Insulin-treated Diabetic Rats Research Articles

Recurrent hypoglycemia exposure increases the risk of platelet activation and thrombosis in insulin-treated diabetic rats.

Diabetes is a widespread disease associated with long-term complications. Treatment of diabetes alleviates these complications but cause an increased risk of recurrent hypoglycemia (RH). Hypoglycemia exposure increases the risk of cardiovascular events by an unknown mechanism. Since the effect of mild/moderate hypoglycemia on thrombosis is unknown, we studied the effect of RH exposure on platelet function and thrombosis in insulin-treated diabetic (ITD) rats. ITD rats were randomized to either control or hypoglycemia groups. First, we determined the minimum duration and frequency of RH exposure that increases the risk of thrombosis and the time window after a single hypoglycemic episode (SH)/RH exposure with increased risk of thrombosis in male ITD rats. Next, we confirmed whether RH exposure increases the risk of thrombosis in female ITD rats. Subsequently, we evaluated the impact of RH exposure on platelet susceptibility to aggregation and platelet gene expression. One hour hypoglycemia increased the clot weight and the effect of SH and RH on thrombosis lasted for at least 1 and 7 days post-exposure, respectively. A minimum frequency of twice-a-week hypoglycemia exposure for 6 weeks increased the risk of thrombosis in male ITD rats. Increased susceptibility of platelet activation was observed in RH-exposed male ITD rats. Lastly, we identified RH-induced alterations in the platelet transcriptome in male ITD rats. We also confirmed that RH exposure increases the risk of thrombosis in female rats. Understanding the mechanism of RH-induced platelet activation and thrombosis may help limit thrombotic complications in diabetes.

Abstract TP392: Prior recurrent hypoglycemia exposure potentiates post-ischemic hypoperfusion in male insulin-treated diabetic rats.

Diabetes is a widespread disease, and stroke is one of the serious complications of diabetes. Antidiabetic therapy increases the risk of recurrent hypoglycemia (RH). We have previously shown that RH exposure leads to severe post-ischemic hypoperfusion at least up to 80 minutes after ischemia and increases the extent of ischemic brain injury in insulin-treated diabetic (ITD) rats. However, the total duration for which these perfusion deficits last is unknown. Thus, we evaluated cerebral blood flow up to 7 days post-ischemia in RH-exposed ITD rats using laser speckle imaging. Diabetic male rats were treated for hyperglycemia using insulin pellets, were assigned randomly to either hyperinsulinemic euglycemia (ITD+RH+Glucose control; n=7) or hyperinsulinemic hypoglycemia (ITD+RH; n=7) groups (3 h duration) (Figure B-C) and were subjected to transient global cerebral ischemia overnight after the last episode of hyperinsulinemic euglycemia or hyperinsulinemic hypoglycemia. We evaluated cerebral perfusion at baseline (pre-ischemia), 1 h, 24 h, 3 d, 5 d, and 7 d post-ischemia. The cerebral ischemia in RH-exposed ITD rats resulted in a significant decrease in percentage change in cerebral blood flow as compared to the control rats when quantified 1 hour (23%, p<0.01) and 1 day (17%, p<0.05) after cerebral ischemia. However, the percentage changes in cerebral blood flow in ITD+RH group were not significantly different from that of the control group at 3, 5, and 7 days after ischemia (Figure A, D). The cerebral blood flow post-ischemia at 1-hour (24%) and 1-day (18%) was significantly lower (p<0.001 each) while at 3 (12%, p<0.05), 5 (25%, p<0.05), and 7 days (29%, p<0.01) was significantly higher than that of the baseline values in RH-exposed ITD rats. The cerebral blood flow at 1 hour, 1 day, and 3 days after ischemia in ITD + RH + glucose rats was not significantly different from the baseline values. The cerebral blood flow at 5 (19%, p<0.05) and 7 (42%, p<0.001) days after ischemia in ITD + RH + glucose rats was significantly higher than the baseline values. Our results show that RH exposure induces severe post-ischemic hypoperfusion for at least ≈24 hours after ischemia in young male ITD rats. Next, we aim to study the duration of the effect of RH on post-ischemic hypoperfusion in female ITD rats and to identify the underlying mechanism causing this effect in the animal model of diabetes. Acknowledgment: NIH (NS122808).

Intestinal Region-Dependent Impact of NFκB-Nrf Crosstalk in Myenteric Neurons and Adjacent Muscle Cells in Type 1 Diabetic Rats.

Type 1 diabetes affects cytokines as potential inducers of NFκB signalling involved in inflammation and neuronal survival. Our goal was to assess the expression of NFκB p65 and its negative regulator, Nrf2, in myenteric neurons and adjacent smooth muscle of different gut segments after chronic hyperglycaemia and immediate insulin treatment. After ten weeks of hyperglycaemia, intestinal samples of control, streptozotocin-induced diabetic and insulin-treated diabetic rats were prepared for fluorescent immunohistochemistry, immunogold electron microscopy, ELISA and qPCR. In the diabetic rats, the proportion of NFκB p65-immunoreactive myenteric neurons decreased significantly in the duodenum and increased in the ileum. The density of NFκB p65-labelling gold particles increased in the ileal but remained unchanged in the duodenal ganglia. Meanwhile, both total and nuclear Nrf2 density increased in the myenteric neurons of the diabetic duodenum. In smooth muscle, NFκB p65 and Nrf2 density increased in the small intestine of diabetic rats. While on the mRNA level, NFκB p65 and Nrf2 were induced, on the protein level, NFκB p65 increased and Nrf2 decreased in muscle/myenteric plexus homogenates. Insulin treatment had protective effects. Our findings reveal a segment-specific NFκB and Nrf expression in myenteric neurons and ganglionic muscular environments, which may contribute to regional neuronal survival and motility disturbances in diabetes.

Hypoglycemic Ability of Sericin-Derived Oligopeptides (SDOs) from Bombyx mori Yellow Silk Cocoons and Their Physiological Effects on Streptozotocin (STZ)-Induced Diabetic Rats.

Patients with diabetes require daily medication to maintain blood sugar levels. Nevertheless, the long-term use of antidiabetics can lose efficacy and cause degeneration in some patients. For long-term diabetes care, integrating natural dietary foods and medicine is being considered. This study investigated the impact of SDOs on blood sugar levels and their physiological effects on diabetic rats. We induced diabetes in male Wistar rats with STZ (50 mg/kg) and then administered an oral glucose tolerance test to determine the SDO dosage comparable to glibenclamide. The rats were divided into nine groups: normal, diabetic, and diabetic with insulin (10 U/kg), glibenclamide (0.6 mg/kg), bovine serum albumin (BSA; 200 mg/kg), soy protein isolate (200 mg/kg), or SDOs (50, 100, and 200 mg/kg). Diabetic rats administered SDOs had a higher body weight and serum insulin but a lower blood sugar than diabetic control rats. Biochemical assays indicated lower AST/SGOT, ALT/SGPT, BUN, and triglycerides but higher HDL in the SDO groups. Immunohistochemistry showed that SDOs reduced damaged islet cells, increased beta-cell size, and improved insulin levels while decreasing alpha cell size and glucagon. The vascular effects of SDOs were like those of normal control treatment and insulin treatment in diabetic rats. SDOs, a yellow silk protein, show potential for long-term diabetes care.

Abstract TMP117: Exposure to Single or Recurrent Hypoglycemia Increases Stroke Risk in Insulin-Treated Female Diabetic Rats

Stroke is a serious complication of diabetes. Intensive treatment of diabetes increases the risk of recurrent hypoglycemia (RH). Hypoglycemia exposure causes prothrombotic effects. Earlier, we demonstrated that exposure to single hypoglycemia (SH), 5-day RH (once every day), and twice-a-week RH for 6 weeks increases stroke risk in male insulin-treated diabetic (ITD) rats. In the present study, we determined the effect of SH and RH on stroke risk/thrombosis in female rats. Streptozotocin diabetic female rats were treated for hyperglycemia using insulin pellets (Figure A). Rats were randomly assigned to either hyperinsulinemic euglycemia (ITD+RH+Glucose, control) or hyperinsulinemic hypoglycemia (ITD+RH) groups (3 h duration) (Figure B). Separate groups tested the effect of SH, RH for 5 days, or RH for 6 weeks. Based on the results of the male studies, stroke risk after SH, 5-day RH, and 6-week RH were assessed on days 1, 7, and 3 post-last hypoglycemia, respectively. Groups were balanced (confirmed by Chi square test) in terms of the proportion of animals at different stages of the estrous cycle. To assess the risk of stroke, the carotid artery and jugular vein were linked with a shunt containing a suture, and the weight of the clot accumulated on the suture following 15 min of blood flow was quantified as a measure of stroke risk/thrombosis. The clot weight in the SH-exposed ITD group was significantly higher by 47% (22±3 mg, n=9, p<0.05) when compared to its respective control (15±1 mg, n=8). The clot weight determined 7 days after the 5-day ITD+RH group (18±1 mg, n=7) was significantly (p<0.05) higher than its control group (15±1 mg, n=9). The clot weight in the 6-week ITD+RH group was significantly higher by 47% (20±2 mg, n=9, p<0.01) when compared to its control (14±1 mg, n=7) (Figure C). Our results show that similar to male rats, SH and RH increase stroke risk in female ITD rats. We are currently evaluating the underlying mechanisms. Acknowledgment: NIH (NS122808).

Examining the Effects of Hyperbaric Oxygen Therapy on the Cardiovascular System and Oxidative Stress in Insulin-Treated and Non-Treated Diabetic Rats.

This study explored the effects of hyperbaric oxygen therapy (HBOT) on the cardiovascular system and oxidative stress in streptozotocin-induced diabetic rats. Wistar albino rats were divided into four groups: DM group (diabetic rats), DM+HBOT group (diabetic rats exposed to HBOT for 1 h daily, five days a week, at 2.8 atmosphere absolute (ATA) with 100% oxygen for two weeks), DM+INS group (diabetic rats treated with neutral protamine hagedorn (NPH) insulin at a dosage of 3-5 U/day), and DM+HBOT+INS group (diabetic rats treated with both NPH insulin and HBOT for two weeks). Evaluations included glycemic control, oxidative stress parameters, and cardiac function measurements. NPH insulin treatment reduced blood glucose levels, although normoglycemia was not achieved. The DM+HBOT+INS group demonstrated the lowest pro-oxidative marker levels. NPH insulin treatment improved cardiac function, and combination therapy effectively restored cardiac function in diabetic animals. NPH insulin treatment reduced hyperglycemia and improved cardiac function in diabetic rats. The combined approach of NPH insulin and HBOT resulted in decreased pro-oxidative markers. These findings provide valuable insights for managing cardiovascular complications and oxidative stress in diabetes.

Melatonin ameliorates serobiochemical alterations and restores the cardio-nephro diabetic vascular and cellular alterations in streptozotocin-induced diabetic rats.

Melatonin possesses a wide range of pharmacological activities, including antidiabetic properties. Diabetes mellitus (DM) induces several physiopathological changes in body organs, which could be observed lately after systemic failure. In the current study, we aimed to investigate the serobiochemical changes and the histopathological picture in the diabetic heart and the kidney early before chronic complications and highlight the association between hyperglycemia, glomerular alterations, and cardiovascular changes. In addition, the role of melatonin in the treatment of cardio-nephro diabetic vascular and cellular adverse changes in streptozotocin-induced diabetic rats was also studied. A total of 40 mature Wistar albino rats were distributed into five groups; (1) control untreated rats, (2) diabetic mellitus untreated (DM) rats, in which DM was induced by the injection of streptozotocin (STZ), (3) control melatonin-treated (MLT), (4) melatonin-treated diabetic (DM + MLT) rats, in which melatonin was injected (10 mg/kg/day, i.p.) for 4 weeks, and (5) insulin-treated diabetic (DM + INS) rats. The serum biochemical analysis of diabetic STZ rats showed a significant (P < 0.05) increase in the concentrations of blood glucose, total oxidative capacity (TOC), CK-MB, endothelin-1, myoglobin, H-FABP, ALT, AST, urea, and creatinine as compared to control rats. In contrast, there was a significant (P < 0.05) decrease in serum concentration of insulin, total antioxidative capacity (TAC), total nitric oxide (TNO), and total protein level in DM rats vs. the control rats. Significant improvement in the serobiochemical parameters was noticed in both (DM + MLT) and (DM + INS) groups as compared with (DM) rats. The histological examination of the DM group revealed a disorder of myofibers, cardiomyocyte nuclei, and an increase in connective tissue deposits in between cardiac tissues. Severe congestion and dilation of blood capillaries between cardiac muscle fibers were also observed. The nephropathic changes in DM rats revealed various deteriorations in glomeruli and renal tubular cells of the same group. In addition, vascular alterations in the arcuate artery at the corticomedullary junction and interstitial congestion take place. Melatonin administration repaired all these histopathological alterations to near-control levels. The study concluded that melatonin could be an effective therapeutic molecule for restoring serobiochemical and tissue histopathological alterations during diabetes mellitus.

Insulin Treatment Affects Brain Protein Synthesis Rate in Streptozotocin-Induced Diabetic Rats

Insulin Treatment Affects Brain Protein Synthesis Rate in Streptozotocin-Induced Diabetic Rats

Intestinal Region-Dependent Alterations of Toll-Like Receptor 4 Expression in Myenteric Neurons of Type 1 Diabetic Rats.

Toll-like receptor 4 (TLR4) can activate pro-inflammatory cascades in the gastrointestinal tract. Our aim was to determine TLR4 expression in myenteric neurons of different gut regions using a type 1 diabetic model. Ten weeks after the onset of hyperglycemia, myenteric whole-mount preparations from the duodenum, ileum and colon of streptozotocin-induced diabetic, insulin-treated diabetic and control rats were prepared for TLR4/peripherin double-labelling fluorescent immunohistochemistry. Immunogold electron microscopy was applied to evaluate TLR4 expression in the myenteric perikaryon and neuropil. Tissue TLR4 levels were measured by enzyme-linked immunosorbent assay. In controls, the number and proportion of the TLR4-immunoreactive myenteric neurons showed an increasing tendency to aboral direction. These values were significantly higher in diabetics compared to controls in the duodenum and ileum, but were significantly lower in the colon. In diabetics, the distribution of TLR4-labelling gold particles between the perikaryon and neuropil of myenteric neurons varied in a different way by intestinal segment. TLR4 tissue concentration changed only in the diabetic duodenum, and it decreased in muscle/myenteric plexus-containing homogenates, while it increased in mucosa/submucosa/submucous plexus-containing samples relative to controls. Insulin had beneficial effects on TLR4 expression. These findings support that chronic hyperglycemia has segment-specific effects on TLR4 expression, contributing to gastrointestinal disorders in diabetic patients.

Expression of GABAAα1, GABAB1, and mGluR2 receptors in the lateral geniculate body of male neonates born to diabetic rats.

Diabetes during gestation is one of the most common pregnancy complications and has adverse effects on offspring, including a negative impact on the offspring's central nervous system (CNS). Diabetes is a metabolic disease associated with visual impairment. Due to the importance of the lateral geniculate body (LGB) in the visual pathway, the present study examined the effect of maternal diabetes on the expression of gamma-aminobutyric acid (GABAAα1 and GABAB1) and metabotropic Glutamate (mGlu2) receptors in the LGB of male neonates of diabetic rats. Diabetes was induced in female adult rats by a single intraperitoneal dose of streptozotocin (STZ) 65 (mg/kg). In the Insulin-treated diabetic rats, diabetes was controlled by subcutaneous NPH-insulin injection daily. After mating and delivery, male offspring were killed by carbon dioxide gas inhalation at P0, P7, and P14 (postnatal days 0, 7, and 14). The expression of GABAAα1, GABAB1, and mGluR2 in the LGB of male neonates was determined using the immunohistochemistry (IHC) method. The expression of GABAAα1 and GABAB1 was significantly reduced, whereas the expression of mGluR2 was markedly increased in the diabetic group compared with the control and insulin-treated groups at P0, P7, and P14. The results of the present study showed that induction of diabetes altered the expression of GABAAα1, GABAB1, and mGluR2 in the LGB of male neonates born to diabetic rats at P0, P7, and P14. Moreover, insulin treatment could reverse these effects of diabetes.

The Effects of Streptozotocin-Induced Diabetes and Insulin Treatment on Carnitine Biosynthesis and Renal Excretion.

Carnitine insufficiency is reported in type 1 diabetes mellitus. To determine whether this is accompanied by defects in biosynthesis and/or renal uptake, liver and kidney were obtained from male Sprague-Dawley rats with streptozotocin-induced diabetes. Diabetic rats exhibited the metabolic consequences of type 1 diabetes, including hypoinsulinemia, hyperglycemia, and increased urine output. Systemic hypocarnitinemia, expressed as free carnitine levels, was evident in the plasma, liver, and kidney of diabetic rats. Compared to control rats, the low free carnitine in the plasma of diabetic rats was accompanied by decreased expression of γ-butyrobetaine hydroxylase in liver and kidney, suggesting impaired carnitine biosynthesis. Expression of organic cation transporter-2 in kidney was also reduced, indicating impaired renal reabsorption, and confirmed by the presence of elevated levels of free carnitine in the urine of diabetic rats. Insulin treatment of diabetic rats reversed the plasma hypocarnitinemia, increased the free carnitine content in both kidney and liver, and prevented urinary losses of free carnitine. This was associated with increased expression of γ-butyrobetaine hydroxylase and organic cation transporter-2. The results of our study indicate that type 1 diabetes induced with streptozotocin disrupts carnitine biosynthesis and renal uptake mechanisms, leading to carnitine insufficiency. These aberrations in carnitine homeostasis are prevented with daily insulin treatment.

Immediate Insulin Treatment Prevents Diabetes-Induced Gut Region-Specific Increase in the Number of Myenteric Serotonergic Neurons

To evaluate the effects of hyperglycemia and insulin treatment on the proportion of serotonin-immunoreactive (5-HT-IR) myenteric neurons, samples were taken from the duodenum, ileum, and colon of diabetic, insulin-treated diabetic, and control rats 10 weeks after the onset of streptozotocin-induced hyperglycemia. Myenteric whole-mount preparations were immunostained with anti-5-HT and pan-neuronal anti-HuCD markers. In controls, the 5-HT-IR myenteric neurons represent a small proportion (~2.5%) of the total neuronal number in the investigated gut segments. The proportion of 5-HT-IR myenteric neurons was significantly higher in the duodenum (p &lt; 0.01) and colon (p &lt; 0.0001) of diabetic rats compared to the controls but exhibited a slight increase in the ileum. Immediate insulin treatment resulted in a significantly lower proportion of myenteric 5-HT-IR neurons in each segment (duodenum p &lt; 0.0001; ileum p &lt; 0.01; and colon p &lt; 0.0001) compared to the untreated diabetics. Our study demonstrates that the proportion of 5-HT-IR myenteric neurons was enhanced in type 1 diabetes in a region-specific manner. Immediate insulin treatment prevents a higher hyperglycemia-induced amount of 5-HT-IR neurons and restores it to the control level in each investigated gut segment. Despite the low proportion of 5-HT-IR myenteric neurons, hyperglycemia-related changes of these neurons may play a crucial role in gastrointestinal symptoms in type 1 diabetes.

Biomarker monitoring and long-acting insulin treatment in a stress model to facilitate personalized diabetic control

Biomarker monitoring and long-acting insulin treatment in a stress model to facilitate personalized diabetic control

Catecholamines production by kidney tissue and mesangial cell culture is differentially modulated by diabetes.

Introduction:According to the International Diabetes Federation, the number of people with diabetes mellitus may reach 700 million in 2045. Catecholamines are involved in the regulation of several kidney functions. This study investigates the effects of hyperglycemia on catecholamines' metabolism in kidney tissue from control, diabetic, and insulin-treated diabetic rats, both in vivo and in vitro.Methods:Male Wistar-Hannover rats were randomized into: control, diabetic, and insulin-treated diabetic groups. Diabetes was induced by a single injection of streptozotocin, and diabetic treated group also received insulin. After 60 days, blood and kidney tissue from all groups were collected for catecholamines' quantification and mesangial cells culture.Results:diabetic rats had lower body weight, hyperglycemia, and increase water intake and diuresis. Additionally, diabetes promoted a sharp decrease in creatinine clearance compared to control group. Regarding the whole kidney extracts, both diabetic groups (treated and non-treated) had significant reduction in norepinephrine concentration. In mesangial cell culture, catecholamines' concentration were lower in the culture medium than in the intracellular compartment for all groups. Norepinephrine, epinephrine, and dopamine medium levels were increased in the diabetic group.Conclusion:The major finding of the present study was that 8 weeks of diabetes induction altered the kidney catecholaminergic system in a very specific manner, once the production of catecholamines in the excised kidney tissue from diabetic rats was differentially modulated as compared with the production and secretion by cultured mesangial cells.

Effects of insulin treatment on hepatic CYP1A1 and CYP2E1 activities and lipid peroxidation levels in streptozotocin-induced diabetic rats.

Reactive oxygen species (ROS) and lipid peroxidation (LPO) levels may increase in diabetic state and lead to oxidative stress, which plays a critical role in the progression of diabetes. There are various sources of ROS, including cytochrome P450 monooxygenases (CYP450s), which may be modulated in terms of their activities and expressions under diabetic conditions. This study is aimed to investigate the effects of streptozotocin-induced diabetes and insulin treatment on hepatic cytochrome P450 1A1 (CYP1A1) and cytochrome P450 2E1 (CYP2E1) activities and LPO levels. Methods: CYP1A1 and CYP2E1 activities were measured with ethoxyresorufin O-deethylase and p-nitrophenol hydroxylase activities, respectively. LPO levels were then corroborated via thiobarbituric acid reactive substances. Results: In diabetic rats, a marked 2.1- and 2.4-fold increase in hepatic CYP1A1 activity and 1.8- and 1.6-fold increase in hepatic CYP2E1 activity were observed compared to controls and insulin-treated diabetic rats, respectively. Hepatic LPO levels in diabetic rats did not significantly change compared to controls. However, in insulin-treated diabetic rats, LPO levels are 0.92- and 0.89-fold remarkably decrease compared to controls and diabetics, respectively. Conclusion: The present study suggests that insulin might have a useful role in the modulation of CYP1A1 and CYP2E1 activities as well as LPO levels in the liver of diabetic rats.

18-LB: Increased Acidosis and Free-Radical Production Causes Recurrent Hypoglycemia–Induced Increase in Post-ischemic ER Stress in Treated Diabetic Rats

Cerebral ischemia is a serious complication of diabetes. Treatment of diabetes causes recurrent hypoglycemia (RH). Prior RH exposure increases ischemic brain damage in insulin-treated diabetic (ITD) rats. However, the mechanism of this injury is not well understood. We showed that ischemic acidosis increases reactive oxygen species (ROS) production in RH-exposed ITD (ITD + RH) rats. As enhanced acidosis and ROS production in normal subjects activate PERK, we hypothesized that increased ischemic acidosis and ROS production in RH-exposed ITD rats activate PERK. Therefore, we determined if the administration of tris-(hydroxymethyl)-aminomethane (THAM; an alkalizing agent) and N-acetylcysteine (NAC; an antioxidant) decrease RH-induced post-ischemic PERK activation in ITD + RH rats. Wistar rats were treated with insulin 2-3 weeks after streptozotocin induced diabetes. After 2 weeks, hyperinsulinemic hypoglycemia was induced for 3 hours for 5 days. Following groups were employed: (I) ITD + vehicle (Veh) (n = 6); (II) ITD + RH + Veh (n = 7); (III) ITD + RH + THAM (0.3 M, 3 ml/kg/hr, i.v.) (n = 6); and (IV) ITD + RH + NAC (150 mg/kg, i.v.) (n = 6). Transient global cerebral ischemia was induced by bilateral carotid artery occlusion with hypotension a day after RH. THAM/Veh was infused from 15 min before to 88 minutes after ischemia. NAC/Veh was administered 5 min before and 12 h after ischemia. Based on Western blot analysis, pPERK to total PERK ratio in the ITD + RH + THAM and ITD + RH + NAC groups were significantly lower by 32% and 26%, respectively, when compared with ITD + RH + Veh group. CHOP levels in the ITD + RH + THAM and ITD + RH + NAC group were lower by 38% and 63%, respectively, when compared with ITD + RH + Veh group. Therefore, ischemic acidosis and ROS production may mediate RH-induced ischemic activation of PERK. Understanding the ischemic damage in RH-exposed ITD rats may help identify new treatments for diabetes induced increase in ischemic injury. Disclosure A.K. Rehni: None. S. Cho: None. K.R. Dave: None. Funding National Institutes of Health (NS073779); American Heart Association (18POST34070061)

Salivary molecular spectroscopy: A sustainable, rapid and non-invasive monitoring tool for diabetes mellitus during insulin treatment

Monitoring of blood glucose is an invasive, painful and costly practice in diabetes. Consequently, the search for a more cost-effective (reagent-free), non-invasive and specific diabetes monitoring method is of great interest. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy has been used in diagnosis of several diseases, however, applications in the monitoring of diabetic treatment are just beginning to emerge. Here, we used ATR-FTIR spectroscopy to evaluate saliva of non-diabetic (ND), diabetic (D) and insulin-treated diabetic (D+I) rats to identify potential salivary biomarkers related to glucose monitoring. The spectrum of saliva of ND, D and D+I rats displayed several unique vibrational modes and from these, two vibrational modes were pre-validated as potential diagnostic biomarkers by ROC curve analysis with significant correlation with glycemia. Compared to the ND and D+I rats, classification of D rats was achieved with a sensitivity of 100%, and an average specificity of 93.33% and 100% using bands 1452 cm-1 and 836 cm-1, respectively. Moreover, 1452 cm-1 and 836 cm-1 spectral bands proved to be robust spectral biomarkers and highly correlated with glycemia (R2 of 0.801 and 0.788, P < 0.01, respectively). Both PCA-LDA and HCA classifications achieved an accuracy of 95.2%. Spectral salivary biomarkers discovered using univariate and multivariate analysis may provide a novel robust alternative for diabetes monitoring using a non-invasive and green technology.

The Effect of Experimental Periodontitis on the Serum Levels of Leptin and IL-18 in Insulin-Treated Diabetic Rats

The aim of this study in rats was to assess the effect of ligature-induced periodontitis on insulin-treated type 1 diabetes in terms of serum levels of leptin and interleukin-18 (IL-18). Twenty five male Wistar rats were studied, out of 40 initially used: (1) 14 rats with insulin-treated diabetes (control, DI) and (2) 11 rats with combination of insulin-treated diabetes and periodontitis (test, DIP). For all rats, type 1 diabetes was streptozotocin-induced and insulin treatment started on day 5. For DIP group, periodontitis was induced by ligation on day 16. Serum levels of leptin and IL-18 were assessed on days 16 (baseline) and 77 (final) by using multiplex immunoassay. All rats were sacrificed on day 77 (end of the study). Insulin treatment in the newly-induced diabetes significantly increased serum leptin levels and significantly reduced serum IL-18 levels. In the newly-induced diabetes, the combination of the continuation of insulin treatment and periodontitis non-significantly increased serum leptin and IL-18 levels. Serum IL-18 levels were significantly higher for the combination of periodontitis and insulin-treated diabetes as compared to insulin-treated diabetes alone. Within its limits, this study in rats showed that experimental periodontitis induced changes in serum biomarker levels suggestive of alterations in the systemic inflammation generated by insulin-treated type 1 diabetes.

Tlr1-13, Nod1/2 and antimicrobial gene expression in the epididymis and testis of rats with alloxan-induced diabetes.

Pattern recognition receptors (PRRs) such as toll-like receptors (TLRs) and nuclear oligomerization domain (NOD) receptors along with antimicrobial proteins and peptides (AMPs) are crucial for innate immunity. The pathology of insulin-dependent diabetes mellitus is associated with the disrupted expression of TLRs, NODs and AMPs in the kidney, lungs and other organs. However, such a relation in the male reproductive tract is not yet investigated. In this study, we analysed the expression pattern of Tlr1-13, Nod1/2 receptors and AMPs (β-defensins and defensin-like proteins of the Sperm-Associated Antigen 11 (Spag11) family) in the male reproductive tissues (caput, cauda and testis) obtained from diabetic or insulin-treated diabetic or untreated control rats. Alterations in the expression pattern of Tlr1-13, Nod 1/ 2, Defb1, 2, 21, 24, 27, 30 and Spag11a/ c/ t were observed under diabetic conditions. Administration of insulin to diabetic rats could modulate the expression pattern of only some these genes. Results of our study indicate perturbed gene expression profile of Tlrs, Nod1/2, Defbs and Spag11 isoforms in the epididymis and testis of diabetic rats, and this could be one of the important reasons for the increased risk of infections in the male genital tract.

The Effect of Experimental Periodontitis on the Serum Levels of Leptin and IL-18 in Insulin-Treated Diabetic Rats

The aim of this study in rats was to assess the effect of ligature-induced periodontitis on insulin-treated type 1 diabetes in terms of serum levels of leptin and interleukin-18 (IL-18). Twenty five male Wistar rats were studied, out of 40 initially used: (1) 14 rats with insulin-treated diabetes (control, DI) and (2) 11 rats with combination of insulin-treated diabetes and periodontitis (test, DIP). For all rats, type 1 diabetes was streptozotocin-induced and insulin treatment started on day 5. For DIP group, periodontitis was induced by ligation on day 16. Serum levels of leptin and IL-18 were assessed on days 16 (baseline) and 77 (final) by using multiplex immunoassay. All rats were sacrificed on day 77 (end of the study). Insulin treatment in the newly-induced diabetes significantly increased serum leptin levels and significantly reduced serum IL-18 levels. In the newly-induced diabetes, the combination of the continuation of insulin treatment and periodontitis non-significantly increased serum leptin and IL-18 levels. Serum IL-18 levels were significantly higher for the combination of periodontitis and insulin-treated diabetes as compared to insulin-treated diabetes alone. Within its limits, this study in rats showed that experimental periodontitis induced changes in serum biomarker levels suggestive of alterations in the systemic inflammation generated by insulin-treated type 1 diabetes.