Normoglycemic Mice Research Articles

Abstract TP374: Spleen Immune Cell Changes in Stroke and Hyperglycemia: Insights from Flow Cytometry

Introduction: The cross-talk between the brain and peripheral immune organs plays a crucial role in the response to stroke injury. Spleen responses are implicated in stroke pathology and inflammatory responses. Hyperglycemia is known to worsen stroke outcomes, with increased immune cell infiltration into the brain. However, it remains unclear whether hyperglycemia exacerbates spleen immune cell responses and whether this contributes to the heightened immune response in hyperglycemic stroke. Method: Male C57/BL6 mice (10-12 weeks) were subjected to transient middle cerebral artery occlusion (MCAO) for 30 minutes, followed by reperfusion to mimic ischemic stroke. Acute hyperglycemia was induced by glucose injection 10 minutes before MCAO. The study included three groups: sham, MCAO only, and MCAO with hyperglycemia (n=6-7 per group). At 24 hours post-stroke, spleen and brain immune cell populations were analyzed using our established 12-color flow cytometry technique, which simultaneously analyzes myeloid and lymphoid subpopulations. Results: Compared with sham controls, stroke significantly decreased the numbers of dendritic cells (2.33 ± 0.48 vs. 1.50 ± 0.42 million; p<0.05), NK cells (2.30 ± 0.37 vs. 1.21 ± 0.37 million; p<0.01), Ly6C+ macrophages (0.29 ± 0.12 vs. 0.11 ± 0.06 million; p<0.01), and total lymphocytes (56.9 ± 16.6 vs. 37.0 ± 5.1 million; p<0.05) at 24 hours post-stroke in the spleen, whereas there were no significant changes in neutrophils and Ly6C+ monocytes cell counts. Hyperglycemia during stroke did not alter spleen immune cell numbers compared to stroke alone. However, hyperglycemia significantly increased brain-infiltrating immune cells at 24 hours after stroke, including neutrophils, B cells, and CD8+ T cells compared to normoglycemic mice. Conclusion: This study provides insights into spleen immune cell responses to acute ischemic stroke and hyperglycemia. Significant changes in spleen immune cell populations after stroke confirm their potential roles in stroke pathology and recovery, warranting further investigation. Acute hyperglycemia, however, does not significantly affect spleen immune cell numbers after stroke, implying that the spleen is not the primary target of hyperglycemia-exacerbated immune cell infiltration into ischemic stroke brains. These results pave the way for exploring other mechanisms driving immune cell infiltration under hyperglycemic conditions.

Pro-healing impact of liraglutide on skin wounds in normoglycemic mice.

Recent studies demonstrated that glucagon-like peptide-1 receptor agonists (GLP-1RA) have promising prospects in promoting wound healing. In this study, we intend to investigate the pro-healing effect and potential molecular mechanism of topical administration of GLP-1RA liraglutide on wounds in normoglycemic mice. Two full-thickness wounds were created on the back of the C57BL/6 mice. The "lower" wounds were topically infiltrated with liraglutide every day after injury; while the "upper" wounds were infiltrated with saline solution. Wound area was measured daily during the 10-day study period. The wound tissue was stained with H&E and immunofluorescence. Western blotting was performed to detect the markers in macrophages. The results showed that topical administration of liraglutide resulted in a rapid reduction of wound size. The capillary density and the expression of vascular endothelial growth factor (VEGF)-A were significantly increased in liraglutide-treated wounds. Findings from immunofluorescence and Western blotting revealed that liraglutide promoted phenotypic polarization of macrophages from M1 to M2. We further identified that M2a macrophages predominantly presented in the early and middle stages of inflammation phase and M2d macrophages presented in the middle and late stages. Our study suggested that GLP-1RA liraglutide could promote wound healing in normoglycemic mice, which is partly attributed to the modulation of the macrophage polarization from M1 subtype to M2 subtype.

Knockout of the Carbohydrate Responsive Element Binding Protein Enhances Proliferation and Tumorigenesis in Renal Tubules of Mice.

Glycogen-storing so-called clear cell kidney tubules (CCTs), precursor lesions of renal cell carcinoma, have been described in diabetic rats and in humans. The lesions show upregulation of the Akt/mTOR-pathway and the related transcription factor carbohydrate responsive element binding protein (ChREBP), which is supposedly pro-oncogenic. We investigated the effect of ChREBP-knockout on nephrocarcinogenesis in streptozotocin-induced diabetic and normoglycemic mice. Diabetic, but not non-diabetic mice, showed CCTs at 3, 6 and 12 months of age. Glycogenosis was confirmed by periodic acid schiff reaction and transmission electron microscopy. CCTs in ChREBP-knockout mice consisted of larger cells and occurred more frequently compared to wildtype mice. Progression towards kidney tumors was observed in both diabetic groups but occurred earlier in ChREBP-knockout mice. Proliferative activity assessed by BrdU-labeling was lower in 1-week-old but higher in 12-month-old diabetic ChREBP-knockout mice. Surprisingly, renal neoplasms occurred spontaneously in non-diabetic ChREBP-knockout, but not non-diabetic wildtype mice, indicating an unexpected tumor-suppressive function of ChREBP. Immunohistochemistry showed upregulated glycolysis and lipogenesis, along with activated Akt/mTOR-signaling in tumors of ChREBP-knockout groups. Immunohistochemistry of human clear cell renal cell carcinomas revealed reduced ChREBP expression compared to normal kidney tissue. However, the molecular mechanisms by which loss of ChREBP might facilitate tumorigenesis require further investigation.

A Specific Collagen Hydrolysate Improves Postprandial Glucose Tolerance in Normoglycemic and Prediabetic Mice and in a First Proof of Concept Study in Healthy, Normoglycemic and Prediabetic Humans.

In response to nutrients, intestinal L- and K-cells naturally secrete glucagon-like peptide 1 (GLP-1). GLP-1 regulates postprandial blood glucose by increasing insulin secretion, slowing down gastric emptying and inducing satiety. A selection of specifically developed collagen hydrolysates was screened for their ability to enhance natural GLP-1 production invitro. The best performing hydrolysate, H80 (Nextida GC), was orally administered at different doses to lean, normoglycemic mice and overweight, prediabetic mice. Lean mice were acutely challenged 45 min before an oral glucose load. While daily supplemented for 6 weeks, prediabetic mice were acutely challenged at day 21 and 34. Oral glucose tolerance, plasma insulin and GLP-1 levels were assessed, and a gastric emptying assay performed in prediabetic mice. H80 significantly lowered the blood glucose response in lean and prediabetic mice, at a 4 g/kg dose (-25% and -36%, respectively), compared to vehicle. In chronically supplemented, prediabetic mice, acute H80 administration slowed down gastric emptying (-60%) after 21 days and increased plasma insulin (+166%) after 35 days of supplementation. H80 increased plasma active GLP-1 in lean (+217%) and prediabetic (+860%) mice. Overall, the data indicate that the specific collagen hydrolysate, H80, has significant GLP-1-mediated effects on oral glucose tolerance in lean and prediabetic mice. Furthermore, effects on postprandial glucose tolerance were evaluated in a small, human, proof of concept study. H80 reduced the postprandial glucose response at a 5 g dose in healthy, normoglycemic and prediabetic participants. Oral supplementation with H80 might thus be a promising strategy to maintain normal glucose tolerance.

8433 Elucidation of the Molecular Basis of Human and Mouse Pancreatic β-Cell Senescence Induced by Hyperglycemia

Abstract Disclosure: K. Iwasaki: None. P. Carapeto: None. C. Aguayo-Mazzucato: None. Background and AIM: Senescent cells accumulate with age and contribute to the development of chronic diseases, including Type 2 Diabetes (T2D). Whereas senescence is an important pathogenic mechanism in diabetes, the role of hyperglycemia as a metabolic driver of pancreatic β-cells has not been clarified. The aim of this study is to elucidate the molecular basis of hyperglycemia-induced pancreatic β-cell senescence using human and rodent islets. Methods: 1) Human islets were cultured for 2 weeks in 20.2 mM high (HG) or 2.8mM low glucose (LG), and analyzed by qPCR and glucose-responsive insulin secretion (GSIS) to explore the molecular basis of senescence in ex vivo. To test the effects of senescent cell removal, senolytics Dasatinib (1μM) and Quercetin (20μM) (DQ) were used. 2) To evaluate the effects of hyperglycemia in vivo, we transplanted mouse and human islets under the kidney capsule of normoglycemic (NG) or streptozocin-induced hyperglycemic (Stz-Db) immunodeficient mice for two weeks. Graft function was evaluated by intraperitoneal glucose tolerance test and cellular identity using qPCR or immunohistochemistry. DQ (5mg/kg/day + 50mg/kg/day) was used to evaluate the effects of removing senescent cells in transplantation (Tx). To investigate the effect of specific removal of p16-positive senescent β-cells in transplanted islets, INK-ATTAC mice islets (p16Ink4a-mediated apoptosis by targeted activation of caspases) were transplanted into Stz-Db and treated with BB homodimer (BB) to remove p16+ cells or vehicle. Results: (1) Human islets from 5 healthy donors cultured in HG had an increased senescence index (mean of senescence markers p16, p21 and HMGB1 mRNA) when compared to LG islets (p<0.05) and lost glucose responsiveness as evaluated by ex vivo GSIS compared to LG (p<0.05). 2) Human islets (1000IEQ) were transplanted to Stz-Db or NG mice. In Stz-Db graft, P21 mRNA significantly increased with decreased expression of β-cell genes when compared to NG. Stz-Db mice treated with DQ (Db-DQ) for 5 days after Tx, significantly decreased expression of senescence index (p<0.05) and increased in β-cell index (mean of β-cell hallmark genes) (p<0.05) at mRNA levels. Stz-Db mice transplanted with INK-ATTAC mice islets had lower fed glucose levels, decreased p16 mRNA, increased expression β-cell hallmark genes and improved insulin secretion ex vivo after removal of p16+ cells. Conclusion: Sustained exposure of human islets to hyperglycemia induced cellular senescence, leading to pancreatic β-cell dysfunction and loss of cellular identity. Furthermore, elimination of senescent cells may protect pancreatic β-cells from hyperglycemia-induced functional decline, which may have potential clinical application in both Type 1 Diabetes and T2D. Presentation: 6/2/2024

Hyperglycemia enhances brain susceptibility to lipopolysaccharide-induced neuroinflammation via astrocyte reprogramming.

Hyperglycemia has been shown to modulate the immune response of peripheral immune cells and organs, but the impact of hyperglycemia on neuroinflammation within the brain remains elusive. In the present study, we provide evidences that streptozotocin (STZ)-induced hyperglycemic condition in mice drives a phenotypic switch of brain astrocytes to a proinflammatory state, and increases brain vulnerability to mild peripheral inflammation. In particular, we found that hyperglycemia led to a significant increase in the astrocyte proliferation as determined by flow cytometric and immunohistochemical analyses of mouse brain. The increased astrocyte proliferation by hyperglycemia was reduced by Glut1 inhibitor BAY-876. Transcriptomic analysis of isolated astrocytes from Aldh1l1CreERT2;tdTomato mice revealed that peripheral STZ injection induced astrocyte reprogramming into proliferative, and proinflammatory phenotype. Additionally, STZ-induced hyperglycemic condition significantly enhanced the infiltration of circulating myeloid cells into the brain and the disruption of blood-brain barrier in response to mild lipopolysaccharide (LPS) administration. Systemic hyperglycemia did not alter the intensity and sensitivity of peripheral inflammation in mice to LPS challenge, but increased the inflammatory potential of brain microglia. In line with findings from mouse experiments, a high-glucose environment intensified the LPS-triggered production of proinflammatory molecules in primary astrocyte cultures. Furthermore, hyperglycemic mice exhibited a significant impairment in cognitive function after mild LPS administration compared to normoglycemic mice as determined by novel object recognition and Y-maze tasks. Taken together, these results demonstrate that hyperglycemia directly induces astrocyte reprogramming towards a proliferative and proinflammatory phenotype, which potentiates mild LPS-triggered inflammation within brain parenchymal regions.

Antihyperglycemic, antiglycation, anti-hypercholesteremic, and toxicity evaluation with gas chromatography mass spectrometry profiling for Aloe armatissima leaves

Abstract Aloe species are known for the treatment of various conditions including diabetes mellitus, hypocholesteremia, and glycation end products. Nevertheless, the biological activity of Aloe armatissima is yet to be reported. It is a first-time report to evaluate the Aloe armatissima leaves (AAL) extract for its antioxidant, anti-glycation, anti-hyperglycemic, and anti-hyperlipidemic potential. In vitro tests of 1,1-diphenyl-2-picrylhydrazyl for the antioxidant and HSA for the antiglycation activity whereas in vivo models were used to assess the toxicity, antihyperglycemic, and anti-hypercholesteremic effects. The volatile profile was determined via gas chromatography-mass spectrometry. The IC50 values of 116 ± 0.66 (μg/mL) for antioxidant activity and 0.21 ± 0.009 (mg/mL) for antiglycation activity were observed for the AAL extract. The acute toxicity in the animal model revealed a lack of toxicity for the extract. The in vivo models exhibited a dose-dependent hypoglycemic and anti-hyperglycemic effects with significant (P < 0.01) blood glucose levels reduction. Moreover, a profound decrease in serum cholesterol, triglyceride, and LDL along with a significant (P < 0.05) increase in HDL and serum insulin levels was recorded. The statistical analysis demonstrated the values of F (24,125) = 23.95, P = 0.001, effect size = 1.95 (normoglycemic mice), F (24,125) = 143.21, P = 0.001, effect size = 4.79 (glucose loaded mice), and F (24,125) = 82.69, P = 0.001, effect size = 3.6 (diabetic model). GCMS showed the presence of eleven compounds with tetratetracontane (100%), β-sitosterol (27.76), and vitamin E (18.68) in major amounts. The results underscore the extract’s capacity to effectively combat various ailments; however, the active phytochemicals need to be isolated and the pharmacological activities may be established at the molecular level.

Abstract WMP115: Rapid Vascular Complement Activation: A Key Driver of Blood-Brain Barrier Disruption in Ischemic Stroke With Acute Hyperglycemia

Background: Acute hyperglycemia, which occurs in over 40% of ischemic stroke patients irrespective of pre-existing diabetes, exacerbates stroke injury. Understanding its mechanisms is vital for innovative treatments. Here we investigate the role of complement in hyperglycemia-exacerbated damage after stroke. Method: Male C57/BL6 mice (10-11 weeks) underwent 30-min suture-induced middle cerebral artery occlusion followed by reperfusion, resembling clinical thrombectomy. Acute hyperglycemia was induced 10 min pre-stroke via glucose injection. Mice were sacrificed at 4.5h and 24h post-stroke to analyze brain swelling, blood-brain barrier (BBB) leakage and hemorrhagic transformation (HT). Mortality, neurological deficits, and motor-sensory functions were assessed at 14 days post-stroke. Complement activation was visualized via C3d immunostaining. Complement C3's role was examined using knock-out mice and the targeted complement inhibitor CR2-Crry fusion protein. Result: Hyperglycemia rapidly worsens BBB leakage (p<0.0001), brain swelling (p<0.05), and HT (p<0.0001) at 4.5 hr after stroke. Hyperglycemic stroke mice exhibit higher mortality (100% vs 25%, p=0.0008), body weight loss, and behavior impairment at the sub-acute phase compared to normoglycemic mice. Notably, acute hyperglycemia rapidly increased plasma C3 levels at 1hr after stroke (p<0.01), accompanied by rapid and time-dependent activation of C3 in ischemic brain vessels, as marked by increased vascular C3d; and this co-localized with autoantibody (IgM/IgG) vessel buildup and vessel leakage. Additionally, C3d levels positively correlated with brain swelling and HT (p<0.01). Vascular C3d and leakage into parenchyma were markedly reduced in the absence of reperfusion. Blocking complement via C3 knock-out or post-reperfusion systemic injection of C3 inhibitor CR2-Crry mitigates hyperglycemia's detrimental effects, including BBB leakage. Conclusion: Rapid vascular activation of complement C3 is a significant driver of hyperglycemic-exacerbated pathology in experimental stroke. Inhibiting C3 activation could be a potential therapeutic approach to improve hyperglycemic stroke outcome.

Phytochemical analysis and antihyperglycemic activity of Castilleja arvensis

Castilleja genus comprises approximately 211 species, some of them exhibiting potential in treating various diseases. Remarkably, despite its abundance, there is a significant lack of scientific studies that explore the chemical composition and/or therapeutic activity of this genus.In this work, the chemical composition of Castilleja arvensis was determined, and its antihyperglycemic activity was evaluated in vivo, in vitro, and ex vivo. Hydroalcoholic extract of C. arvensis (HECa) was obtained from the maceration of aerial parts. HECa was fractionated by liquid–liquid extractions to obtain the CH2Cl2 fraction (DF), EtOAc fraction (EF), n-BuOH fraction (BF) and aqueous residue (AR). The antihyperglycemic activity was determined in vivo through oral glucose and sucrose tolerance tests in normoglycemic CD-1 mice. Ex vivo assays were performed to determine intestinal glucose absorption, muscular glucose uptake and hepatic glucose production. α-glucosidase inhibitory activity was evaluated in vitro. Phytochemical screening was carried out through conventional chromatography techniques. Structure elucidation of the isolated compounds was performed by GC–MS and NMR experiments.HECa, its fractions and AR showed significant antihyperglycemic activity in vivo. According to the in vitro and ex vivo assays, this effect can be attributed to different mechanisms of action, including a delay in intestinal glucose absorption, an improvement in insulin sensitivity, and the regulation of hepatic glucose production. These effects may be due to different metabolites identified in fractions from the HECa, including genkwanin, acacetin, verbascoside and ipolamiide. Thus, current research shows that C. arvensis is an important source of bioactive compounds for the management of glycemia.

Systemic Glucose Regulation by a Hindbrain Inhibitory Circuit in a Mouse Model of Type 1 Diabetes

Introduction: Previous work showed that increasing the electrical activity of inhibitory neurons in the dorsal vagal complex (DVC) is sufficient to increase whole-body glucose concentration in normoglycemic mice. Here we tested the hypothesis that deactivating GABAergic neurons in the dorsal hindbrain of hyperglycemic mice decreases synaptic inhibition of parasympathetic motor neurons in the dorsal motor nucleus of the vagus (DMV) and reduces systemic glucose levels. Methods: Chemogenetic activation or inactivation of GABAergic neurons in the nucleus tractus solitarius (NTS) was used to assess effects of modulating parasympathetic output on blood glucose concentration in normoglycemic and hyperglycemic mice. Patch-clamp electrophysiology in vitro was used to assess cellular effects of chemogenetic manipulation of NTS GABA neurons. Results: Chemogenetic activation of GABAergic NTS neurons in normoglycemic mice increased their action potential firing, resulting in increased inhibitory synaptic input to DMV motor neurons and elevated blood glucose concentration. Deactivation of GABAergic DVC neurons in normoglycemic mice altered their electrical activity but did not alter systemic glucose levels. Conversely, stimulation of GABAergic DVC neurons in mice that were hyperglycemic subsequent to treatment with streptozotocin changed their electrical activity but did not alter whole-body glucose concentration, while deactivation of this inhibitory circuit significantly decreased circulating glucose concentration. Peripheral administration of a brain impermeant muscarinic acetylcholine receptor antagonist abolished these effects. Conclusion: Disinhibiting vagal motor neurons decreases hyperglycemia in a mouse model of type 1 diabetes. This inhibitory brainstem circuit emerges as a key parasympathetic regulator of whole-body glucose homeostasis that undergoes functional plasticity in hyperglycemic conditions.

Antidiabetic phytodrug from Maerua angolensis DC: Formulation, standardization, in vitro and in vivo evaluations

Maerua angolensis is a shrub or small tree widely distributed in tropical Africa. The plant materials are used for various ethnomedicinal applications across the region, including the prevention and treatment of diabetes. This study was aimed to evaluate the antidiabetic potential of formulated freeze-dried aqueous extract of Maerua angolensis leaves using standard in vitro and in vivo experimental models. Infusion extract of the plant's powdered dried leaves was prepared, the resultant extract was freeze-dried and formulated (denoted FIEMa). The physicochemical, quantitative and qualitative phytochemical constituents of FIEMa were determined using analytical techniques (UV-visible and HPLC). The antidiabetic activity of the reconstituted formulation was investigated using in vitro and in vivo models. The in vitro activity was assessed using Phosphomolybdenum assay and DPPH (2,2-Diphenyl-1-picrylhydrazyl) radical scavenging activity for antioxidant effect, and α-amylase inhibition and α- glycosidase inhibition assays. The assays of the normoglycemic, oral glucose load tolerance, and single-dose alloxan (150 mg/kg) induced diabetes in rats were used for in vivo antidiabetic activity. The results show that FIEMa contains valuable phytochemicals. It exhibited antioxidant effect, α-amylase and α- glucosidase inhibition activities. FIEMa did not significantly reduce blood glucose levels (BGL) in normoglycemic mice but produced a significant reduction of BGL in the oral glucose tolerance test. In alloxan-induced diabetic rats, significant reductions of BGL were observed in groups treated with high doses of FIEMa in the single-dose study and from the second week onwards in the repeated dose study. It also exhibited less reduction in body weight compared to the control group. The findings indicate that FIEMa has an antidiabetic activity, low risk of hypoglycemia, and body weight loss. The valuable phytochemicals plausibly mediate this effect, especially the dominant betulinic acid detected in FIEMa, most likely acting in synergy.

Pharmacological screening of antidiabetic activity of ethanolic extracts of justicia tranquebarensis on streptozocin induced rats.

Non-communicable diseases like diabetes mellitus are becoming major health problem these days. There is an increasing demand by patients to use the natural products (plants) with anti-diabetic activity due to the severe side effects associated with the use of insulin and oral anti-diabetic drugs. One such plant is Justicia tranquebareinsis which has been traditionally used for the treatment of diabetes mellitus. The aim of this study was, therefore, to evaluate hypoglycemic activity of ethanol extract of Justicia tranquebareinsis in streptozotocin-induced diabetic rats, respectively and to carry out acute toxicity test. Two doses (200 mg/kg and 400 mg/kg) of the ethanol extract of Justicia tranquebareinsis were administered to normal glucose loaded and diabetic rat to study blood glucose lowering effect. Ethanol extract of Justicia tranquebareinsis (200mg/kg and 400mg/kg, p.o.) showed significant tolerance (P < 0.05) to oral glucose load at 1 and 2 hrs after glucose load. The extract also produced significant (P < 0.05) blood glucose reduction at 4 hours after its administration in normoglycemic mice. The extract at 400mg/kg dose level produced significant (P < 0.05) reduction in blood glucose level at 2, 3 and 4 hours of treatment in streptozotocin (45 mg/kg) induced diabetic mice. Acute oral toxicity studies of ethanol extract of Justicia tranquebareinsis in rats showed no death or signs of toxicity at the dose of 2000 mg/kg indicating the safety nature of the extract. The results of the experiments showed that ethanol extract of Justicia tranquebareinsis has significant antihyperglycemic activity in streptozotocin induced diabetic rat and improvement in glucose tolerance and slight hypoglycemic activity in normal rat justifying the traditional claim for its use in diabetes.

“Green” synthesized versus chemically synthesized zinc oxide nanoparticles: In vivo antihyperglycemic activity and pharmacokinetics

Zinc is one of the most studied trace elements, commonly used as supplement in diabetes treatment. By its involvement in the synthesis, secretion of insulin, promotion of insulin sensitivity and its multiple enzymatic functions it is known to contribute to reduce hyperglycemia. Researchers have shown that zinc administered under the form of zinc oxide nanoparticles (ZnONPs) is more effective than under its ionic form. Studies evaluating the antihyperglycemic activity of these nanocarriers include both ZnONPs synthesised using plants (i.e. green synthesized) or chemically synthesized. The present work aims to compare green synthesized ZnONPs with the marketed chemically synthesized ones. Green ZnONPs were synthesized using the aqueous extract of the stem bark of the medicinal plant Panda oleosa and zinc nitrate hexahydrate. Both nanocarriers were compared in terms of optical properties, morphology, composition, chemical functions, resistance to oxidation, in vivo antihyperglycemic activity via oral glucose tolerance test (OGTT) and pharmacokinetics in relation to zinc in C57BL/6J mice. A UV absorption peak was observed at 354 nm and 374 nm for the green and marketed ZnONPs, respectively. The shape and hydrodynamic diameters were anisotropic and of 228.8 ± 3.0 nm for the green ZnONPs and spherical and of 225.6 ± 0.9 nm for the marketed ZnONPs. Phenolic compounds accounted for 2.58 ± 0.04% of the green ZnONPs and allowed them to be more stable and unaffected by an oxidizing agent during the experiment, while the marketed chemically synthesized ZnONPs aggregated with or without contact with an oxidizing agent. No significant differences were observed on the amounts of zinc absorbed when comparing green ZnONPs, chemically synthesized ZnONPs and zinc sulfate in a pharmacokinetics study in normoglycemic mice. When evaluating the in vivo hypoglycemic activity of the nanocarriers in obese/diabetic mice, green synthesized ZnONPs displayed a significant hypoglycemic effect compared with the chemically synthesized nanoparticles following an OGTT. Altogether, these data indicate that phytocompounds, as catechin derivatives and polyphenols, attached to the green synthesized ZnONPs’ surface, could contribute to their hypoglycemic activity. The comparison thus demonstrated that green synthesized ZnONPs are significantly more efficient than chemically ones at reducing hyperglycemia regardless of their absorption.

Sex-dependent effects of finerenone on hemostasis in normoglycemic and streptozotocin-induced diabetic mice

Diabetes is associated with aldosterone excess and the overactivation of its mineralocorticoid receptor (MR) which leads to the development of many cardiovascular dysfunctions. Therefore, MR antagonists have been found to exert favorable effects on the cardiovascular system. Finerenone is a new nonsteroidal MR antagonist approved for the treatment of chronic kidney disease associated with type 2 diabetes. Clinical studies have demonstrated that finerenone improves cardiovascular outcomes. However, its influence on hemostasis in the cardioprotective effect is unknown. Therefore, the main aim of our study was to evaluate the effects of finerenone (10 mg/kg, p.o.) on selected hemostasis parameters in streptozotocin (180 mg/kg, i.p.)-induced diabetes. Since regulation of the MR activity is sex-dependent, the study was conducted in both female and male mice. The most beneficial effects of finerenone were observed in diabetic female mice which included a decrease in thrombus formation, attenuation of platelet activity, inhibition of the coagulation system, and activation of fibrinolysis. In contrast, in male diabetic mice only an attenuation of the coagulation system was observed. Furthermore, finerenone also exerted unfavorable effects, but only in normoglycemic mice, manifested as a slight increase in platelet activity in males and an enhancement of the coagulation system activity in females. Our study is the first to show the sex-dependent and glycemia-dependent effects of finerenone on hemostasis in diabetes. The occurrence of beneficial effects only in female diabetic mice requires in-depth study.

Empagliflozin restores the deregulated cardiac endothelial cell transcriptome in vivo upon myocardial ischemia reperfusion injury and reduces the infiltration of immune cells

Abstract Background/Introduction Empagliflozin (EMPA) is a sodium-glucose co-transporter 2 (SGLT-2) inhibitor with established cardioprotective effects in heart failure but its use in myocardial ischemia-reperfusion (IR) injury is still under investigation. We have shown that chronic administration of EMPA reduces infarct size in normoglycemic mice subjected to IR and that endothelial cells (ECs) are implicated in the protective mechanism. Purpose We aimed to determine EMPA’s distinct effect on three cell populations: cardiac ECs, fibroblasts (FBs) and cardiomyocytes (CMs) in vivo. Methods C57Bl6 male mice were randomized as follows: 1) Control-Sham, 2) Control-IR and 3) EMPA-IR. EMPA group received EMPA 10mg/kg/day for 6 weeks which is a clinically relevant dose and Control groups received vehicle (5% DMSO in normal saline). At the end of the administrations, mice were subjected to 30’I/2hR or sham operation and the ischemic heart was collected to isolate CMs. From the non-CMs populations the cardiac ECs and FBs were sorted. RNA was obtained from the distinct cell populations and 3’ RNA sequencing was applied to investigate the pathways affected by IR and EMPA treatment. An additional cohort of mice was employed to validate the results at mRNA level. In order to examine the effects of EMPA upon prolonged reperfusion, we repeated the experimental protocol, and the mice were sacrificed upon 7 days of R. Based on the RNA sequencing data, the infiltration of the heart with immune cells was examined. Results Comparison between IR and Sham groups revealed that IR alters the transcriptomic profile of ECs and FBs with 576 and 571 genes respectively being significantly deregulated (FDR<0.05). CMs transcriptome was not altered by IR. The comparison between Control-IR and EMPA-IR groups revealed that EMPA does not affect the FBs’ gene expression profile while in CMs, EMPA affects only 3 genes expression (FDR<0.05) related to mitochondrial metabolism. The ECs are mostly affected by EMPA treatment with 211 genes being significantly altered (FDR<0.05). Pathway enrichment analysis in ECs revealed that the deregulated pathways by IR related to extracellular matrix organization, matrix degradation and immune cell adhesion are restored by EMPA treatment. The expression levels of several significant genes of our dataset including Mmp-2, Timp-1, Icam, Vecam, etc was validated in the independent cohort of mice. On the 7th day of R, EMPA reduced the infiltration of neutrophils and inflammatory (Ly6C high) monocytes in the myocardium compared to the Control group indicating that EMPA may affect the remodeling process upon IR. Conclusions We identified ECs and FBs as cell target populations for therapeutic interventions at 2h of R. EMPA pre-treatment reverses the deregulated transcriptome related to extracellular matrix organization, matrix degradation and immune cell adhesion in ECs and eventually reduces the infiltration of immune cells in the ischemic myocardium.

Regulation of Smooth Muscle Cell Proliferation by Mitochondrial Ca2+ in Type 2 Diabetes.

Type 2 diabetes (T2D) is associated with increased risk of atherosclerotic vascular disease due to excessive vascular smooth muscle cell (VSMC) proliferation. Here, we investigated the role of mitochondrial dysfunction and Ca2+ levels in VSMC proliferation in T2D. VSMCs were isolated from normoglycemic and T2D-like mice induced by diet. The effects of mitochondrial Ca2+ uptake were studied using mice with selectively inhibited mitochondrial Ca2+/calmodulin-dependent kinase II (mtCaMKII) in VSMCs. Mitochondrial transition pore (mPTP) was blocked using ER-000444793. VSMCs from T2D compared to normoglycemic mice exhibited increased proliferation and baseline cytosolic Ca2+ levels ([Ca2+]cyto). T2D cells displayed lower endoplasmic reticulum Ca2+ levels, reduced mitochondrial Ca2+ entry, and increased Ca2+ leakage through the mPTP. Mitochondrial and cytosolic Ca2+ transients were diminished in T2D cells upon platelet-derived growth factor (PDGF) administration. Inhibiting mitochondrial Ca2+ uptake or the mPTP reduced VSMC proliferation in T2D, but had contrasting effects on [Ca2+]cyto. In T2D VSMCs, enhanced activation of Erk1/2 and its upstream regulators was observed, driven by elevated [Ca2+]cyto. Inhibiting mtCaMKII worsened the Ca2+ imbalance by blocking mitochondrial Ca2+ entry, leading to further increases in [Ca2+]cyto and Erk1/2 hyperactivation. Under these conditions, PDGF had no effect on VSMC proliferation. Inhibiting Ca2+-dependent signaling in the cytosol reduced excessive Erk1/2 activation and VSMC proliferation. Our findings suggest that altered Ca2+ handling drives enhanced VSMC proliferation in T2D, with mitochondrial dysfunction contributing to this process.

FOXO 1 deletion in chondrocytes rescues diabetes-impaired fracture healing by restoring angiogenesis and reducing apoptosis.

Diabetes mellitus is associated with higher risks of long bone and jaw fractures. It is also associated with a higher incidence of delayed union or non-union. Our previous investigations concluded that a dominant mechanism was the premature loss of cartilage during endochondral bone formation associated with increased osteoclastic activities. We tested the hypothesis that FOXO1 plays a key role in diabetes-impaired angiogenesis and chondrocyte apoptosis. Closed fractures of the femur were induced in mice with lineage-specific FOXO1 deletion in chondrocytes. The control group consisted of mice with the FOXO1 gene present. Mice in the diabetic group were rendered diabetic by multiple streptozotocin injections, while mice in the normoglycemic group received vehicle. Specimens were collected 16 days post fracture. The samples were fixed, decalcified, and embedded in paraffin blocks for immunostaining utilizing anti cleaved caspase-3 or CD31 specific antibodies compared with matched control IgG antibody, and apoptosis by the TUNEL assay. Additionally, ATDC5 chondrocytes were examined in vitro by RT-PCR, luciferase reporter and chromatin immunoprecipitation assays. Diabetic mice had ~ 50% fewer blood vessels compared to normoglycemic mice FOXO1 deletion in diabetic mice partially rescued the low number of blood vessels (p < 0.05). Additionally, diabetes increased caspase-3 positive and apoptotic chondrocytes by 50%. FOXO1 deletion in diabetic animals blocked the increase in both to levels comparable to normoglycemic animals (p < 0.05). High glucose (HG) and high advanced glycation end products (AGE) levels stimulated FOXO1 association with the caspase-3 promoter in vitro, and overexpression of FOXO1 increased caspase-3 promoter activity in luciferase reporter assays. Furthermore, we review previous mechanistic studies demonstrating that tumor necrosis factor (TNF) inhibition reverses impaired angiogenesis and reverses high levels of chondrocyte apoptosis that occur in fracture healing. New results presented here, in combination with recent studies, provide a comprehensive overview of how diabetes, through high glucose levels, AGEs, and increased inflammation, impair the healing process by interfering with angiogenesis and stimulating chondrocyte apoptosis. FOXO1 in diabetic fractures plays a negative role by reducing new blood vessel formation and increasing chondrocyte cell death which is distinct from its role in normal fracture healing.

Effects of Topical 1,25 and 24,25 Vitamin D on Diabetic, Vitamin D Deficient and Vitamin D Receptor Knockout Mouse Corneal Wound Healing.

Delayed or prolonged corneal wound healing and non-healing corneas put patients at risk for ocular surface infections and subsequent stromal opacification, resulting in discomfort or visual loss. It is important to enhance corneal wound healing efficiency and quality. Vitamin D (Vit D) is both a hormone and a vitamin, and its insufficiency has been linked to immune disorders and diabetes. For this study, wound healing and recruitment of CD45+ cells into the wound area of normoglycemic and diabetic mice were examined following corneal epithelial debridement and treatment with 1,25-dihyroxyvitamin D (1,25 Vit D) or 24,25-dihydroxyvitamin D (24,25 Vit D). Treatment with topical 1,25-dihyroxyvitamin D (1,25 Vit D) resulted in significantly increased corneal wound healing rates of normoglycemic, diabetic and diabetic Vit D deficient mice. Furthermore, 24,25-dihydroxyvitamin D (24,25 Vit D) significantly increased corneal wound healing of diabetic Vit D deficient and Vit D receptor knockout (VDR KO) mice. In addition, CD45+ cell numbers were reduced in diabetic and VDR KO mouse corneas compared to normoglycemic mice, and 24,25 Vit D increased the recruitment of CD45+ cells to diabetic mouse corneas after epithelial debridement. CD45+ cells were found to infiltrate into the corneal basal epithelial layer after corneal epithelial debridement. Our data indicate that topical Vit D promotes corneal wound healing and further supports previous work that the Vit D corneal wound healing effect is not totally VDR-dependent.

Comparative effect of resveratrol, carnosic acid and hernandulcin on target enzymes and biochemical markers linked to carbohydrate and lipid metabolism in mice

Resveratrol (RV), carnosic acid (CA) and hernandulcin (HE; a non-caloric sweetener) are envisioned as promising nutraceuticals to designnew functional foods for improving lipid and carbohydrate metabolism. This study aimed to investigate the in vitro inhibitory effectof these molecules on specific enzyme targets and their capacity to improve distinctive markers associated to carbohydrate and lipidmetabolism in murine model. The enzymes explored were alpha-amylase, alpha-glucosidase, and pancreatic lipase whereas ICR malemice were used for in vivo testing. Saturation curves (10-200 µM mL-1) and Lineweaver-Burk regressions suggested that RV, CA andHE exerts non-competitive inhibition on pancreatic lipase, alpha-glucosidase and alpha-amylase but, CA produced a strong competitiveactivity on alpha-amylase. RV was more effective to inhibit alpha-glucosidase (IC50, 22.1 µM) whereas CA was the most effective toinhibit both alpha-amylase (IC50, 11.7 µM) and pancreatic lipase (IC50, 31.5 µM). The effects of the oral administration of RV (300 mg/kg) HE (100 mg/kg) and CA (100 mg/kg) as well as the simultaneous administration of the three compounds at the same concentration was also explored in normoglycemic and diabetic mice. In addition, the prolonged administration of these substances combined with hypercaloric/atherogenic diet for 30 days was performed. Our results revealed a clear modulatory activity in both postprandial glucose and triglyceride levels as well an improvement in biochemical markers of mice treated with hypercaloric/atherogenic diet. The administration of HE produced a notable change (p &lt; 0.01) in postprandial glucose assimilation at 60 min post-treatment in diabetic mice, whereas the other two compounds exerted a stronger depletion of glucose levels from 30 to 120 min post-treatment. A similar trend was recorded by RV and CA in postprandial triglyceride content, however, the latter compound was more effective (p &lt; 0.05) at lower doses than RV. The simultaneous administration of the three compounds produced a significant improvement (p &lt; 0.01) in biochemical parameters associated to carbohydrate (insulin and glucose) and lipid metabolism (total cholesterol, LDL-c, HDL-c, triglycerides, leptin, and adiponectin).Outstandingly, the mixture of the three compounds was more effective (p &lt; 0.01) than the administration of sole compounds to amelioratethe side effects of the hypercaloric/atherogenic diet. Finally, the body weight of treated mice significantly decreased (from 5 to 20%; p &lt; 0.05) in comparison with mice only fed with hypercaloric/atherogenic diet. Our results suggest that mixtures of RV, HE and CA may work better than their sole administration in mice and part of their biological activity could be associated with their inhibitory properties on the enzyme targets evaluated in this investigation.&#x0D; Keywords: Carnosic acid; Hernandulcin; Resveratrol; Hypoglycemic; Hypolipidemic; Murine model; Target enzymes

Hypoglycemic Activity of Hydro-Acetonic and HydroMethanolic Leaf and Bark Extracts of Bauhinia rufescens Lam in Mice

The purpose of this study was to determine the hypoglycaemic effects of leaf and bark extracts of Bauhinia rufescens Lam, used in traditional medicine for diabetes in Senegal. Phytochemical screening of hydromethanolic and hydro-acetonic extracts obtained from leaves and barks was carried out according to standard analytical methods. Experiments were carried out on normoglycemic and diabetic mice. These were obtained after the injection of a single dose of glucose (4 g/kg of body weight). The results showed that polyphenolic compounds, tannins, and flavonoids were the main phytochemical constituents of the extracts. Then the oral administration of the different extracts of Bauhinia rufescens Lam at doses of 500, 700, and 1000 mg/kg of body weight led to a significant reduction in blood sugar (p &lt; 0.05), similar to the antidiabetic action of glibenclamide (10 mg/kg of body weight). Whatever the organ and the extraction solvent, the doses of 500 and 700 mg/kg of body weight induced dose-dependent hypoglycemia, just like glibenclamide. These results show that the metabolites revealed in the extracts could be responsible for their hypoglycemic effects. Thus, supported by a complete chemical study with a view to isolating the active substances responsible for the antidiabetic effect is possible. It will be allowed to formulate from a phytomedicine with potential for diabetes which makes possible their uses by populations for treatment of diabetes.