Multiple Low-dose Streptozotocin Injections Research Articles

Human umbilical cord-derived mesenchymal stromal cells improve myocardial fibrosis and restore miRNA-133a expression in diabetic cardiomyopathy

BackgroundDiabetic cardiomyopathy (DCM) is a serious health-threatening complication of diabetes mellitus characterized by myocardial fibrosis and abnormal cardiac function. Human umbilical cord mesenchymal stromal cells (hUC-MSCs) are a potential therapeutic tool for DCM and myocardial fibrosis via mechanisms such as the regulation of microRNA (miRNA) expression and inflammation. It remains unclear, however, whether hUC-MSC therapy has beneficial effects on cardiac function following different durations of diabetes and which mechanistic aspects of DCM are modulated by hUC-MSC administration at different stages of its development. This study aimed to investigate the therapeutic effects of intravenous administration of hUC-MSCs on DCM following different durations of hyperglycemia in an experimental male model of diabetes and to determine the effects on expression of candidate miRNAs, target mRNA and inflammatory mediators.MethodsA male mouse model of diabetes was induced by multiple low-dose streptozotocin injections. The effects on severity of DCM of intravenous injections of hUC-MSCs and saline two weeks previously were compared at 10 and 18 weeks after diabetes induction. At both time-points, biochemical assays, echocardiography, histopathology, polymerase chain reaction (PCR), immunohistochemistry and enzyme-linked immunosorbent assays (ELISA) were used to analyze blood glucose, body weight, cardiac structure and function, degree of myocardial fibrosis and expression of fibrosis-related mRNA, miRNA and inflammatory mediators.ResultsSaline-treated diabetic male mice had impaired cardiac function and increased cardiac fibrosis after 10 and 18 weeks of diabetes. At both time-points, cardiac dysfunction and fibrosis were improved in hUC-MSC-treated mice. Pro-fibrotic indicators (α-SMA, collagen I, collagen III, Smad3, Smad4) were reduced and anti-fibrotic mediators (FGF-1, miRNA-133a) were increased in hearts of diabetic animals receiving hUC-MSCs compared to saline. Increased blood levels of pro-inflammatory cytokines (IL-6, TNF, IL-1β) and increased cardiac expression of IL-6 were also observed in saline-treated mice and were reduced by hUC-MSCs at both time-points, but to a lesser degree at 18 weeks.ConclusionIntravenous injection of hUC-MSCs ameliorated key functional and structural features of DCM in male mice with diabetes of shorter and longer duration. Mechanistically, these effects were associated with restoration of intra-myocardial expression of miRNA-133a and its target mRNA COL1AI as well as suppression of systemic and localized inflammatory mediators.

The Effects of Resistance Exercise Training on Skeletal Muscle Metabolism and Insulin Resistance Development in Female Rodents with Type 1 Diabetes.

The etiology of insulin resistance (IR) development in type 1 diabetes mellitus (T1DM) remains unclear; however, impaired skeletal muscle metabolism may play a role. While IR development has been established in male T1DM rodents, female rodents have yet to be examined in this context. Resistance exercise training (RT) has been shown to improve IR and is associated with a lower risk of hypoglycemia onset in T1DM compared to aerobic exercise. The purpose of this study was to investigate the effects of RT on IR development in female T1DM rodents. Forty Sprague Dawley eight-week-old female rats were divided into four groups: control sedentary (CS; n = 10), control trained (CT; n = 10), T1DM sedentary (DS; n = 10), and T1DM trained (DT; n = 10). Multiple low-dose streptozotocin injections were used to induce T1DM. Blood glucose levels were maintained in the 4-9 mmol/l range with intensive insulin therapy. CT and DT underwent weighted ladder climbing 5 days/week for six weeks. Intravenous glucose tolerance tests (IVGTT) were conducted on all animals following the six-week period. Results demonstrate that DS animals exhibited significantly increased weekly blood glucose measures compared to all groups including DT (p < 0.0001), despite similar insulin dosage levels. This was concomitant with a significant increase in insulin-adjusted area under the curve following IVGTT in DS (p < 0.05), indicative of a reduction in insulin sensitivity. Both DT and DS exhibited greater serum insulin concentrations compared to CT and CS (p < 0.05). DS animals also exhibited significantly greater glycogen content in white gastrocnemius muscle compared to CS and DT (p < 0.05), whereas DT and DS animals exhibited greater p-Akt: Akt ratio in the white vastus lateralis muscle and citrate synthase activity in the red vastus lateralis muscle compared to CS and CT (p < 0.05). These results indicate that female rodents with T1DM develop poor glycemic control and IR which can be attenuated with RT, possibly related to differences in intramyocellular glycogen content.

Deletion of carboxypeptidase E in beta cells disrupts proinsulin processing but does not lead to spontaneous development of diabetes in mice.

Carboxypeptidase E (CPE) facilitates the conversion of prohormones into mature hormones and is highly expressed in multiple neuroendocrine tissues. Carriers of CPE mutations have elevated plasma proinsulin and develop severe obesity and hyperglycemia. We aimed to determine whether loss of Cpe in pancreatic beta cells disrupts proinsulin processing and accelerates development of diabetes and obesity in mice. Pancreatic beta cell-specific Cpe knockout mice (βCpeKO; Cpefl/fl x Ins1Cre/+) lack mature insulin granules and have elevated proinsulin in plasma; however, glucose-and KCl-stimulated insulin secretion in βCpeKO islets remained intact. High fat diet-fed βCpeKO mice showed comparable weight gain and glucose tolerance compared to Wt littermates. Notably, beta-cell area was increased in chow-fed βCpeKO mice and beta-cell replication was elevated in βCpeKO islets. Transcriptomic analysis of βCpeKO beta cells revealed elevated glycolysis and Hif1α-target gene expression. Upon high glucose challenge, beta cells from βCpeKO mice showed reduced mitochondrial membrane potential, increased reactive oxygen species, reduced MafA, and elevated Aldh1a3 transcript levels. Following multiple low-dose streptozotocin treatment, βCpeKO mice had accelerated hyperglycemia with reduced beta-cell insulin and Glut2 expression. These findings suggest that Cpe and proper proinsulin processing are critical in maintaining beta cell function during the development of hyperglycemia.

581-P: The Effects of Resistance Training on Insulin Resistance Development in Female Rodents with Type 1 Diabetes

The etiology of insulin resistance (IR) development in type 1 diabetes (T1D) remains unclear; however, impaired glucose metabolism in skeletal muscle may play a role. While IR development has been established in male rodents with T1D, female rodents have yet to be examined in this context. Resistance exercise training (RT) has been shown to improve IR and is associated with a lower risk of hypoglycemia onset in T1D compared to aerobic exercise. The purpose of this study was to investigate the effects of RT on IR development in female rodents with T1D. Forty Sprague-Dawley 8-week-old female rats were divided into four groups: control sedentary (CS; n=10), control trained (CT; n=10), T1D sedentary (DS; n=10), T1D trained (DT; n=10). Multiple low-dose Streptozotocin injections (20 mg/kg each day for 7 consecutive days) were used to induce T1D. Blood glucose levels were maintained in normal range (4-9mmol/L) with one implanted insulin pellet (2IU/day). CT and DT underwent weighted ladder climbing 5 days/week for 6 weeks. Intravenous glucose tolerance tests (IVGTT) were conducted on all animals during weeks 4 and 7. Results demonstrate that DS animals exhibited significantly increased weekly blood glucose measures compared to all groups including DT (p&amp;lt;0.05), despite similar insulin dosage levels. This was concomitant with a significant increase in area under the curve following IVGTT from week 4 to week 7 in DS (p&amp;lt;0.05), indicative of a reduction in insulin sensitivity. DS animals also exhibited significantly greater glycogen content in white gastrocnemius muscle compared to all groups (p&amp;lt;0.05). These results indicate that female rodents with T1D develop poor glycemic control and IR which can be attenuated with RT, possibly related to differences in glycogen content within muscle. This data supports the negative role of elevated muscle glycogen on insulin sensitivity in T1D and the potential role of RT in ameliorating these metabolic changes. Disclosure M.Sammut: None. D.Mcbey: None. C.Melling: None.

Assessment of executive function in a rodent model of Type 1 diabetes

This study examined the impact of Type 1 Diabetes Mellitus (T1DM) on executive function using a series of operant conditioning-based tasks in rats. Sprague Dawley rats were randomized to either non-diabetic (n = 12; 6 male) or diabetic (n = 14; 6 male) groups. Diabetes was induced using multiple low-dose streptozotocin injections. All diabetic rodents were insulin-treated using subcutaneous insulin pellet implants (9–15 mM). At week 14 of the study, rats were placed on a food restricted diet to induce 5–10 % weight loss. Rodents were familiarized and their set-shifting ability was tested on a series of tasks that required continuous adjustments to novel stimulus-reward paradigms in order to receive food rewards. Results showed no differences in the number of trials, nor number and type of errors made to successfully complete each task between groups. Therefore, we report no differences in executive function, or more specifically set-shifting abilities between non-diabetic and diabetic rodents that receive insulin.

15-OR: Mitochondrial NRF2 in ß-cell Oxidative Stress Response

β-cells are highly metabolic and vulnerable to a variety of exogenous stressors, including excess glucose and proinflammatory cytokines. Many of these stressors elicit the accumulation of reactive oxygen species (ROS) , which can cause oxidative damage and lead to cell death. To restore homeostasis in the face of accumulating ROS, all cells, including β-cells, are equipped with adaptive stress response mechanisms such as the antioxidant response and autophagy. The antioxidant response is primarily driven by nuclear translocation of the transcription factor, NRF2. However, recently we observed a pool of NRF2 in the mitochondria that is associated with both the antioxidant response and a selective form of autophagy, known as mitophagy. We therefore hypothesized that NRF2 mediates coupling of mitophagy with the antioxidant response in the β-cell and is essential for restoration of β-cell homeostasis under conditions of increased ROS. We first asked whether loss of murine NRF2 was associated with increased susceptibility to β-cell damage induced by the toxic glucose analog, streptozotocin (STZ) . Multiple low dose STZ injections (45mg/kg for 5 consecutive days) rendered whole-body NRF2 knockout mice diabetic more quickly than wild type controls. To study the role of β-cell NRF2 in autophagy/antioxidant response coupling, we then generated a β-cell specific NRF2 knockout mouse (NRF2Δβ) by breeding NRF2 floxed mice with mice bearing the Ins1Cre/wt allele. NRF2Δβ mice appear to be phenotypically normal until at least 20 weeks of age and do not develop spontaneous diabetes. We then set out to determine how loss of β-cell NRF2 affects the mitochondria by both immunofluorescence analysis and transmission electron microscopy of isolated pancreata. We observed rounded β-cell mitochondrial morphology in the absence of NRF2. Collectively, our data are consistent with a role for NRF2 in β-cell mitochondrial function and suggest that NRF2 is required for proper response to β-cell damage. Disclosure A.Novak: n/a. J.Crowder: None. M.Marasco: Other Relationship; Eli Lilly and Company. A.K.Linnemann: Consultant; Janssen Research &amp; Development, LLC. Funding NIDDK (R01DK124380)

Serum Amyloid A3 Promoter-Driven Luciferase Activity Enables Visualization of Diabetic Kidney Disease.

The early detection of diabetic nephropathy (DN) in mice is necessary for the development of drugs and functional foods. The purpose of this study was to identify genes that are significantly upregulated in the early stage of DN progression and develop a novel model to non-invasively monitor disease progression within living animals using in vivo imaging technology. Streptozotocin (STZ) treatment has been widely used as a DN model; however, it also exhibits direct cytotoxicity to the kidneys. As it is important to distinguish between DN-related and STZ-induced nephropathy, in this study, we compared renal responses induced by the diabetic milieu with two types of STZ models: multiple low-dose STZ injections with a high-fat diet and two moderate-dose STZ injections to induce DN. We found 221 genes whose expression was significantly altered during DN development in both models and identified serum amyloid A3 (Saa3) as a candidate gene. Next, we applied the Saa3 promoter-driven luciferase reporter (Saa3-promoter luc mice) to these two STZ models and performed in vivo bioluminescent imaging to monitor the progression of renal pathology. In this study, to further exclude the possibility that the in vivo bioluminescence signal is related to renal cytotoxicity by STZ treatment, we injected insulin into Saa3-promoter luc mice and showed that insulin treatment could downregulate renal inflammatory responses with a decreased signal intensity of in vivo bioluminescence imaging. These results strongly suggest that Saa3 promoter activity is a potent non-invasive indicator that can be used to monitor DN progression and explore therapeutic agents and functional foods.

Towards the Development of a Female Animal Model of T1DM Using Hyaluronic Acid Nanocoated Cell Transplantation: Refinements and Considerations for Future Protocols.

Female mice (Black 6 strain) (C57BL/6) aged 6 weeks were subject to low dose streptozotocin (STZ) treatment for five consecutive days to mimic type 1 diabetes mellitus (T1DM) with insulitis. At two weeks after STZ injections, evaluation of the elevated glucose levels was used to confirm diabetes. The diabetic mice were then subject to the transplantation of pancreatic β-cells (MIN-6 line). Four groups of mice were studied. The first group was injected with saline-only acting as the placebo surgery control, also known as SHAM group, the second and third groups were injected with MIN-6 single cells and polyethylene glycol-modified dipalmitoyl-glycerol-phosphatidyl ethanolamine (PEG-DPPE) modified MIN-6 single cells (500 µg per 1.106 cells), respectively, while the fourth group was injected with hyaluronic acid (HA)-coated MIN-6 single cells (5 bilayers). At seven- and fourteen-days following transplantation, the mice were euthanised. The renal and pancreatic tissues were then collected and histologically analysed. The induction of diabetes in female mice, through five-consecutive daily STZ injections resulted in inconsistent glycaemic levels. Interestingly, this shows an incomplete diabetes induction in female mice, of which we attribute to sex dimorphism and hormonal interferences. Transplantation failure of free-floating encapsulated cells was unable to decrease blood glucose hyperglycaemia to physiological ranges. The result is attributed to deprived cell–cell interactions, leading to decreased β-cells functionality. Overall, we highlight the necessity of refining T1DM disease models in female subjects when using multiple low-dose STZ injections together with transplantation protocols. Considerations need to be made regarding the different developmental stages of female mice and oestrogen load interfering with pancreatic β-cells susceptibility to STZ. The use of pseudo islets, cell aggregates and spheroids are sought to improve transplantation outcome in comparison to free-floating single cells.

Myeloid‐Specific Inhibition of GHS‐R on Metabolic Dysregulation in Skeletal Muscle During Experimental Type 2 Diabetes

Skeletal muscle plays a significant role in insulin-stimulated glucose disposal, which is a focal point in type 2 diabetes (T2D) research.The complex and highly regulated relationship between insulin secretion and insulin-mediated glucose disposal continue to be debated in the literature.Emerging evidence suggests that chronic-low grade inflammation and its associated inflammatory responses play important roles in T2D pathophysiology.Macrophages are a major source of inflammatory cytokines and have been demonstrated to be responsible for the inflammation of many tissues. Growth Hormone Secretagogue receptor (GHS-R), the known receptor of hormone ghrelin, has roles in both glucose homeostasis and inflammation. Insulin resistance is a major risk factor for T2D and muscle is a key tissue associated with insulin resistance. Little is known whether macrophage GHS-R affects the metabolic dysregulation of skeletal muscle in T2D. METHODS This study utilized a high fat diet and multiple low dose streptozotocin injections to generate a T2D model to evaluate the effects of myeloid-specific inhibition of GHS-R (LysM-Cre;Ghsrf/f) on signaling pathways associated with metabolic dysregulation in skeletal muscle.Gastrocnemius muscles were examined for common markers of insulin signaling and protein anabolism.Gene expression and phosphorylation state of proteins were measured using Western blotting. RESULTS Our data indicate that the GHS-R deficiency in myeloid cells resulted in an increase of mTORC1 activity, showing a suppression of phosphorylation on AKT at Ser473 (â25%; p<0.05).AKT is phosphorylated at Ser473 by mTORC2, and the formation/activity of mTORC2 activity is generally suppressed by mTORC1.Although not statistically significant, mTORC1 activity appeared to be elevated, supported by downstream targets of mTORC1 of higher phosphorylation ratios in both S6K ribosomal at Ser235/236 (P=0.11) and 4EBP1 at Thr37/46 (P=0.18).It is to note that upstream canonical AKT activation was not affected by treatment based on the phosphorylation of its substrate GSK3B (P=0.99).In addition, the knockout model also showed a lower phosphorylation of P-ACC suggesting a lower activity of AMPK, an insulin-independent pathway of glucose uptake known to suppresses mTORC1 activity. CONCLUSION Results from the present study suggests that a myeloid-specific knockout of GHSR1a plays a role in the metabolic shift of skeletal muscle towards significant decrease in mTORC2, likely due to augmented mTORC1 activity.

Spleen-Kidney Supplementing Formula Alleviates Renal Fibrosis in Diabetic Rats via TGF-β1-miR-21-PTEN Signaling Pathway.

Background Spleen-Kidney Supplementing Formula (SKSF), composed of 6 raw Chinese herbs and proposed based on the therapeutic principle of supplementing spleen-kidney and clearing the extra heat of stomach-lung, is effective in the treatment of type 2 diabetes mellitus (T2DM). AimThis study aims to investigate the mechanism of SKSF to alleviate renal fibrosis in diabetic rats. MethodsT2DM model was induced by high-fat diet and multiple injections of low-dose streptozotocin. After 8-week intervention, samples were collected for detection. ResultsSKSF decreased fasting blood glucose, glycosylated hemoglobin A1c, blood urea nitrogen, uric acid, urea, 24-hour urine protein, and KW/BW ratio, while it increased creatinine clearance rate of T2DM rats. Meanwhile, SKSF attenuated the renal fibrosis and improved the morphology and structure of renal tissue. Furthermore, SKSF significantly reduced the expression level of plasma miR-21 and TGF-β1 protein level and increased PTEN protein level. ConclusionSKSF could attenuate the renal damage and renal fibrosis induced in T2DM, which may be related to its regulation on the expressions of TGF-β1, PTEN, and miR-21.

Sodium Butyrate Ameliorates Streptozotocin-Induced Type 1 Diabetes in Mice by Inhibiting the HMGB1 Expression.

Type 1 diabetes (T1D) is an autoimmune disease characterized by the immune cell-mediated progressive destruction of pancreatic β-cells. High-mobility group box 1 protein (HMGB1) has been recognized as a potential immune mediator to enhance the development of T1D. So we speculated that HMGB1 inhibitors could have anti-diabetic effect. Sodium butyrate is a short fatty acid derivative possessing anti-inflammatory activity by inhibiting HMGB1. In the current study, we evaluated the effects of sodium butyrate in streptozotocin (STZ)-induced T1D mice model. Diabetes was induced by multiple low-dose injections of STZ (40 mg/kg/day for 5 consecutive days), and then sodium butyrate (500 mg/kg/day) was administered by intraperitoneal injection for 7 consecutive days after STZ treatment. Blood glucose, incidence of diabetes, body weight, pancreatic histopathology, the amounts of CD4+T cell subsets, IL-1β level in serum and pancreatic expressions levels of HMGB1, and NF-κB p65 protein were analyzed. The results showed that sodium butyrate treatment decreased blood glucose and serum IL-1β, improved the islet morphology and decreased inflammatory cell infiltration, restored the unbalanced Th1/Th2 ratio, and down-regulated Th17 to normal level. In addition, sodium butyrate treatment can inhibit the pancreatic HMGB1 and NF-κB p65 protein expression. Therefore, we proposed that sodium butyrate should ameliorate STZ-induced T1D by down-regulating NF-κB mediated inflammatory signal pathway through inhibiting HMGB1.

Optimal LDR to Protect the Kidney From Diabetes: Whole-Body Exposure to 25 mGy X-rays Weekly for 8 Weeks Efficiently Attenuates Renal Damage in Diabetic Mice.

To explore an optimal frequency of whole-body low-dose radiation (LDR) to protect the kidney from diabetes, type 1 diabetic mice were induced with multiple injections of low-dose streptozotocin in male C57BL/6J mice. Diabetic or age-matched normal mice received whole-body exposure to 12.5 or 25 mGy either every other day or weekly for 4 or 8 weeks. Diabetes decreased the urinary creatinine and increased the microalbumin in urine, renal accumulation of 3-nitrotyrosine and 4-hydroxynonenal, and renal expression of collagen IV and fibronectin. All these renal pathological and functional changes in diabetic mice were significantly attenuated by exposure to LDR at all regimens. However, whole-body exposure of diabetic mice to 25 mGy weekly and to 12.5 mGy every other day for 8 weeks provided a better prevention of diabetic nephropathy than other LDR regimens. Furthermore, whole-body exposure to 25 mGy weekly for 8 weeks showed no detectable effect on the kidney of normal mice, but whole-body exposure to normal mice at 12.5 mGy every other day for 8 weeks increased urinary microalbumin and renal expression of collagen IV and fibronectin. These results suggest that whole-body exposure to LDR at 25 mGy weekly is the optimal condition of LDR to protect the kidney from diabetes.

Glycosphingolipids promote pro-atherogenic pathways in the pathogenesis of hyperglycemia-induced accelerated atherosclerosis.

Three out of four people with diabetes will die of cardiovascular disease. However, the molecular mechanisms by which hyperglycemia promotes atherosclerosis, the major underlying cause of cardiovascular disease, are not clear. Three distinct models of hyperglycemia-associated accelerated atherosclerosis were used to identify commonly altered metabolites and pathways associated with the disease. Normoglycemic apolipoprotein-E-deficient mice served as atherosclerotic control. Hyperglycemia was induced by multiple low-dose streptozotocin injections, or by introducing a point-mutation in one copy of insulin-2 gene. Glucosamine-supplemented mice, which experience accelerated atherosclerosis to a similar extent as hyperglycemia-induced models without alterations in glucose or insulin levels, were also included in the analysis. Untargeted plasma metabolomics were used to investigate hyperglycemia-associated accelerated atherosclerosis in three disease models. The effect of specific significantly altered metabolites on pro-atherogenic processes was investigated in cultured human vascular cells. Hyperglycemic and glucosamine-supplemented mice showed distinct metabolomic profiles compared to controls. Meta-analysis of three disease models revealed 62 similarly altered metabolite features (FDR-adjusted p < 0.05). Identification of shared metabolites revealed alterations in glycerophospholipid and sphingolipid metabolism, and pro-atherogenic processes including inflammation and oxidative stress. Post-multivariate and pathway analyses indicated that the glycosphingolipid pathway is strongly associated with hyperglycemia-induced accelerated atherosclerosis in these atherogenic mouse models. Glycosphingolipids induced oxidative stress and inflammation in cultured human vascular cells. Glycosphingolipids are strongly associated with hyperglycemia-induced accelerated atherosclerosis in three distinct models. They also promote pro-atherogenic processes in cultured human cells. These results suggest glycosphingolipid pathway may be a potential therapeutic target to block or slow atherogenesis in diabetic patients.

Potential Protective Mechanism in the Cardiac Microvascular Injury.

Cardiac microvascular injury often occurs in patients with type 2 diabetes mellitus (T2DM) who develop hyperglycemia and hyperlipidemia. However, besides reported contradictory roles in cardiac diseases, the function of TRPV1 (transient receptor potential vanilloid 1) in cardiac microvessels is not well defined. This study was performed to determine the detailed role of TRPV1 in cardiac microvascular endothelial cells (CMECs) in T2DM. T2DM mice were established by multiple injections of low-dose streptozotocin and high-fat feeding. CMECs were cultured separately in mediums of normal glucose, high glucose (HG), high fatty acid (HF), and HG plus HF (HG-HF). HG-HF inhibited TRPV1 expression in CMECs, reducing cellular Ca2+ content ([Ca2+]i). T2DM impaired cardiac function, disturbed glucose uptake, and damaged microvascular barrier, which were further aggravated by TRPV1-/- Exposure to HG-HF, particularly in TRPV1-/- CMECs, led to a higher level of apoptosis and a lower level of nitric oxide production in viable CMECs. HG-HF markedly enhanced generation of reactive oxygen species and nitrotyrosine, especially in the absence of TRPV1. H2O2 administration reduced TRPV1 expression in CMECs. HG-HF significantly depressed expression of PGC-1α (peroxisome proliferator-activated receptor-γ coactivator-1α) and OPA1 (optic atrophy 1) by reducing [Ca2+]i, whereas OPA1 supplementation partly reversed those detrimental effects induced by TRPV1-/- Furthermore, capsaicin treatment not only attenuated CMECs injury induced by HG-HF but also mitigated cardiac microvascular injury induced by T2DM. Collectively, T2DM leads to cardiac microvascular injury by exacerbating the vicious circle of TRPV1 blockage and reactive oxygen species overload. Long-term capsaicin can protect cardiac microvessels against T2DM via suppressing oxidative/nitrative stress mediated by TRPV1/Ca2+/PGC-1α/OPA1 pathway in CMECs.

Abstract 215: Glycosphingolipids Contribute to Pro-atherogenic Pathways in the Pathogenesis of Hyperglycemia-induced Atherosclerosis

Three out of every four people with diabetes will die of cardiovascular disease. However, the molecular mechanisms by which diabetes promotes atherosclerosis are not clear. In this study, comprehensive metabolomic techniques were used to investigate the molecular mechanisms by which hyperglycemia promotes accelerated atherogenesis in three distinct disease mouse models. Normoglycemic apolipoprotein E-deficient mice served as the atherosclerotic control. Hyperglycemia was induced by multiple low-dose streptozotocin injections or by introducing a point mutation in one copy of the insulin-2 gene. Glucosamine-supplemented mice, which experience accelerated atherosclerosis to a similar extent as the hyperglycemia-induced models, without alterations in the levels of glucose or insulin, were also included in the analysis. Mice with accelerated atherosclerosis showed distinct metabolomic profiles compared to the controls. We detected 6369 metabolite features in the plasma of each mouse. Second-order analysis of pair comparisons between each disease model and the control resulted in 62 commonly altered features (p&lt;0.05). Identification of shared metabolites revealed alterations in glycerophospholipid and sphingolipid metabolisms, and pro-atherogenic processes including inflammation and oxidative stress. Post-multivariate and pathway analyses indicated glycosphingolipid metabolism is the most significantly altered pathway. Glycosphingolipid metabolites induced oxidative stress and inflammation in cultured human vascular cells including macrophages, endothelial and smooth muscle cells. Treatment with a known antioxidant, α-tocopherol, reduced oxidative stress and inflammation induced by glycosphingolipids. Our findings suggest that the glycosphingolipid pathway contributes to pro-atherogenic pathways in the pathogenesis of hyperglycemia-induced atherosclerosis, making it a potential therapeutic target to block or slow atherogenesis in diabetic patients.

Investigation of insulin resistance in the popularly used four rat models of type-2 diabetes

Animal models are widely used to develop drugs for treating diabetes mellitus (DM). Insulin resistance (IR) is one of the main problems in type-2 DM (T2DM). Streptozotocin (STZ) is used to damage pancreatic cells for induction of DM. Many rat models were applied in research as T2DM. However, the degree of IR in each model is unknown. In the present study, IR and insulin signaling were compared in four models of type 2 diabetes: rats fed a fructose-rich chow for 8 weeks, rats feed high-fat chow for 4 weeks followed by injection with streptozotocin (35 mg/kg, i.p.), rats injected with a single low dose streptozotocin (45 mg/kg, i.p.), and rats injected with a single dose of nicotinamide followed by a single high dose of streptozotocin (60 mg/kg, i.p.). Values from these determinations in diabetic rats showing the order that insulin resistance is most marked in rats received fructose-rich chow followed by high-fat diet before STZ injection induced model (HFD/STZ rats), and rats injected with low dose of STZ but it is less marked in rats induced by nicotinamide and STZ. Additionally, insulin secretion was reduced in three rat models except the rats receiving fructose-rich chow. Western blots also showed the same changes in phosphorylation of IRS-1 or Akt using soleus muscle from each model. The obtained data suggest a lack of pronounced IR in the rats with acute diabetes induced by nicotinamide and STZ while IR is markedly identified in rats fed fructose-rich chow. However, the increase of plasma glucose levels in fructose-rich chow-fed rats was not so significant as other groups. Therefore, HFD/STZ rats is an appropriate and stable animal model which is analogous to the human T2DM through a combination of high-fat diet with multiple low-dose STZ injections.

Diabetes Activates Periodontal Ligament Fibroblasts via NF-κB In Vivo

Diabetes mellitus increases periodontitis and pathogenicity of the oral microbiome. To further understand mechanisms through which diabetes affects periodontitis, we examined its impact on periodontal ligament fibroblasts in vivo and in vitro. Periodontitis was induced by inoculation of Porphyromonas gingivalis and Fusobacterium nucleatum in normoglycemic and diabetic mice. Diabetes, induced by multiple low-dose injections of streptozotocin increased osteoclast numbers and recruitment of neutrophils to the periodontal ligament, which could be accounted for by increased CXC motif chemokine 2 (CXCL2) and receptor activator of nuclear factor kappa B ligand (RANKL) expression by these cells. Diabetes also stimulated a significant increase in nuclear factor kappa B (NF-κB) expression and activation in periodontal ligament (PDL) fibroblasts. Surprisingly, we found that PDL fibroblasts express a 2.3-kb regulatory unit of Col1α1 (collagen type 1, alpha 1) promoter typical of osteoblasts. Diabetes-enhanced CXCL2 and RANKL expression in PDL fibroblasts was rescued in transgenic mice with lineage-specific NF-κB inhibition controlled by this regulatory element. In vitro, high glucose increased NF-κB transcriptional activity, NF-κB nuclear localization, and RANKL expression in PDL fibroblasts, which was reduced by NF-κB inhibition. Thus, diabetes induces changes in PDL fibroblast gene expression that can enhance neutrophil recruitment and bone resorption, which may be explained by high glucose–induced NF-κB activation. Furthermore, PDL fibroblasts express a regulatory element in vivo that is typical of committed osteoblasts.

Combination of high-fat/high-fructose diet and low-dose streptozotocin to model long-term type-2 diabetes complications

The epidemic of type 2 diabetes mellitus (T2DM) is fueled by added fructose consumption. Here, we thus combined high-fat/high-fructose diet, with multiple low-dose injections of streptozotocin (HF/HF/Stz) to emulate the long-term complications of T2DM. HF/HF/Stz rats, monitored over 56 weeks, exhibited metabolic dysfunctions associated with the different stages of the T2DM disease progression in humans: an early prediabetic phase characterized by an hyperinsulinemic period with modest dysglycemia, followed by a late stage of T2DM with frank hyperglycemia, normalization of insulinemia, marked dyslipidemia, hepatic fibrosis and pancreatic β-cell failure. Histopathological analyses combined to [18F]-FDG PET imaging further demonstrated the presence of several end-organ long-term complications, including reduction in myocardial glucose utilization, renal dysfunction as well as microvascular neuropathy and retinopathy. We also provide for the first time a comprehensive µ-PET whole brain imaging of the changes in glucose metabolic activity within discrete cerebral regions in HF/HF/Stz diabetic rats. Altogether, we developed and characterized a unique non-genetic preclinical model of T2DM adapted to the current diet and lifestyle that recapitulates the major metabolic features of the disease progression, from insulin resistance to pancreatic β-cell dysfunction, and closely mimicking the target-organ damage occurring in type 2 diabetic patients at advanced stages.

Effects of Modified Sanzi Yangqin Decoction on Tyrosine Phosphorylation of IRS-1 in Skeletal Muscle of Type 2 Diabetic Rats.

This study aimed to investigate the effect of Modified Sanzi Yangqin Decoction on tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) in skeletal muscle of type 2 diabetic rats. The rat model of type 2 diabetes was induced by high-fat diet and multiple low-dose streptozotocin injections. Diabetic model rats were randomly divided into 5 groups: the model control group, the metformin group, and Modified Sanzi Yangqin Decoction groups of low, medium, and high doses. OGTT was conducted every two weeks during treatment period. At the end of the treatment, the fasting blood glucose (FBG) level and the fasting C-peptide level were measured to calculate insulin resistance index. The levels of IRS-1, p-IRS-1(Tyr895), and protein tyrosine phosphates 1B (PTP1B) in skeletal muscle were also measured. Modified Sanzi Yangqin Decoction significantly reduced the FBG level, increased the fasting C-peptide level, and lowered the insulin resistance index in type 2 diabetic rats. It also significantly increased the protein level of p-IRS-1(Tyr895) and reduced the PTP1B protein level in skeletal muscle of type 2 diabetic rats. Modified Sanzi Yangqin Decoction increases tyrosine phosphorylation of IRS-1 in skeletal muscle of type 2 diabetic rats, which results from the increase of p-IRS-1(Tyr895) protein and is related to the suppression of PTP1B protein.

200 Nitric oxide promotes insulin-independent glucose uptake and preserves cardiac function and energetics in diabetes

Introduction In the presence of diabetes (DM), myocardial glucose uptake and glycolysis are impaired and the heart rapidly adapts to use exclusively fatty acids (FA) for ATP generation. This maladaptation is believed to play a key role in the development of a cardiomyopathy over time. Here, we show that stimulating myocardial nitric oxide synthase (NOS) activity is sufficient to alleviate myocardial metabolic inflexibility, improve energy metabolism and prevent LV dysfunction in DM by increasing myocardial insulin-independent glucose transport. Methods Myocardial-specific overexpression of GTP cyclohydrolase I (mGCH1) was used to increase both tetrahydrobiopterin (BH4) and NOS activity in cardiomyocytes. Diabetes mellitus (DM) was induced by multiple low-dose streptozotocin injections (vs sham). PCr/ATP ratio was measured in perfused hearts using 31 P-MRS, glucose transport estimated by deoxy-glucose uptake, and oxygen consumption rate (OCR) of intact cardiomyocytes using a phosphorescent probe. Results As expected, sham-injected mGCH1 transgenic hearts had higher BH4 levels and constitutive NOS activity compared with WT. 12 weeks after DM induction, LV dysfunction developed in WT mice but not in mGCH1 mice, in the absence of changes in myocardial BH4 content and NOS activity in either group. WT diabetic hearts had a lower PCr/ATP ratio (1.32±0.1 vs 1.73±0.1, p Myocardial GCH1 overexpression was associated with a higher protein levels of the insulin-independent glucose transporter, GLUT-1 (p Conclusions Our study reveals that a myocardial increase in BH4 and NOS activity is sufficient to maintain a favourable substrate utilisation and preserve cardiac mitochondrial function in the presence of DM. This work provides new insight into the potential metabolic triggers of diabetic cardiomyopathy and suggests exciting new targets for BH4-based therapeutics.