, at both protein and messenger RNA (mRNA) levels, have been characterized using the techniques of immunocytochemistry, Western blotting, and ribonuclease protection assay. We also used Western blotting of pro-collagen-I C-peptide to demonstrate that the rate of fibrogenesis was highest over the first 2 weeks of diabetes. TGF-β1, TGF-β2, and receptor mRNA and protein were detected in the control nondiabetic kidney. It was found that dramatic and dynamic changes occur in all parts of the renal TGF-β axis in both models of experimental diabetes, but TGF-β2 and TGF-βRII proteins were the predominant responsive element, particularly during the acute phase of disease. For example, during the acute phase of disease (0–30 days), although renal TGF-β1 mRNA levels were elevated, no increases in the corresponding protein were detected in the kidney. By contrast, in the absence of changes in TGF-β2 mRNA levels, twice as much TGF-β2 protein was measured in the kidney by day 30 of STZ-induced diabetes compared with day 0 controls analyzed by Western blotting (P < 0.05), and the protein was localized both to the nuclei and cytoplasm of glomerular cells, analyzed by immunocytochemistry. In addition, three times as much TGF-βRII protein was found by day 90 of STZ-induced diabetes compared with day 0 controls, making this the most responsive receptor type. These results suggest that the entire TGF-β axis has a role in the etiology of kidney fibrosis and could be manipulated therapeutically to preserve kidney function.

The renal expression of transforming growth factor-beta isoforms and their receptors in acute and chronic experimental diabetes in rats.

Transforming growth factors-beta (TGF-beta) are fibrogenic factors that have been strongly implicated in the development of diabetic nephropathy. Our aim was to use two animal models [the streptozotocin (STZ)-induced diabetic rat and the genetically prone biobreeding (BB) rat] to fully characterize the responses of the renal TGF-beta system in both short- and long-term diabetes. In this study changes in the entire renal TGF-beta system, at both protein and messenger RNA (mRNA) levels, have been characterized using the techniques of immunocytochemistry, Western blotting, and ribonuclease protection assay. We also used Western blotting of pro-collagen-I C-peptide to demonstrate that the rate of fibrogenesis was highest over the first 2 weeks of diabetes. TGF-beta1, TGF-beta2, and receptor mRNA and protein were detected in the control nondiabetic kidney. It was found that dramatic and dynamic changes occur in all parts of the renal TGF-beta axis in both models of experimental diabetes, but TGF-beta2 and TGF-betaRII proteins were the predominant responsive element, particularly during the acute phase of disease. For example, during the acute phase of disease (0-30 days), although renal TGF-beta1 mRNA levels were elevated, no increases in the corresponding protein were detected in the kidney. By contrast, in the absence of changes in TGF-beta2 mRNA levels, twice as much TGF-beta2 protein was measured in the kidney by day 30 of STZ-induced diabetes compared with day 0 controls analyzed by Western blotting (P < 0.05), and the protein was localized both to the nuclei and cytoplasm of glomerular cells, analyzed by immunocytochemistry. In addition, three times as much TGF-betaRII protein was found by day 90 of STZ-induced diabetes compared with day 0 controls, making this the most responsive receptor type. These results suggest that the entire TGF-beta axis has a role in the etiology of kidney fibrosis and could be manipulated therapeutically to preserve kidney function.

Contractile responses to various stimuli in isolated resistance vessels from simultaneously hypertensive and streptozotocin-diabetic rats.

Diabetes mellitus and hypertension are common chronic diseases that frequently occur simultaneously. The induction of streptozotocin (STZ) diabetes mellitus in spontaneously hypertensive rats (SHR) offers the opportunity to investigate the influence of both entities in a reproducible manner. We investigated the effects of various vasoconstrictors on isolated small arteries from the mesenteric vascular bed of normotensive rats (Wistar-Kyoto rats, WKY) and SHR with chronic (8 weeks), STZ-induced diabetes mellitus. No consistent changes in hemodynamic parameters of the (STZ-) normotensive and (STZ-) hypertensive rats were noted. The K(+)-normalization procedure yields the individual optimal lumen diameter, which was the same for the arteries of the four groups of rats. The passive wall tension resulting from this normalization procedure was higher only in preparations from the control hypertensive group as compared with those from the control normotensive rats. Morphological investigations showed that small arteries from control SHR had an increased tunica media thickness as compared with those of control WKY; the STZ-WKY had an increased tunica media thickness as compared with preparations from control WKY. The vasoconstriction caused by alpha 1-adrenoceptor stimulation [norepinephrine (NE), methoxamine] and serotonin is unchanged in chronic experimental diabetes. The diabetic state reduced the sensitivity [-log EC50(M)] for the concentration-response curves (CRC) of calcium chloride. The CRC of potassium chloride indicated the same sensitivities, but maximal active wall tensions of vessels from STZ-SHR were reduced as compared with those from STZ-WKY. The well-known enhancement of the effects of various contractile stimuli caused by hypertension could not be demonstrated for the isolated small arteries used in the present study, although a nonsignificant tendency was observed. However, the STZ-diabetic state did not cause important additional pharmacodynamic changes, despite the morphological alterations in those vessels.

Partial correction of impaired creatine kinase activity in diabetic rat heart by physical training

The effect of physical training on total creatine kinase (CK), CK-MM, and CK-MB isoenzyme activity was studied in hearts of diabetic and control rats. Diabetes was induced with streptozotocin (50 mg/kg), and only rats with blood glucose levels between 14 and 22 mmol/L 1 week later were kept in the protocol. Exercise training was performed on a treadmill in a progressive 10-week program. Physical training did not induce any significant changes in plasma glucose or insulin levels in diabetic rats. Total CK, CK-MM, and CK-MB activity was decreased in diabetic rat heart by 27%, 22%, and 56%, respectively. Physical training did not induce any important changes in CK activity in heart of nondiabetic rats. However, in diabetic rat heart, training increased total CK activity by 13%, CK-MM activity by 12%, and CK-MB activity by 31%. We conclude that the decrease in cardiac CK activity observed in chronic experimental diabetes mellitus can be partly alleviated by a program of physical training. This may be one of the mechanisms whereby physical conditioning improves cardiac function in experimental diabetes.

Nerve blood flow in early experimental diabetes in rats: relation to conduction deficits

A reduction in nerve blood flow in chronic experimental diabetes has been linked to impaired conduction. Recently, there have been reports that this is preceded by a period of functional hyperemia. The present investigation explored early changes in sciatic nerve endoneurial blood flow and function in streptozocin-treated rats with durations of diabetes from 1 wk to 4 mo. Blood flow was monitored by microelectrode polarography and hydrogen clearance in thiobutabarbital (Inactin)-anesthetized animals. It was reduced by 41% as early as 1 wk after diabetes induction. There was no evidence of an early functional hyperemia, flow remaining 44% depressed up to 4 mo. In another investigation, similar reductions in blood flow were acutely induced in normal rats rendered hyperglycemic by glucose infusion. In diabetic animals, conduction velocity in sciatic branches supplying gastrocnemius and tibialis anterior muscles was correlated with blood flow. The link was further tested using a group of 2-mo diabetic rats treated with guanethidine. Treatment caused a functional adrenergic sympathectomy, and blood flow increased to within the normal range. Conduction velocity, depressed by 26% with diabetes, was normalized by treatment. These observations support the hypothesis that hyperglycemia-induced blood flow reductions and resultant endoneurial hypoxia are important factors underlying nerve conduction deficits early in the development of diabetic neuropathy.

Alterations in phospholipid N-methylation of cardiac subcellular membranes due to experimentally induced diabetes in rats.

Phosphatidylethanolamine N-methylation was examined in cardiac subcellular membranes after inducing chronic experimental diabetes in rats (65 mg streptozotocin/kg, i.v.). The incorporation of radiolabeled methyl groups from S-adenosyl-L-methionine in diabetic sarcolemma was significantly depressed at all three catalytic sites (I, II, and III) of the methyltransferase system. An increase in methyl group incorporation was evident at site I without any changes at sites II and III in diabetic sarcoplasmic reticulum and mitochondria. Similar changes were also seen for the individual N-methylated lipids (monomethyl-, dimethylphosphatidylethanolamine, and phosphatidylcholine) specifically formed at each catalytic site in all cardiac membranes from diabetic animals. These alterations in N-methylation were reversible by a 14-d insulin therapy to the diabetic animals. In the presence of 10 microM ATP and 0.1 microM Ca2+, N-methylation was maximally activated at site I in both control and diabetic sarcolemma and sarcoplasmic reticulum, but not in mitochondria. Incubation of cardiac membranes with of S-adenosyl-L-methionine showed that Ca2(+)-stimulated ATPase activities in both sarcolemma and sarcoplasmic reticulum were augmented; however, the activation of diabetic sarcolemma was lesser and that of diabetic sarcoplasmic reticulum was greater in comparison with the control preparations. These results identify alterations in phosphatidylethanolamine N-methylation in subcellular membranes from diabetic heart, and it is suggested that these defects may be crucial in the development of cardiac dysfunction in chronic diabetes.

Na(+)-H+ exchange in cardiac sarcolemmal vesicles isolated from diabetic rats

Intracellular pH and [Na+] in the heart are regulated by the sarcolemmal membrane Na(+)-H+ exchange pathway. No data are currently available regarding the adaptation of this system to pathological conditions in the heart. Because ionic interactions with the heart are altered in cardiomyopathy during chronic experimental diabetes, it was hypothesized that Na(+)-H+ exchange may become abnormal. In addition, the effects of treating diabetic rats with daily injection of L-propionylcarnitine were investigated to determine whether alterations in lipid metabolism may be involved in any potential changes in ion transport. Rats were injected with streptozotocin (65 mg/kg) and killed 8-10 wk later, and sarcolemmal membrane vesicles were isolated from pooled ventricles. Significant depressions in Na(+)-K(+)-adenosinetriphosphatase (ATPase) activity and Na(+)-Ca2+ exchange were observed in the diabetic preparations in comparison to control. L-Propionylcarnitine treatment of the diabetic rats partially normalized these activities. A striking depression in cardiac sarcolemmal Na(+)-H+ exchange was observed in the diabetic animals in comparison to control, and this was not a result of a nonspecific increase in membrane permeability. L-Propionylcarnitine treatment of the diabetic rats did not improve sarcolemmal Na(+)-H+ exchange.

Sensitivity changes to inotropic agents in rabbit atria after chronic experimental diabetes.

The effect of experimental diabetes on the sensitivity of isolated left atrial strips to inotropic agents was investigated in rabbits made diabetic with alloxan. After 4 weeks of diabetes no change in sensitivity was detected in response to isoproterenol or ouabain. In contrast, 15 weeks of diabetes induced a decreased sensitivity to beta-adrenergic stimulation, exhibited as a shift to the right in concentration-response curves obtained in response to isoproterenol and noradrenaline. In addition, after 15 weeks of diabetes the inotropic response to ouabain was depressed, and a small decrease in sensitivity was detected in response to forskolin. In contrast, no significant changes in the concentration-response curves obtained from alpha-adrenergic stimulation by phenylephrine or calcium chloride were detected. Unlike the streptozotocin diabetic rat, which exhibits low serum thyroid hormone levels, no changes in serum thyroid hormones were detected in the alloxan diabetic rabbit. It is suggested that the increased inotropic sensitivity to alpha-adrenergic agonists observed in the diabetic rat, but not in the rabbit, may be due to low serum thyroid hormone levels. In contrast, the deleterious effects of diabetes on beta-adrenergic and ouabain sensitivity occur independently of changes in serum thyroid hormones.

The quantification of cerebral infarction following focal ischemia in the rat: influence of strain, arterial pressure, blood glucose concentration, and age.

Focal cerebral ischemia was induced by occlusion of the middle cerebral artery in rats. The volumetric assessment of infarcted tissue, 2 days following occlusion, was calculated from the examination of eight preselected coronal sections. Five differing rat strains were examined. A small and variable infarcted volume was seen in Wistar-Kyoto rats; Sprague-Dawley rats had a relatively large, but still variable, infarcted volume. Of the normotensive rat strains, the most reproducible volume of infarcted tissue was seen in Fischer-344 rats; also the absolute value of the infarcted volume did not vary from one series to another in this strain. Chronic arterial hypertension, studied in both normal and stroke-prone spontaneously hypertensive rats, was associated with significantly larger infarction volumes. Age does not change the volume of necrosis: Fischer-344 rats were studied at 3, 9, and 20 months of age, and no significant differences were noted between these ages. Experimental diabetes was induced by the administration of streptozotocin 3 days prior to middle cerebral artery occlusion. Severe hyperglycemia (greater than 400 mg/dl) was associated with a considerably increased volume of infarction. The variability of the resultant lesion is high in the most commonly studied strains, but our results suggest that, for studies in normotensive rats, the use of the Fischer-344 strain produces a standardized and repeatable infarction that may be significantly modified by experimental interventions. Age is not a factor that affects the occlusion-induced infarction; in contrast, both chronic arterial hypertension and experimental diabetes aggravate the histological consequences of middle cerebral artery occlusion in the rat. We conclude that quantitative histological evaluation of infarct size allows a meaningful assessment of the gravity of focal cerebral ischemia.