Normal Glucose Concentrations Research Articles

Targeting the histone methyltransferase SETD7 rescues diabetes-induced impairment of angiogenic response by transcriptional repression of semaphorin 3G

Abstract Background Peripheral artery disease (PAD) is highly prevalent in patients with diabetes (DM) and associates with a high rate of limb amputation and poor prognosis. Surgical and catheter-based revascularization have failed to improve outcome in DM patients with PAD. Hence, a need exists to develop new treatment strategies able to promote blood vessel growth in this setting. Mono-methylation of histone 3 at lysine 4 (H3K4me1) - a specific epigenetic signature induced by the histone methyltransferase SETD7 - favours an open chromatin thus enabling gene transcription. Purpose To investigate whether SETD7-dependent epigenetic changes modulate angiogenic response in diabetes. Methodology Primary human aortic endothelial cells (HAECs) were exposed to normal glucose (NG, 5 mM) or high glucose (HG, 25 mM) concentrations for 48 hours. Unbiased gene expression profiling was performed by RNA sequencing (RNA-seq) followed by Ingenuity Pathway Analysis (IPA). In vitro assays, namely cell migration and tube formation were employed to study angiogenic properties in HAECs. SETD7 and H3K4me1 levels were investigated by Western blot and Chromatin immunoprecipitation (ChIP). Pharmacological blockade of SETD7 was achieved by using the highly selective inhibitor (R)-PFI-2. Mice with streptozotocin-induced diabetes were orally treated with (R)-PFI-2 or vehicle and underwent hindlimb ischemia by femoral artery ligation for 14 days. Blood flow recovery was analysed at 30 minutes, 7 and 14 days by laser Doppler imaging. Our experimental findings were also translated in gastrocnemius muscle samples from patients with and without diabetes. Results RNA-seq in HG-treated HAECs revealed a profound upregulation of the methyltransferase SETD7, an enzyme involved in mono-methylation of lysine 4 at histone 3 (H3K4me1). SETD7 upregulation in HG-treated HAECs was associated with increased H3K4me1 levels as well as with impaired endothelial cell migration and tube formation. Both SETD7 gene silencing and pharmacological inhibition by (R)PFI-2 rescued hyperglycemia-induced impairment of HAECs migration and tube formation, while SETD7 overexpression blunted the angiogenic response. RNA-seq and ChIP assays showed that SETD7-dependent H3K4me1 regulates the transcription of the angiogenesis inhibitor semaphorin-3G (SEMA-3G). Moreover, SEMA-3G overexpression blunted migration and tube formation in SETD7-depleted HAECs. In diabetic mice with hindlimb ischemia, treatment with (R)-PFI-2 improved limb vascularization and perfusion as compared to vehicle. Finally, SETD7/SEMA3G axis was upregulated in muscle specimens from T2D patients as compared to controls. Conclusion Targeting SETD7 represents a novel epigenetic-based therapy to boost neovascularization in diabetic patients with PAD.

Thioredoxin Interacting Protein Expressed in Osteoblasts Mediates the Anti-Proliferative Effects of High Glucose and Modulates the Expression of Osteocalcin.

Hyperglycemia is associated with impaired bone health in patients with diabetes mellitus. Although a direct detrimental effect of hyperglycemia on the bone has been previously reported, the specific molecular mediator(s) responsible for the inhibitory effect of high glucose levels on the bone remains unclear. We hypothesized that thioredoxin-interacting protein (Txnip), an essential mediator of oxidative stress, is such a mediator. We cultured MG-63 cells (immortalized human osteoblasts) with normal or high glucose concentrations and transfected them with scrambled or Txnip-specific small interfering RNA (siRNA). High glucose levels increased Txnip expression and reduced MG-63 cell proliferation. The high-glucose level mediated reduction in cell proliferation was prevented in Txnip siRNA-transfected cells. In addition, we demonstrated that silencing Txnip mRNA expression in osteoblasts reduced the expression of the osteocalcin gene. Our results suggest that high glucose levels or silencing of Txnip mRNA expression may induce apoptosis in osteoblasts. Our findings indicate that Txnip is an intracellular mediator of the anti-proliferative effects of extracellular high glucose levels on osteoblasts.

In vitro anti-inflammatory activity of Malus × domestica var. Mela Rosa Marchigiana pulp callus extract contrasting high glucose conditions

The anti-inflammatory activity of the “Mela Rosa Marchigiana” (MRM) pulp callus ethanol Extract (MRME) was tested in different cellular models including (i) LPS-treated RAW 264.7; (ii) HUVEC exposed to short-term high Glucose (HG, 45 mM) or normal glucose (NG, 5 mM) concentrations; and (iii) HG (30 mN) and LPS-treated U937. To evaluate the anti-inflammatory effect of the extract, Nitric Oxide (NO) production was measured in RAW 264.6 cells, while IL-8, IL-1ß and MCP-1 expressions, along with modulation of some inflammation- or senescence-associated miRNAs (miR-21, miR-126, miR-17 and miR-217) were assessed in HUVECs and/or U937. MRME treatment reduced pro-inflammatory markers amount, suggesting a decreased generalised inflammatory response. Present findings indicate that MRME can contrast senescence- or HG-induced inflammation. Moreover, in HG- and LPS-induced inflammation, MRME reduced U937 monocyte activation by inhibiting mitochondrial respiration and inflammatory markers. Finally, the inhibitory potential of MRME on the digestive enzymes α-glucosidase, α-amylase and lipase activity was investigated. Our results support the idea that MRME has a positive effect on inhibition of endothelial/macrophage dysfunction under HG/senescence inflammatory conditions. Furthermore, the obtained results showed a modest inhibitory power of the extract (IC50 values: 2.98 ± 0.24, 1.77 ± 0.15, and 2.06 ± 0.31 mg/ml, respectively), which, however, could be of some help in decreasing the absorption of glucose and triglycerides.

Effect of bovine beta-casomorphins on rat pancreatic beta cells (RIN-5F) under glucotoxic stress

Beta-casomorphins (BCMs) are the bio-active peptides having opioid properties which are formed by the proteolytic digestion of β-caseins in milk. BCM-7 forms when A1 milk is digested in the small intestine due to a histidine at the 67th position in β-casein, unlike A2 milk, which has proline at this position and produces BCM-9. BCM-7 has further degraded into BCM-5 by the dipeptidyl peptidase-IV (DPP-IV) enzyme in the intestine. The opioid-like activity of BCM-7 is responsible for eliciting signaling pathways which enable a wide range of physiological effects. The aim of our study was to find out the differential role of BCMs (BCM-7, BCM-9 and BCM-5) on pancreatic β-cell proliferation, insulin secretion, and opioid peptide binding receptors from β-cells (RIN-5F cell line) in normal (5.5 mM) and high glucose (27.5 mM) concentrations. Our results showed that BCM-7/9/5 did not affect β-cell viability, proliferation, and insulin secretion at normal glucose level. However, at higher glucose concentration, BCMs significantly protected β-cells from glucotoxicity but did not affect the insulin secretion. Interestingly, in the presence of Mu-opioid peptide receptor antagonist CTOP, BCMs did not protect β-cells from glucotoxicity. The results suggest that BCMs protect β-cells from glucotoxicity via non-opioid mediated pathways because BCMs did not modulate the gene expression of the mu, kappa and delta opioid peptide receptors.

Modeling blood vessel dynamics: Effects of glucose variations on HUVECs in a hollow fiber bioreactor under laminar shear stress

This study aimed to establish a blood vessel model within a hollow fiber bioreactor to evaluate the impact of high and fluctuating glucose levels on human umbilical vein endothelial cells (HUVECs) under laminar shear stress (LSS). HUVECs were cultured for 48 h in normal (5 mM), high (20 mM), and variable (20 mM / 5 mM alternating every 24 h) glucose concentrations under LSS of 0.66 Pa. An automated medium replacement system was developed. The control cultures remained static. The analysis included cell viability via cytometric analysis, glucose consumption, lactate production via electroenzymatic methods, and the expression of 21 genes via qPCR. The percentage of apoptotic cells did not significantly differ across glucose concentrations under LSS. HUVECs favor glycolysis for energy regardless of LSS. Under LSS, the IL1B, CCL2, and SELE genes were upregulated under high-glucose conditions and downregulated under variable-glucose conditions. A few other genes related to inflammation, oxidative stress, cell adhesion and apoptosis were upregulated under high-glucose conditions. In conclusion, using the blood vessel model we effectively examined the impact of glucose profiles on HUVECs under LSS in a device replicating the cylindrical geometry of blood vessels. LSS and tubular cell arrangement might mitigate the adverse effects of variable glucose on endothelial cells.

TLR3-mediated Astrocyte Responses in High and Normal Glucose Adaptation Differently Regulated by Metformin.

Toll-like receptors 3 (TLR3) are innate immune receptors expressed on a wide range of cell types, including glial cells. Inflammatory responses altered by hyperglycemia highlight the need to explore the molecular underpinnings of these changes in cellular models. Therefore, here we estimated TLR3-mediated response of astrocytes cultured at normal (NG, 5 mM) and high (HG, 22.5 mM) glucose concentrations for 48 h before stimulation with polyinosinic:polycytidylic acid Poly(I:C) (PIC) for 6 h. Seahorse Extracellular Flux Analyzer (Seahorse XFp) was used to estimate the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). Although adaptation to HG affected ECAR and OCR, the stimulation of cells with PIC had no effect on ECAR. PIC reduced maximal OCR, but this effect disappeared upon adaptation to HG. PIC-stimulated release of cytokines IL-1β, IL-10 was reduced, and that of IL-6 and iNOS was increased in the HG model. Adaptation to HG reduced PIC-stimulated synthesis of COX-derived oxylipins measured by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Adaptation to HG did not alter PIC-stimulated p38 activity, ERK mitogen-activated protein kinase, STAT3 and ROS production. Metformin exhibited anti-inflammatory activity, reducing PIC-stimulated synthesis of cytokines and oxylipins. Cell adaptation to high glucose concentration altered the sensitivity of astrocytes to TLR3 receptor activation, and the hypoglycemic drug metformin may exert anti-inflammatory effects under these conditions.

Long non-coding RNA PANDA drives diabetic vascular dysfunction by promoting endothelial senescence and oxidative damage

Abstract Funding Acknowledgements None. Background ROS and inflammation are major features of diabetic vasculopathy, yet the underlying mechanisms remain elusive. Long non-coding RNAs (lncRNAs) are emerging as important players in the pathogenesis of cardiovascular disease. Recent work has shown that PANDA, a newly identified lncRNA, is a key regulator of cellular senescence and apoptosis. Purpose To elucidate the role and molecular mechanism of lncRNA PANDA in diabetic vascular disease. Methods Human aortic endothelial cells (HAECs) were exposed to normal (NG, 5 mmol/L) and high glucose concentrations (HG, 25 mmol/L). PANDA depletion in HG-treated HAECs was obtained by siRNA transfection. Expression of PANDA was assessed by real time PCR. RNAs sequencing (RNA-seq) and bioinformatic analysis (network perturbation amplitude, NPA) were leveraged to unveil transcriptional changes upon PANDA depletion. PANDA RNA immuno-precipitation (RIP) was performed to check its binding to relevant transcriptional factor. Western Blots was used to investigate on NRF2 protein and its products. Beta-galactosidase staining was used to detect endothelial senescence while, migration and tube formation were employed to evaluate angiogenic properties of HAECs. ROS production and NRF2 localization were investigated by fluorescence staining. The ex vivo effect of PANDA siRNA on endothelial function was assessed in aortic rings from diabetic mice. Results PANDA expression was significantly increased in HAECs exposed to HG as compared to NG. Transcriptomic analysis revealed dysregulation of several genes upon PANDA silencing with the antioxidant gene heme oxygenase-1 as the top-ranking transcript in HG-treated cells (Fig). NPA analysis showed a strong involvement of PANDA in senescence, DNA damage, NRF2 signaling, hypoxic stress response and proliferation. Under HG conditions PANDA perturbed the pathway of the transcription factor NRF2, thus inhibiting the expression of NRF2-dependent pro-survival and anti-aging gene. Indeed, silencing of PANDA in HG-treated HAECs reduced the apotosis and senescence (Figure 1). PANDA depletion in HG improved endothelial migration and tube formation, while ROS production was reduced. In HG, there was an increasing of PANDA-NRF2 binding (Fig). Interesting, in HG NRF2 was more present in the cytosol, while PANDA siRNA allowed its translocation into the where it can promote the transcription of HMOX1. Of interest, PANDA levels were increased in aortas from diabetic mice while depletion of PANDA rescued endothelial dysfunction (Figure 2). Conclusions The hyperglycemia induced the upregulation of PANDA driving endothelial senescence and impairing angiogenic properties. PANDA depletion in HG-treated HAECs rescued maladaptive transcriptional changes enhancing NRF2 pathway. Of note, targeting PANDA in the diabetic vasculature was able to rescue endothelial dysfunction. These results indicate PANDA as a novel molecular target in the setting of diabetic vascular disease.

METHODS AND ACTIVITIES OF HEALTH COMPETENCE DEVELOPMENT IN NON-FORMAL ENVIRONMENT: EXPERIENCE OF CHILDREN WITH TYPE 1 DIABETES MELLITUS

Diabetes mellitus is a disease when certain processes in the body, which maintain a normal blood glucose concentration, get imbalanced. Research object is educational methods and activities used for the development and training of health competence. The aim of the research is to reveal educational methods and activities used for the development of health competence in non-formal environment, based on the experience of children with type 1 diabetes mellitus. Research methods: theoretical – analysis, comparison, and generalization of scientific literature; empirical – the use of focus group discussions with children with diabetes mellitus for data collection; Empirical research revealed that various educational methods and activities aimed at developing health competences are carried out for children with type 1 diabetes mellitus in non-formal educational environments (medical facilities, summer camps and health camps). The following activities and classes are used in medical facilities: lessons, conversations, questionnaires, and games. Activities used in summer camps are as follows: walks, cycling, games designed to develop physical activity skills. In the educational environment of health cams, the following educational methods and activities are used for the development of health competence for children with type 1 diabetes mellitus: lessons, games, educational activities, trips, and creative activities.

Нарушения углеводного обмена у пациентов с боевыми травмами

Aim. To investigate the prevalence and some aspects of the carbohydrate metabolism pathology in patients with combat limb injuries. Materials and methods. 210 patients who suffered from combat injuries of the lower and/or upper extremities from 4 to 58 days (mean 15.9±8.7 days) before admission to the hospital were enrolled in the study. All study participants were men. The mean age of the patients was 36.0±8.4 years (19–59 years). Fasting venous blood glucose concentrations were determined in all patients. Patients with hyperglycemia underwent a study of the glycemic profile and HbA1c. Stress-induced hyperglycemia, estimated mean glucose level over the past 3 months (ADAG), and glycemic gap were calculated. Insulin in fasting blood plasma was determined and the HOMA-IR insulin resistance index was calculated. Results. The incidence of hyperglycemia was 23.3%. In the general group of patients, the mean glucose concentration was 5.5±1.0 mmol/l (3.1–9.73 mmol/l). In the group of patients with hyperglycemia, the mean blood glucose level reached 6.7±0.9 mmol/l, in patients with normal glucose concentration – 5.1±0.5 mmol/l (p<0.000001). The mean fasting blood glucose concentration in patients aged 40 years and older reached 5.7±1.1 mmol/l, those under 40 years old – 5.2±0.8 mmol/l (p=0.005). A significant direct correlation was registered between the fasting glucose level upon admission to the hospital and the age of the patients (r=0.26; p=0.00003). The relative risk of hyperglycemia in patients aged 40 years and older was 1.84 (confidence interval 1.005–3.37; p=0.01). The mean time elapsed since injury in patients with hyperglycemia reached 13.2±5.7 days, with normal glucose concentration – 16.7±9.2 days (p=0.02). Patients with hyperglycemia had lower levels of total protein (58.3±10.2 and 62.1±6.9 g/l, respectively; p=0.004), albumin (31.2±4.6 and 33.2±4.9 g/l; p=0.04), hemoglobin (97.7±18.9 and 105.9±16.9 g/l; p=0.004), but higher levels of C-reactive protein (61.0±55.4 and 32.9±38.1 mg/l; p=0.0001), leukocytes (10.6±4.4 and 9.3±3.4×109/l; p=0.02), count of neutrophils (7.8±4.3 and 6.4±2.9×109/l; p=0.01), proportion of neutrophils (71.6±9.6% and 67.7±7.7%; p=0.004), as well as fibrinogen concentration (5.7±1.4 and 4.8±1.2 g/l; p=0.0002) than patients with normal blood glucose levels. In patients with hyperglycemia, the mean HbA1c level was 5.3±0.6%, stress-induced hyperglycemia – 1.4±0.2 mmol/l. The median glycemic gap reached 1.51 mmol/l (Q1–Q3: 0.88–2.1 mmol/l). The median insulin concentration was 12.9 µIU/ml (Q1–Q3: 9.2–27.0 µIU/ml), the insulin resistance index was 3.3 (Q1–Q3: 2.1–8.8). The mean fasting blood glucose concentration at the last determination in patients with initial hyperglycemia reached 5.8±1.3 mmol/l. During observation, in 64.6% of the patients with hyperglycemia upon admission, glucose levels returned to normal, while in 35.4% mild hyperglycemia remained. The median time to normalization of fasting blood glucose concentration or to the last determination of this indicator was 17.5 days (Q1–Q3: 8–30 days). Conclusions. The study results indicate a frequent detection of hyperglycemia in patients with combat limb injuries. In most cases the increase in fasting glucose levels is small and in most patients this indicator returns to normal within a relatively short time. It is necessary to continue research of stress-induced hyperglycemia in the patients with combat injuries and to elucidate the pathogenetic basis of this pathology.

Reframing prediabetes: A call for better risk stratification and intervention.

Prediabetes is an intermediate state of glucose homeostasis whereby plasma glucose concentrations are above normal but below the threshold of diagnosis for diabetes. Over the last several decades, criteria for prediabetes have changed as the cut points for normal glucose concentration and diagnosis of diabetes have shifted. Global consensus does not exist for prediabetes criteria; as a result, the clinical course and risk for type 2 diabetes vary. At present, we can identify individuals with prediabetes based on three glycemic tests (hemoglobin A1c, fasting plasma glucose, and 2-h plasma glucose during an oral glucose tolerance test). The majority of individuals diagnosed with prediabetes meet only one of these criteria. Meeting one, two, or all glycemic criteria changesrisk for type 2 diabetes, but this information is not widely known and does not currently guide intervention strategies for individuals with prediabetes. This review summarizes current epidemiology, prognosis, and intervention strategies for individuals diagnosed with prediabetes and suggests a call for more precise risk stratification of individuals with prediabetes as elevated (one prediabetes criterion), high risk (two prediabetes criteria), and very high risk (three prediabetes criteria). In addition, the roles of oral glucose tolerance testing and continuous glucose monitoring in the diagnostic criteria for prediabetes need reassessment. Finally, we must reframe our goals for prediabetes and prioritize intensive interventions for those at high and very high risk for type 2 diabetes.

Evidence that tirzepatide protects against diabetes-related cardiac damages

BackgroundGlucagon-like peptide-1 receptor agonists (GLP-1RAs) are effective antidiabetic drugs with potential cardiovascular benefits. Despite their well-established role in reducing the risk of major adverse cardiovascular events (MACE), their impact on heart failure (HF) remains unclear. Therefore, our study examined the cardioprotective effects of tirzepatide (TZT), a novel glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) receptor agonist.MethodsA three-steps approach was designed: (i) Meta-analysis investigation with the primary objective of assessing major adverse cardiovascular events (MACE) occurrence from major randomized clinical trials.; (ii) TZT effects on a human cardiac AC16 cell line exposed to normal (5 mM) and high (33 mM) glucose concentrations for 7 days. The gene expression and protein levels of primary markers related to cardiac fibrosis, hypertrophy, and calcium modulation were evaluated. (iii) In silico data from bioinformatic analyses for generating an interaction map that delineates the potential mechanism of action of TZT.ResultsMeta-analysis showed a reduced risk for MACE events by TZT therapy (HR was 0.59 (95% CI 0.40–0.79, Heterogeneity: r2 = 0.01, I2 = 23.45%, H2 = 1.31). In the human AC16 cardiac cell line treatment with 100 nM TZT contrasted high glucose (HG) levels increase in the expression of markers associated with fibrosis, hypertrophy, and cell death (p < 0.05 for all investigated markers). Bioinformatics analysis confirmed the interaction between the analyzed markers and the associated pathways found in AC16 cells by which TZT affects apoptosis, fibrosis, and contractility, thus reducing the risk of heart failure.ConclusionOur findings indicate that TZT has beneficial effects on cardiac cells by positively modulating cardiomyocyte death, fibrosis, and hypertrophy in the presence of high glucose concentrations. This suggests that TZT may reduce the risk of diabetes-related cardiac damage, highlighting its potential as a therapeutic option for heart failure management clinical trials. Our study strongly supports the rationale behind the clinical trials currently underway, the results of which will be further investigated to gain insights into the cardiovascular safety and efficacy of TZT.Graphical

A case of pediatric enteroviral meningitis with a travel history

Enterovirus meningitis represents a common cause of meningitis worldwide. In this case study, a 5-year-old male with a travel history presenting with persistent nausea, vomiting, fever and headache for 3 days is reported. The patient described a fever of 39.5°C on the first day of symptom onset as well as decreased appetite and pain in the front right side of head. In physical examination, initially there was no sign of stiff neck, however he had neck stiffness the following day, Kernig's sign and Brudzinski's signs were positive. On admission, blood tests showed a slightly increased C-reactive protein and a normal white cell count. He was admitted to the pediatrics service and a lumbar puncture was performed. Intravenous ceftriaxone and acyclovir were empirically administered. Cerebrospinal fluid (CSF) analysis revealed lymphocytic pleocytosis, with normal protein and glucose concentration. CSF molecular analysis was positive for enterovirus RNA. Cranial magnetic resonance imaging with contrast was normal. Following confirmed diagnosis, ceftriaxone and acyclovir treatments were discontinued and he was given supportive care. He successfully recovered and was discharged without any complication. This case report highlights that rapid molecular testing favorably impacts patient management by improving antimicrobial stewardship through the reduction of intravenous therapy, side effects and inpatient bed-days.

High Glucose Is a Stimulation Signal of the Salt-Tolerant Yeast Zygosaccharomyces rouxii on Thermoadaptive Growth.

The salt-tolerant yeast Zygosaccharomyces rouxii is a typical aroma-producing yeast used in food brewing, but its mechanism of high temperature tolerance is still unclear. In this study, the response mechanism of Z. rouxii to glucose under high temperature stress at 40 °C was explored, based on the total synthetic lowest-nutrient medium. The results of the growth curves and scanning electron microscopy showed that high glucose was necessary for Z. rouxii to restore growth under high temperature stress, with the biomass at 300 g/L of glucose (OD600, 120h = 2.44 ± 0.26) being 8.71 times higher than that at 20 g/L (OD600, 120h = 0.28 ± 0.08). The results of the transcriptome analysis, combined with RT-qPCR, showed that the KEGG analysis of differentially expressed genes was enriched in pathways related to glucose metabolism, and high glucose (300 g/L) could effectively stimulate the gene expression of glucose transporters, trehalose synthesis pathways, and xylitol synthesis pathways under a high temperature, especially the expression of the glucose receptor gene RGT2 (up-regulated 193.7 times at 12 h). The corresponding metabolic characteristics showed that the contents of intracellular metabolites, such as glucose (Cmax, 6h = 6.50 ± 0.12 mg/g DCW), trehalose (Cmax, 8h = 369.00 ± 17.82 μg/g DCW), xylitol (Cmax, 8h = 1.79 ± 0.27 mg/g DCW), and glycerol (Cmax, 8h = 268.10 ± 44.49 μg/g DCW), also increased with time. The accumulation of acetic acid, as the main product of overflow metabolism under high temperature stress (intracellular Cmax, 2h = 126.30 ± 10.96 μg/g DCW; extracellular Cmax, 12h = 499.63 ± 27.16 mg/L), indicated that the downstream glycolysis pathway was active. Compared with the normal physiological concentration of glucose, a high glucose concentration can effectively stimulate the gene expression and metabolism of salt-tolerant Z. rouxii under high-temperature conditions to restore growth. This study helps to deepen the current understanding of the thermoadaptive growth mechanism of salt-tolerant Z. rouxii.

Tirzepatide prevents neurodegeneration through multiple molecular pathways

BackgroundSeveral evidence demonstrated that glucagon-like peptide 1 receptor agonists (GLP1-RAs) reduce the risk of dementia in type 2 diabetes patients by improving memory, learning, and overcoming cognitive impairment. In this study, we elucidated the molecular processes underlying the protective effect of Tirzepatide (TIR), a dual glucose-dependent insulinotropic polypeptide receptor agonist (GIP-RA)/ GLP-1RA, against learning and memory disorders.MethodsWe investigated the effects of TIR on markers of neuronal growth (CREB and BDNF), apoptosis (BAX/Bcl2 ratio) differentiation (pAkt, MAP2, GAP43, and AGBL4), and insulin resistance (GLUT1, GLUT4, GLUT3 and SORBS1) in a neuroblastoma cell line (SHSY5Y) exposed to normal and high glucose concentration. The potential role on DNA methylation of genes involved in neuroprotection and epigenetic modulators of neuronal growth (miRNA 34a), apoptosis (miRNA 212), and differentiation (miRNA 29c) was also investigated. The cell proliferation was detected by measuring Ki-67 through flow cytometry. The data were analysed by SPSS IBM Version 23 or GraphPad Prism 7.0 software and expressed as the means ± SEM. Differences between the mean values were considered significant at a p-value of < 0.05. GraphPad Prism software was used for drawing figures.ResultsFor the first time, it was highlighted: (a) the role of TIR in the activation of the pAkt/CREB/BDNF pathway and the downstream signaling cascade; (b) TIR efficacy in neuroprotection; (c) TIR counteracting of hyperglycemia and insulin resistance-related effects at the neuronal level.ConclusionsWe demonstrated that TIR can ameliorate high glucose-induced neurodegeneration and overcome neuronal insulin resistance. Thus, this study provides new insight into the potential role of TIR in improving diabetes-related neuropathy.Graphical

Sodium-Glucose Cotransporter-2 Inhibitors in Odontogenic Infections

On top of improving glycemic control, sodium-glucose cotransporter-2 inhibitors have been shown to reduce cardiovascular mortality &amp; heart failure hospitalization. Sodium-glucose cotransporter-2 inhibitors have also been gaining momentum as effective reno-protective agents. Recent evidences have shown that sodium-glucose cotransporter-2 inhibitors are transforming the management of heart failure and chronic kidney disease in patients without type 2 diabetes mellitus. In view of the cardioprotective and reno-protective outcomes, as well as the potential benefits that outweigh adverse effects, it is no doubt that there will be continued increased use of sodium-glucose cotransporter-2 inhibitors. However, with use of sodium-glucose cotransporter-2 inhibitors comes risk of adverse effects, in particular diabetic ketoacidosis. Although uncommon, diabetic ketoacidosis is a potentially life-threatening acute metabolic complication. Diabetic ketoacidosis developing during sodium-glucose cotransporter-2 inhibitors use can present with normal blood glucose concentrations (euglycemia). This atypical presentation can delay diagnosis and hence, treatment. It is therefore crucial for dental practitioners to be cognizant of the increased risk of euglycemic diabetic ketoacidosis associated with sodium-glucose cotransporter-2 inhibitors use, especially during periods of reduced oral intake, such as in patients with odontogenic infection. Euglycemic diabetic ketoacidosis is a diagnosis of exclusion and should be considered as a differential in an ill patient on sodium-glucose cotransporter-2 inhibitors, despite normal blood glucose or absent urine ketones. We report a case of starvation ketosis in a patient with well-controlled type 2 diabetes mellitus, after excisional biopsy of right cervical lymph node and extractions of two lower right molars. Although the patient did not develop euglycemic diabetic ketoacidosis peri-operatively, it was an important diagnosis to exclude considering his high-risk profile of developing euglycemic diabetic ketoacidosis and the potential sequelae of missing the diagnosis.

Effects of bioavailability of macronutrients on overall control of plasma glucose: a review

Macronutrients play a crucial role in management of type 2 diabetes. This is seen in their ability to modulate plasma glucose concentrations. However, the ideal proportions of macronutrients to be consumed in order to maintain ideal plasma glucose concentrations remains elusive. Therefore, this paper set out to conduct a review to investigate the relationship between macronutrients and plasma glucose concentrations from a physiological perspective. The review was conducted using papers obtained from various databases such as MEDLINE (Pubmed), Open Access Journals Elsevier, Free Medical Journals and Google Scholar. The research papers included general reviews, systemic reviews, meta-analyses, and randomized control trials that examined the effect of macronutrients on plasma glucose concentration as well as papers on mathematical models describing the relationship between macronutrient bioavailability and plasma glucose concentration. The review assessed the effect of various macronutrients on post-prandial plasma glucose concentration, post-prandial plasma insulin, post-prandial glucose-dependent insulinotropic peptide plasma concentration, and post-prandial glucose-like peptide-1 plasma concentration. The results of the review showed that carbohydrates influence plasma glucose concentration in a dose dependant manner but this is dependent on their bioavailability. This bioavailability was shown to be subject to fluctuations determined by food processing techniques, food structure, and food matrix. The results also showed that some specific types of fats and proteins indirectly influence plasma glucose concentration through their effect on incretin hormones. The effect of fats and proteins on incretin hormones was through different mechanisms and pathways. In-lieu of the findings, the review concludes that the macronutrient composition of diets designed for type 2 diabetic patients should take into consideration the physiological relationship between the macronutrients and plasma glucose concentrations. In this way, diet proportions can be made in such a manner as to determine the exact amounts that will realize near normal plasma glucose concentrations for a type 2 diabetic patient. Key words: bioavailability, glucose-insulin system, macronutrients, incretin hormones, type 2 diabetes, glucose absorption models

Histone Mono-methylation by SETD7 Orchestrates Angiogenic Response in Diabetic Limb Ischemia

Abstract Background Peripheral artery disease (PAD) is highly prevalent in patients with diabetes (DM) and associates with a high rate of limb amputation and poor prognosis. Surgical and catheter-based revascularization have failed to improve outcome in DM patients with PAD. Hence, a need exists to develop new treatment strategies able to promote blood vessel growth in this setting. Mono-methylation of histone 3 at lysine 4 (H3K4me1) - a specific epigenetic signature induced by the histone methyltransferase SETD7 - favours an open chromatin thus enabling gene transcription. Purpose: To investigate whether SETD7-dependent epigenetic changes modulate angiogenic response in diabetes. Methodology: Primary human aortic endothelial cells (HAECs) were exposed to normal glucose (NG, 5 mM) or high glucose (HG, 25 mM) concentrations for 48 hours. Unbiased gene expression profiling was performed by RNA sequencing (RNA-seq) followed by Ingenuity Pathway Analysis (IPA). In vitro assays, namely cell migration and tube formation were employed to study angiogenic properties in HAECs. SETD7 and H3K4me1 levels were investigated by Western blot and Chromatin immunoprecipitation (ChIP). Pharmacological blockade of SETD7 was achieved by using the highly selective inhibitor (R)-PFI-2. Mice with streptozotocin-induced diabetes were orally treated with (R)-PFI-2 or vehicle and underwent hindlimb ischemia by femoral artery ligation for 14 days. Blood flow recovery was analysed at 30 minutes, 7 and 14 days by laser Doppler imaging. Our experimental findings were also translated in gastrocnemius muscle samples from patients with and without diabetes. Results RNA-seq in HG-treated HAECs revealed a profound upregulation of the methyltransferase SETD7, an enzyme involved in mono-methylation of lysine 4 at histone 3 (H3K4me1). SETD7 upregulation in HG-treated HAECs was associated with increased H3K4me1 levels as well as with impaired endothelial cell migration and tube formation. Both SETD7 gene silencing and pharmacological inhibition by (R)PFI-2 rescued hyperglycemia-induced impairment of HAECs migration and tube formation, while SETD7 overexpression blunted the angiogenic response. RNA-seq and ChIP assays showed that SETD7-dependent H3K4me1 regulates the transcription of the angiogenesis inhibitor semaphorin-3G (SEMA-3G, (Figure 1). Moreover, SEMA-3G overexpression blunted migration and tube formation in SETD7-depleted HAECs. In diabetic mice with hindlimb ischemia, treatment with (R)-PFI-2 improved limb vascularization and perfusion as compared to vehicle (Figure 2). Finally, SETD7/SEMA3G axis was upregulated in muscle specimens from T2D patients as compared to controls (Figure 2) Conclusion: Targeting SETD7 represents a novel epigenetic-based therapy to boost neovascularization in diabetic patients with PAD.

Long non-coding RNA PANDA drives diabetic vascular dysfunction by promoting endothelial senescence and oxidative damage

Abstract Background Accumulation of reactive oxygen species and inflammation are major features of diabetic vasculopathy, yet the underlying mechanisms remain elusive. Long non-coding RNAs (lncRNAs) are emerging as important players in the pathogenesis of cardiovascular disease. Recent work has shown that PANDA, a newly identified lncRNA, is a key regulator of cellular senescence and apoptosis. Purpose To investigate the role of lncRNA PANDA in diabetic vascular disease. Methods Human aortic endothelial cells (HAECs) were exposed to normal (NG, 5 mmol/L) and high glucose concentrations (HG, 25 mmol/L). PANDA depletion in HG-treated HAECs was obtained by siRNA transfection while a scrambled RNA was used as a negative control. Expression of PANDA was assessed by real time PCR. PANDA RNA immuno-precipitation (RIP) was performed to check its binding to relevant transcriptional factors (NF-YA). Beta-galactosidase staining was used to detect endothelial senescence while, migration and tube formation were employed to evaluate angiogenic properties of HAECs. Finally, RNAs sequencing (RNA-seq) and bioinformatic analysis (network perturbation amplitude, NPA) were leveraged to unveil transcriptional changes upon PANDA depletion. Finally, the ex vivo effect of PANDA siRNA on endothelial function was assessed in aortic rings from diabetic mice. Results PANDA expression was significantly increased in HAECs exposed to HG as compared to NG (Fig. 1A). We found that under HG conditions PANDA sequesters the transcription factor NF-YA, thus inhibiting the expression of NF-YA-dependent pro-survival and anti-aging genes (Fig. 1B). Indeed, silencing of PANDA in HG-treated HAECs restored the expression of anti-apoptotic genes Bcl-X(L) and Bcl-2 while blunting senescence-related genes such as p53 and p16INK (Fig. 1C). PANDA depletion also abolished HG-induced cellular apoptosis and senescence, as assessed by caspase-3 activity assay and b-gal staining (Fig. 1D-E), while improving endothelial migration and tube formation (Fig.1F-G). Transcriptomic analysis revealed dysregulation of several genes upon PANDA silencing with the antioxidant gene heme oxygenase-1 (HMOX1) as the top-ranking transcript in HG-treated cells (Fig.1H). NPA analysis showed a strong involvement of PANDA in senescence, DNA damage, NRF2 signaling, hypoxic stress response and proliferation (Fig. 1I). Of interest, PANDA levels were increased in aortas from diabetic mice (Fig. 2A) while depletion of PANDA rescued endothelial dysfunction (Fig. 2B). Conclusions Hyperglycemia-induced upregulation of PANDA drives endothelial senescence while impairing angiogenic properties. PANDA depletion in HG-treated HAECs rescues maladaptive transcriptional changes by restoring expression of the antioxidant gene HMOX1. Of note, targeting PANDA in the diabetic vasculature was able to rescue endothelial dysfunction. Our results indicate PANDA as a novel molecular target in the setting of diabetic vascular disease.

Wound-healing Processes After Pulpotomy in the Pulp Tissue of Type 1 Diabetes Mellitus Model Rats

IntroductionPatients with type 1 diabetes mellitus (DM1) tend to have delayed wound healing, even in the pulp tissue. We hypothesized that hyperglycemia affects odontoblast-like cell (OLC) differentiation and is involved in macrophage polarization. Accordingly, we evaluated dental pulp stem cell differentiation and macrophage phenotypes after pulpotomy. MethodsAfter modifying DM1 rat models by streptozotocin, 8-week-old rats’ upper left first molars were pulpotomized with mineral trioxide aggregate. Meanwhile, the control group was administered saline. Immunohistochemical localization of nestin, osteopontin, α-smooth muscles (α-SMAs), and CD68 (pan-macrophage marker) was conducted 7 days after pulpotomy. The OLC differentiation stage was determined using double immunofluorescence of nestin and α-SMA. Double immunofluorescence of CD68 and iNOS was counted as M1 macrophages and CD68 and CD206 as M2 macrophages. Proliferating cell nuclear antigen and Thy-1 (CD90) were evaluated by immunofluorescence. ResultsIn DM1 rats, the reparative dentin bridge was not complete; however, the osteopontin-positive area did not differ significantly from that in controls. Proliferating cell nuclear antigen, indicative of cell proliferation, increased in positive cells in DM1 rats compared with controls. Double-positive cells for α-SMA and nestin indicated many immature OLCs in DM1. CD90 was positive only in controls. CD68-positive cells, especially M1 macrophages, were increased in DM1 rats, allowing the inflammatory stage to continue 7 days after pulpotomy. ConclusionsThe condition of DM1 model rats can interfere at various stages of the wound healing process, altering OLC differentiation and macrophage polarization. These findings highlight the importance of normal blood glucose concentrations during pulp wound healing.

In fission yeast, 65 non-essential mitochondrial proteins related to respiration and stress become essential in low-glucose conditions.

Mitochondria perform critical functions, including respiration, ATP production, small molecule metabolism, and anti-oxidation, and they are involved in a number of human diseases. While the mitochondrial genome contains a small number of protein-coding genes, the vast majority of mitochondrial proteins are encoded by nuclear genes. In fission yeast Schizosaccharomyces pombe, we screened 457 deletion (del) mutants deficient in nuclear-encoded mitochondrial proteins, searching for those that fail to form colonies in culture medium containing low glucose (0.03-0.1%; low-glucose sensitive, lgs), but that proliferate in regular 2-3% glucose medium. Sixty-five (14%) of the 457 deletion mutants displayed the lgs phenotype. Thirty-three of them are defective either in dehydrogenases, subunits of respiratory complexes, the citric acid cycle, or in one of the nine steps of the CoQ10 biosynthetic pathway. The remaining 32 lgs mutants do not seem to be directly related to respiration. Fifteen are implicated in translation, and six encode transporters. The remaining 11 function in anti-oxidation, amino acid synthesis, repair of DNA damage, microtubule cytoskeleton, intracellular mitochondrial distribution or unknown functions. These 32 diverse lgs genes collectively maintain mitochondrial functions under low (1/20-1/60× normal) glucose concentrations. Interestingly, 30 of them have homologues associated with human diseases.