Diabetic Apolipoprotein E-deficient Mice Research Articles

Macular pigments produced from microalga Chlorella sp. and applied to alleviate the pathogenic process in diabetic mice

Macular pigments (MP), which consist of lutein and zeaxanthin, have antioxidant capabilities and are widely used in eye healthcare. However, few studies have focused on the protective mechanisms of MP against diabetes mellitus. In addition, the advantages of MP produced by microalgae include non-competition with grains for land, year-round harvesting, and rapid growth. Therefore, this study aims to investigate the MP produced and extracted from the microalga Chlorella sp. AT1 and assess its impact on mitigating retinopathy and atherosclerotic damage in diabetic apolipoprotein E-deficient mice (ApoE−/−) subjected to high-fat diet and streptozotocin treatment. The results showed that the major components of MP were lutein and zeaxanthin in a 5:1 ratio. MP supplementation significantly reduced glucose tolerance and insulin resistance by 25 and 41 %, respectively, in comparison to the diabetic group. MP supplementation resulted in the moderation of various inflammatory cytokines and lowered the serum levels of disease markers in comparison to the control group. In addition, the expression levels of cellular antioxidant enzymes were significantly elevated, aligning with a reduction in lipid peroxidation after MP supplementation, in comparison to the diabetic group. Pathological changes in the outer nuclear layer and aortic plaques in diabetic ApoE−/− mice were significantly alleviated by MP supplementation. In summary, microalgal MP alleviated the symptoms of type 2 diabetes mellitus in the diabetic ApoE−/− mice. Consequently, MP holds the potential to serve as a beneficial ingredient in healthy food, contributing to biological efficacy by scavenging free radicals and stabilizing blood glucose levels.

The Synthetic Flavonoid Hidrosmin Improves Endothelial Dysfunction and Atherosclerotic Lesions in Diabetic Mice.

In diabetes, chronic hyperglycemia, dyslipidemia, inflammation and oxidative stress contribute to the progression of macro/microvascular complications. Recently, benefits of the use of flavonoids in these conditions have been established. This study investigates, in two different mouse models of diabetes, the vasculoprotective effects of the synthetic flavonoid hidrosmin on endothelial dysfunction and atherogenesis. In a type 2 diabetes model of leptin-receptor-deficient (db/db) mice, orally administered hidrosmin (600 mg/kg/day) for 16 weeks markedly improved vascular function in aorta and mesenteric arteries without affecting vascular structural properties, as assessed by wire and pressure myography. In streptozotocin-induced type 1 diabetic apolipoprotein E-deficient mice, hidrosmin treatment for 7 weeks reduced atherosclerotic plaque size and lipid content; increased markers of plaque stability; and decreased markers of inflammation, senescence and oxidative stress in aorta. Hidrosmin showed cardiovascular safety, as neither functional nor structural abnormalities were noted in diabetic hearts. Ex vivo, hidrosmin induced vascular relaxation that was blocked by nitric oxide synthase (NOS) inhibition. In vitro, hidrosmin stimulated endothelial NOS activity and NO production and downregulated hyperglycemia-induced inflammatory and oxidant genes in vascular smooth muscle cells. Our results highlight hidrosmin as a potential add-on therapy in the treatment of macrovascular complications of diabetes.

P722Glycemic control with canagliflozin, a SGLT-2 inhibitor, attenuates atherosclerosis and endothelial dysfunction in diabetic apolipoprotein e-deficient mice

Abstract Background Canagliflozin is a SGLT-2 inhibitor, a novel type of drug for type 2 diabetes mellitus treatment. Recent studies have shown that SGLT-2 inhibitors reduce cardiovascular events, although the mechanism is still unknown. Purpose The aim of our study was to examine the effect of canagliflozin on vascular endothelial cell. Method Eight-week-old apolipoprotein E-deficient (ApoE−/−) mice were treated with streptozotocin (STZ, 75 mg/kg/day) in three consecutive days by intraperitoneal injection to induce diabetes. Diabetic ApoE−/− mice were treated with canagliflozin (30 mg/kg/day) by gavage for 12 weeks or 8 weeks to examine its effect on atherosclerosis or endothelial function, respectively. Results Canagliflozin significantly decreased blood glucose level (P<0.001), triglyceride level (P<0.05), and total cholesterol level (P<0.05). Sudan IV staining on the aortic arch showed that canagliflozin decreased atherosclerotic lesion progression (P<0.05). Histological analyses using atherosclerotic lesions in the aortic root showed that canagliflozin reduced lipid disposition (P<0.01), macrophage accumulation (P<0.001, and expression of adhesion molecules such as ICAM-1, and VCAM-1 (P<0.01, and P<0.05 respectively). Canagliflozin also attenuated the development of endothelial dysfunction as determined by acetylcholine-dependent vasodilation (P<0.05), and reduced the expression of inflammatory molecules, such as ICAM-1 and VCAM-1 (P<0.01), also MCP-1, F4/80, IL6, and iNOS (P<0.05) in the aorta. Canagliflozin reduced oxidative stress as determined by the reduction of the expression of NOX2, NOX4, p22phox, p47phox in the aorta and by the urinary excretion of 8-OHdG. In in vitro experiment using human umbilical vein endothelial cells (HUVEC), methylglyoxal (MGO), a precursor of advanced glycation end products, significantly increased the expression of inflammatory molecules such as ICAM-1, MCP-1, and p22phox in (P<0.05, respectively). MGO also decreased the phosphorylation of eNOSser1177 and Akt, and increased phosphorylation of P38 MAPK in HUVEC. Conclusion Glucose lowering effect by canagliflozin attenuates the development of endothelial dysfunction and atherogenesis in diabetic ApoE−/− mice. Anti-inflammatory effect due to the reduction of glucose toxicity on endothelial cells might be one of the mechanisms.

Interplay between HSP90 and Nrf2 pathways in diabetes-associated atherosclerosis

IntroductionOxidative stress and inflammation are determinant processes in the development of diabetic vascular complications. Heat shock protein 90 (HSP90) overexpression in atherosclerotic plaques plays a role in sustaining inflammatory mechanisms, and its specific inhibition prevents atherosclerosis. The present work investigates, in a mouse model of diabetes-driven atherosclerosis, whether atheroprotection by pharmacological HSP90 inhibition is accomplished by bolstering antioxidant defense mechanisms headed by nuclear factor erythroid-derived 2-like 2 (Nrf2). MethodsStreptozotocin-induced diabetic apolipoprotein E-deficient mice were randomized to receive vehicle or HSP90 inhibitor (17-dimethylaminoethylamino-17-demethoxygeldanamycin, 4mg/kg) for 10 weeks. Aortic root sections were analyzed for plaque size and composition, transcription factor activity, and expression of inflammatory and antioxidant markers. In vitro studies were performed in murine macrophages cultured under hyperglycemic conditions. ResultsTreatment with HSP90 inhibitor promoted the activation of Nrf2 in the aortic tissue of diabetic mice (predominantly localized in macrophages and smooth muscle cells) and also in cultured cells. Nrf2 induction was associated with a concomitant inhibition of nuclear factor-κB (NF-κB) in atherosclerotic plaques, thus resulting in a significant reduction in lesion size and inflammatory component (leukocytes and cytokines). Furthermore, atheroprotection by HSP90 inhibition was linked to the induction of cytoprotective HSP70, antioxidant enzymes (heme oxygenase-1, superoxide dismutase and catalase) and autophagy machinery (LC3 and p62/SQSTM1) in aortic tissue. ConclusionHSP90 inhibition protects from atherosclerosis in experimental diabetes through the induction of Nrf2-dependent cytoprotective mechanisms, reinforcing its therapeutic potential.

Interplay between HSP90 and Nrf2 pathways in diabetes-associated atherosclerosis

IntroductionOxidative stress and inflammation are determinant processes in the development of diabetic vascular complications. Heat shock protein 90 (HSP90) overexpression in atherosclerotic plaques plays a role in sustaining inflammatory mechanisms, and its specific inhibition prevents atherosclerosis. The present work investigates, in a mouse model of diabetes-driven atherosclerosis, whether atheroprotection by pharmacological HSP90 inhibition is accomplished by bolstering antioxidant defense mechanisms headed by nuclear factor erythroid-derived 2-like 2 (Nrf2). MethodsStreptozotocin-induced diabetic apolipoprotein E-deficient mice were randomized to receive vehicle or HSP90 inhibitor (17-dimethylaminoethylamino-17-demethoxygeldanamycin, 4mg/kg) for 10 weeks. Aortic root sections were analyzed for plaque size and composition, transcription factor activity, and expression of inflammatory and antioxidant markers. In vitro studies were performed in murine macrophages cultured under hyperglycemic conditions. ResultsTreatment with HSP90 inhibitor promoted the activation of Nrf2 in the aortic tissue of diabetic mice (predominantly localized in macrophages and smooth muscle cells) and also in cultured cells. Nrf2 induction was associated with a concomitant inhibition of nuclear factor-κB (NF-κB) in atherosclerotic plaques, thus resulting in a significant reduction in lesion size and inflammatory component (leukocytes and cytokines). Furthermore, atheroprotection by HSP90 inhibition was linked to the induction of cytoprotective HSP70, antioxidant enzymes (heme oxygenase-1, superoxide dismutase and catalase) and autophagy machinery (LC3 and p62/SQSTM1) in aortic tissue. ConclusionHSP90 inhibition protects from atherosclerosis in experimental diabetes through the induction of Nrf2-dependent cytoprotective mechanisms, reinforcing its therapeutic potential.

MD‐2 is involved in the stimulation of matrix metalloproteinase‐1 expression by interferon‐γ and high glucose in mononuclear cells – a potential role of MD‐2 in Toll‐like receptor 4‐independent signalling

We reported recently that treatment of diabetic apolipoprotein E-deficient mice with the Toll-like receptor 4 (TLR4) antagonist Rs-LPS, a lipopolysaccharide isolated from Rhodobacter sphaeroides, inhibited atherosclerosis. Since it is known that Rs-LPS antagonizes TLR4 by targeting TLR4 co-receptor MD-2, this finding indicates that MD-2 is a potential target for the treatment of atherosclerosis. In this study, we determined if MD-2 is involved in the gene expression regulated by signalling pathways independent of TLR4. Given that interferon-γ (IFNγ) and hyperglycaemia play key roles in atherosclerosis, we determined if MD-2 is involved in IFN-γ and high-glucose-regulated gene expression in mononuclear cells. Results showed that IFN-γ and high glucose synergistically stimulated matrix metalloproteinase 1 (MMP-1), a proteinase essential for vascular tissue remodelling and atherosclerosis, in U937 mononuclear cells, but Rs-LPS inhibited the MMP-1 stimulation. To provide more evidence for a role of MD-2 in IFN-γ-stimulated MMP-1, studies using antibodies and small interfering RNA demonstrated that MD-2 blockade or knockdown attenuated the effect of IFN-γ on MMP-1. Furthermore, studies using PCR arrays showed that MD-2 blockade had a similar effect as IFN-γ receptor blockade on the inhibition of IFN-γ-stimulated pro-inflammatory molecules. Although these findings indicate the involvement of MD-2 in IFN-γ signalling, we also observed that MD-2 was up-regulated by IFN-γ and high glucose. We found that MD-2 up-regulation by IFN-γ played an essential role in the synergistic effect of IFN-γ and LPS on MMP-1 expression. Taken together, these findings indicate that MD-2 is involved in IFN-γ signalling and IFN-γ-augmented MMP-1 up-regulation by LPS.

Anti-Atherogenic Effects of the Combination Therapy with Olmesartan and Azelnidipine in Diabetic Apolipoprotein E-Deficient Mice

Many studies have aimed to identify anti-atherogenic agents in cardiovascular medicine. We have recently demonstrated that the combination therapy with olmesartan (OLM), an angiotensin II receptor blocker, and azelnidipine (AZL), a dihydroprydine calcium-channel blocker, improves endothelial function in diabetic Apolipoprotein-deficient (ApoE(-/-)) mice. In the present study, we examined whether this combination therapy also inhibits atherosclerosis in mice. We used male control and streptozocin-induced diabetic ApoE(-/-) mice. Diabetic ApoE(-/-) mice were orally treated for 5 weeks with vehicle (Untreated), OLM (30 mg/kg/day), AZL (10 mg/kg/day), their combination (OLM+AZL), or hydralazine (HYD, 5 mg/kg/day) as an antihypertensive control. At 5 weeks, systolic blood pressure was significantly elevated in Untreated but was normalized in OLM+AZL and HYD. The atherosclerosis area in the thoracic aorta, perivascular fibrosis and medial thickness of the coronary arteries were increased in Untreated and were ameliorated in OLM+AZL but not in HYD. Staining with a fluorescent probe dihydroethidium showed that production of reactive oxygen species was increased in Untreated, and ameliorated in OLM+AZL. Consistent with these findings, macrophage infiltration in the kidney and the expression of receptor for advanced glycation end-products in the heart, kidney and liver were increased in Untreated and were all ameliorated in OLM+AZL, associated with up-regulation of endothelial NO syntheses (eNOS). In conclusion, the combination therapy with OLM and AZL exerts anti-atherogenic effect in diabetic ApoE(-/-) mice through suppression of oxidative stress and activation of eNOS, independent of its blood pressure-lowering effects. Clinically, this combination therapy may be useful for patients with hypertension, hyperlipidemia and diabetes.

Abstract 9659: Combination Therapy with Olmesartan and Azelnidipine Exerts Anti-Atherogenic Effects in Diabetic Apolipoprotein E-Deficient Mice

Background: We have recently demonstrated that combination therapy with olmesartan (OLM), an angiotensin type 1 receptor blocker, and azelnidipine (AZL), an L-type Ca channel blocker, improves endothelial function more than each monotherapy in diabetic Apolipoprotein-deficient (ApoE -/- ) mice. In this study, we further examined whether this also is the case for atherosclrosis. Method and Results: We used male C57BL/6N (control) and streptozocin-induced diabetic ApoE -/- mice. The diabetic ApoE -/- mice were administered oral vehicle (untreated), OLM (30 mg/kg/day, PO), AZL (10 mg/kg/day, PO), their combination (OLM+AZL), or hydralazine (HYD, 5 mg/kg/day) for 5 weeks (n=8 each). Blood pressure was significantly elevated in the untreated group but was normalized by OLM+AZL or HYD. At 5 weeks, the extent of oil-red O staining in the aorta was markedly increased in the untreated group and was significantly ameliorated in the OLM+AZL group (P<0.05). In the HYD group, despite the normalization of blood pressure, the extent of atherosclrosis in the aorta was comparable with that in the untreated group. Similarly, both perivascular fibrosis and medial thickness of the epicardial coronary arteries were increased in the untreated group, both of which were ameliorated only in the OLM+AZL group (both P<0.05). In addition, cross-sectional area of cardiomyocyte was significantly increased in the untreated group and was significantly ameliorated only in the OLM+AZL group (P<0.05). To examine the mechanisms involved, dihydroethidium staining was performed, which showed that aortic ROS production was significantly increased in the untreated group and was significantly inhibited only in the OLM+AZL group (both P<0.05). Furthermore, both eNOS expression and activity (as examined by its phosphorylation at serine 1177) were increased only in the OLM+AZL group (P<0.05). The expression of RAGE (receptor for advanced glycation end-products) also tended to decrease in the AZL and OLM+AZL groups compared with the untreated group (both P=0.05). Conclusion: These results indicate that the combination therapy with OLM and AZL exerts anti-atherogenic effects more than each monotherapy in ApoE -/- mice in vivo, for which reduction of oxidative stress may be involved.

DPP-4 (CD26) Inhibitor Alogliptin Inhibits Atherosclerosis in Diabetic Apolipoprotein E–Deficient Mice

Dipeptidyl peptidase-4 (DPP-4 or CD26) inhibitors, a new class of antidiabetic compounds, are effective in the treatment of hyperglycemia. Because atherosclerosis-related cardiovascular diseases are the major complications of diabetes, it is important to determine the effect of DPP-4 inhibitors on atherosclerosis. In this study, nondiabetic and diabetic apolipoprotein E-deficient mice were treated with DPP-4 inhibitor alogliptin for 24 weeks, and atherosclerotic lesions in aortic origins were examined. Results showed that diabetes significantly increased atherosclerotic lesions, but alogliptin treatment reduced atherosclerotic lesions in diabetic mice. Metabolic studies showed that diabetes increased plasma glucose and that alogliptin treatment reduced glucose. Furthermore, immunohistochemistry study showed that diabetes increased interleukin-6 (IL-6) and IL-1β protein expression in atherosclerotic plaques, but alogliptin treatment attenuated diabetes-augmented IL-6 and IL-1β expression. In consistence with the observations from the mouse models, our in vitro studies showed that alogliptin-inhibited toll-like receptor 4 (TLR-4)-mediated upregulation of IL-6, IL-1β, and other proinflammatory cytokines by mononuclear cells. Taken together, our findings showed that alogliptin-inhibited atherosclerosis in diabetic apolipoprotein E-deficient mice and that the actions of alogliptin on both glucose and inflammation may contribute to the inhibition.

α-Lipoic acid protects diabetic apolipoprotein E-deficient mice from nephropathy

Aim Both hyperglycemia and hyperlipidemia increase oxidative stress and contribute to the development of diabetic nephropathy (DN). We investigated the effects of α-lipoic acid, a natural antioxidant and a cofactor in the multienzyme complexes, on the development of DN in diabetic apolipoprotein E-deficient mice. Methods Twelve-week-old male apoE−/− mice on C57BL/6J genetic background were made diabetic with injections of streptozotocin (STZ). STZ-treated diabetic apoE−/− mice and non-diabetic control were fed with a synthetic high-fat (HF) diet with or without lipoic acid (LA) supplementation. Multiple parameters including plasma glucose, cholesterol, oxidative stress markers, cytokines, and kidney cortex gene expression, and glomerular morphology were evaluated. Results LA supplementation markedly protected the β cells, reduced cholesterol levels, and attenuated albuminuria and glomerular mesangial expansion in the diabetic mice. Renoprotection by LA was equally effective regardless of whether the dietary supplementation was started 4 weeks before, simultaneously with, or 4 weeks after the induction of diabetes by STZ. LA supplementation significantly improved DN and oxidative stress in the diabetic mice. Severity of albuminuria was positively correlated with level of thiobarbituric acid reactive substances (TBARs) in the kidney ( r 2=0.62, P<.05). Diabetes significantly changed the kidney expression of Rage, Sod2, Tgfb1 and Ctgf, Pdp2, nephrin, and Lias. LA supplementation corrected these changes except that it further suppressed the expression of the Lias gene coding for lipoic acid synthase. Conclusions Our data indicate that LA supplementation effectively attenuates the development and progression of DN through its antioxidant effect as well as enhances glucose oxidation.

In Skeletal Muscle Advanced Glycation End Products (AGEs) Inhibit Insulin Action and Induce the Formation of Multimolecular Complexes Including the Receptor for AGEs

Chronic hyperglycemia promotes insulin resistance at least in part by increasing the formation of advanced glycation end products (AGEs). We have previously shown that in L6 myotubes human glycated albumin (HGA) induces insulin resistance by activating protein kinase Calpha (PKCalpha). Here we show that HGA-induced PKCalpha activation is mediated by Src. Coprecipitation experiments showed that Src interacts with both the receptor for AGE (RAGE) and PKCalpha in HGA-treated L6 cells. A direct interaction of PKCalpha with Src and insulin receptor substrate-1 (IRS-1) has also been detected. In addition, silencing of IRS-1 expression abolished HGA-induced RAGE-PKCalpha co-precipitation. AGEs were able to induce insulin resistance also in vivo, as insulin tolerance tests revealed a significant impairment of insulin sensitivity in C57/BL6 mice fed a high AGEs diet (HAD). In tibialis muscle of HAD-fed mice, insulin-induced glucose uptake and protein kinase B phosphorylation were reduced. This was paralleled by a 2.5-fold increase in PKCalpha activity. Similarly to in vitro observations, Src phosphorylation was increased in tibialis muscle of HAD-fed mice, and co-precipitation experiments showed that Src interacts with both RAGE and PKCalpha. These results indicate that AGEs impairment of insulin action in the muscle might be mediated by the formation of a multimolecular complex including RAGE/IRS-1/Src and PKCalpha.

Receptor for advanced glycation endproducts (RAGE) and vascular inflammation: insights into the pathogenesis of macrovascular complications in diabetes.

The incidence and severity of atherosclerosis is increased in patients with diabetes. Indeed, accelerated macrovascular disease in diabetic patients has emerged as a leading cause of morbidity and mortality in the United States and worldwide. Multiple investigations have suggested that there are numerous potential contributory factors that underlie these observations. Our laboratory has focused on the contribution of receptor for advanced glycation endproducts (RAGE) and its proinflammatory ligands, advanced glycation endproducts (AGEs) and S100/calgranulins in vascular perturbation, manifested as enhanced atherogenesis or accelerated restenosis after angioplasty. In rodent models of diabetic complications, blockade of RAGE suppressed vascular hyperpermeability, accelerated atherosclerotic lesion area and complexity in diabetic apolipoprotein E-deficient mice, and prevented exaggerated neointimal formation in hyperglycemic fatty Zucker rats subjected to injury of the carotid artery. In this review, we summarize these findings and provide an overview of distinct mechanisms that contribute to the development of accelerated diabetic macrovascular disease. Insights into therapeutic strategies to prevent or interrupt these processes are presented.