Effects of tumor necrosis factor-α rs1800629 and interleukin-10 rs1800872 genetic variants on type 2 diabetes mellitus susceptibility and metabolic parameters among Jordanians.

OBJECTIVES: Diabetes mellitus (DM) is a complex chronic illness with diverse pathogenesis and associations with health complications. Genetic factors significantly contribute to DM development, and tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) genes play major roles. This study aims to explore the influence of TNF-α rs1800629 and IL-10 rs1800872 genetic variants on T2DM development in Jordanian patients at Jordan University Hospital. METHODS: One-hundred and 60 diabetic and 159 non-diabetic subjects were genotyped for TNF-α rs1800629. Additionally, 181 diabetic and 191 non-diabetic subjects were genotyped for IL-10 rs1800872 using PCR-RFLP genotyping method. The demographic, lipid, and glycemic parameters of the patients were obtained from the computer records in the hospital. RESULTS: TNF-α rs1800629 and IL-10 rs1800872 genetic variants exhibited significant different frequencies in non-T2DM subjects and T2DM patients. The difference in TNF-α rs1800629 genotype frequency between non-T2DM and T2DM participants was significant under the dominant model, while the IL-10 rs1800872 genotype frequency was significant under the recessive model. A significant association (p<0.05) was observed between TNF-α rs1800629 and total cholesterol levels, and between IL-10 rs1800872 polymorphism and glycosylated hemoglobin (HbA1c) and creatinine levels among T2DM patients. CONCLUSIONS: TNF-α rs1800629 and IL-10 rs1800872 are identified as genetic risk factors for T2DM. These variants also correlate with variations in cholesterol, HbA1c, and creatinine levels among T2DM patients. Larger clinical studies are warranted to validate these findings.

Alterations in the gene expression of SARS-COV-2 entry receptors and enzymes in lungs and hearts of controlled and uncontrolled diabetic mice.

BACKGROUND: The entry of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into the host cell is carried out by specific receptors and enzymes, including human angiotensin-converting enzyme 2 receptor (ACE2), transmembrane serine protease 2 (TMPRSS2), and cathepsin-L (CTSL). COVID-19 patients with comorbidities, such as diabetes mellitus (DM), are more prone to severe symptoms and have a higher risk of mortality. AIMS: The present study aimed to investigate the impact of controlled and uncontrolled type 1 DM (T1DM) on the gene expression of mouse Ace2, Tmprss2, and Ctsl and correlate it with the pathological alterations in the lungs and the heart of DM mice. METHODS: Balb/c mice were administered a single dose of 240 mg/kg streptozocin to induce T1DM. The blood glucose level was measured to confirm the induction of DM. Normalization of blood glucose levels in T1DM mice was achieved using 0.1 mL/kg Mixtard® insulin therapy. The mice's lungs and hearts were harvested, and the mRNA was extracted and converted to cDNA. The gene expression of Ace2, Tmprss2, Ctsl, Cyp4a11, and Adrb1 genes, which play a role in the homeostasis of lungs and hearts, were measured using quantitative real-time polymerase chain reaction (RT-PCR). The pathological alterations in the hearts and lungs induced by T1DM were evaluated using the relative heart and lung weights, in addition to the pathohistological examination. RESULTS: After inducing T1DM for 14 days, we observed a significant reduction in the total weight of uncontrolled DM (UDM) mice (P < 0.05). Pathohistological examination of UDM lung tissues revealed thickening of the alveolar walls with narrowing of the surface of the alveolar sacs. Additionally, we found that UDM mice exhibited downregulation of Ace2 gene expression (P < 0.05) in their lungs, while both UDM and control DM (CDM) mice showed upregulation of Ctsl gene expression in their hearts (P < 0.05). Notably, Cyp4a12 gene expression was significantly downregulated (P < 0.05) in UDM mice but returned to normal levels in CDM mice. CONCLUSIONS: We conclude from this study that T1DM downregulates Ace2 receptor and Cyp4a12 gene expression, which is correlated with the thickening of alveolar walls and narrowing of the surface of alveolar sacs in the lungs. Insulin administration for controlling T1DM ameliorated these pathological alterations. These results can help increase our understanding of the impact of controlled and uncontrolled T1DM on the lungs and may explain, at least in part, why DM patients with COVID-19 experience exacerbation of symptoms.

Upregulation of Beta 1 and Arachidonic Acid Metabolizing Enzymes in the Mouse Hearts and Kidneys after Sub Chronic Administration of Rofecoxib.

BACKGROUND: An imbalance in the levels of arachidonic acid (ARA) metabolites in cardiovascular disorders and drug-induced cardiotoxicity have been previously described. AIMS: This study aimed to investigate the influence of cyclooxygenase-2 (COX-2) selective inhibitors on the gene expression of ARA-metabolizing genes and beta1 gene in the hearts and kidneys of experimental mice. METHODS: Thirty-five balb/c mice were divided into five groups with seven mice per group. The groups were then given two distinct types of COX-2 selective inhibitors, rofecoxib and celecoxib, in two different doses equivalent to those used in human treatment for 30 days. The mRNA expression of beta1, ace2, and ARA-metabolizing genes, coxs, lipoxygenases (aloxs), and cytochrome p450 (cyp450s) in mice heart and kidneys were assessed. Genes were analyzed using real-time polymerase chain reaction analysis. In addition, rofecoxib-induced histological alterations were examined. RESULTS: It was found that only the high dose of rofecoxib (5 mg/kg) caused toxicological alterations, a finding that was indicated by a significant increase (P < 0.05) in the relative weight of the mouse hearts and increase in the ventricle wall thickness as observed through pathohistological examination. This increase was associated with a significant increase in the mRNA expression level of the beta1 receptor in both the heart and kidneys of the mice (53- and 12-fold, respectively). The expression of both cox1 and 2 genes was increased 4-fold in the kidneys. In addition, the expression of the alox12 gene increased significantly (by 67-fold in the heart and by 21-fold in the kidney), while alox15 gene expression was upregulated in the heart by 8-fold and 5-fold in the kidney. The genes responsible for synthesizing 20- Hydroxyeicosatetraenoic acid (cyp4a12 and cyp1a1) were significantly upregulated (P < 0.05) in the hearts of high-dose rofecoxib-treated mice by 7- and 17 -fold, respectively. In addition, the expression of epoxyeicosatrienoic acid-synthesizing genes, cyp2c29 and cyp2j5, was increased significantly (P < 0.05) in the hearts of high-dose rofecoxib-treated mice by 4- and 16-fold, respectively. CONCLUSION: Rofecoxib caused upregulation of the mRNA expression of the beta 1 gene in association with increased expression of ARA-metabolizing genes in mouse hearts and kidneys. These findings may help us understand the molecular cardiotoxic mechanism of rofecoxib.

Variability of CYP2C8 Polymorphisms in Three Jordanian Populations: Circassians, Chechens and Jordanian-Arabs.

CYP2C8 is a member of Cytochrome P450 enzymes system. It plays an important role in metabolizing a wide range of exogenous and endogenous compounds. CYP2C8 is involved in the metabolism of more than 100 drugs, typical substrates include: anticancer agents, antidiabetic agents, antimalarial agents, lipid lowering drugs and many others that constitute 20% of clinically prescribed drugs. Genetic variations of CYP2C8 have been reported with different frequencies in different populations. These genetic polymorphisms can lead to differences in the efficacy and safety of different types of medications metabolized by CYP2C8. The aim of this study was to investigate the allele frequencies of CYP2C8*3 (rs10509681 and rs11572080) and CYP2C8*4 (rs1058930) polymorphisms in three populations living in Jordan; Circassians and Chechens and Jordanian-Arabs and compare those frequencies with other populations. A total of 200 healthy Jordanians, 93 Circassians and 88 Chechens were included in this study. Genotyping of CYP2C8*3 and CYP2C8*4 polymorphisms was done by using polymerase chain reaction (PCR) followed by Restriction Fragment Length Polymorphism (RFLP). Using the Chi-square test, we found that the prevalence of CYP2C8*3 and *4 among the three populations were significantly different. Moreover, the mutant allele CYP2C8*3 (416A) was only detected in the Jordanian-Arab population with an allele frequency of 0.082, while the mutant allele CYP2C8*4 (792G) was detected with frequencies of 0.065, 0.122, 0.017 in Jordanian-Arabs, Circassians and Chechens, respectively. As our results show, CYP2C8*3 was undetectable in our Circassians and Chechens samples, on the other hand, Circassians had the highest allele frequency of CYP2C8*4 compared to Chechens and Jordanian-Arabs. These genetic variations of the gene encoding the CYP2C8 drug metabolizing enzymes can lead to clinical differences in drug metabolism and ultimately variations in drug effectiveness and toxicities. This study provides evidence for the importance of personalized medicine in these populations and can be the foundation for future clinical studies.

The Association between Apolipoprotein E Polymorphism and Response to Statins in Group of Hyperlipidemic Patients.

BACKGROUND AND OBJECTIVE: APOE has an important role in lipids metabolism, and in the variability in low density lipoprotein (LDL) response to statins treatment between individuals. In this study, we aim to investigate the association between APOE polymorphism and response to statins in Jordanian hyperlipidemic patients at the diabetic clinic of Jordan University Hospital. METHODS: One hundred and fifty two Jordanian Hyperlipidemic patients (52 males and 100 females) aged between 35-75 years were enrolled in this study. This study was approved by the Institutional Review Board (IRB) of Jordan University Hospital. The genotypes of the patients were identified by polymerase chain reaction followed by restriction fragment length polymorphism assay method (PCRRFLP). RESULTS AND CONCLUSION: The study showed that there is an association between APOE polymorphism and response to statin therapy. Patients who were APOE ε4 carriers had lower response to statins compared to ε3 and ε2 carriers (p=0.002). In addition, we found that there was no significant association between APOE polymorphism and LDL baseline (p=0.214). No significant differences were found in APOE genotypes distribution between males and females (p=0.06). No significant association was found between age and APOE genotypes (p=0.347). A genotype screening test for dyslipidemic Jordanian patients is recommended to choose the appropriate treatment decisions, dosage, and to recognize the potential side effects of statin therapy.

L-Citrulline Supplementation Increases Plasma Nitric Oxide Levels and Reduces Arginase Activity in Patients With Type 2 Diabetes.

Type 2 diabetes mellitus (T2DM) is becoming a major contributor to cardiovascular disease. One of the early signs of T2DM associated cardiovascular events is the development of vascular dysfunction. This dysfunction has been implicated in increasing the morbidity and mortality of T2DM patients. One of the important characteristics of vascular dysfunction is the impaired ability of endothelial cells to produce nitric oxide (NO). Additionally, decreases in the availability of NO is also a major contributor of this pathology. NO is produced by the activity of endothelial NO synthase (eNOS) on its substrate, L-arginine. Reduced availability of L-arginine to eNOS has been implicated in vascular dysfunction in diabetes. Arginase, which metabolizes L-arginine to urea and ornithine, competes directly with NOS for L-arginine. Hence, increases in arginase activity can decrease arginine levels, reducing its availability to eNOS and decreasing NO production. Diabetes has been linked to elevated arginase and associated vascular endothelial dysfunction. We aimed to determine levels of plasma NO and arginase activity in (T2DM) patients and the effects of L-citrulline supplementation, a natural arginase inhibitor, on inhibiting arginase activity in these patients. Levels of arginase correlated with HbA1c levels in diabetic patients. Twenty-five patients received L-citrulline supplements (2000 mg/day) for 1 month. Arginase activity decreased by 21% in T2DM patients after taking L-citrulline supplements. Additionally, plasma NO levels increased by 38%. There was a modest improvement on H1Ac levels in these patients, though not statistically significant. The effect of L-citrulline on arginase activity was also studied in bovine aortic endothelial cells (BAECs) grown in high glucose (HG) conditions. HG (25 mM, 72 h) caused a 2-fold increase in arginase activity in BAECs and decreased NO production by 30%. L-citrulline (2.5 mM) completely prevented the increase in arginase activity and restored NO production levels. These data indicate that L-citrulline can have therapeutic benefits in diabetic patients through increasing NO levels and thus maintaining vascular function possibly through an arginase inhibition related pathway.

TPMT Genotype and Adverse Effects of Azathioprine among Jordanian Group.

BACKGROUND: Inflammatory Bowel Disease (IBD) is a common disease affecting many patients. This disease is treated by azathioprine and TPMT genetic polymorphism affecting the patient's tolerance. The aim of this study is to investigate the importance of TMPT genotyping in reducing the incidence of adverse effects of azathioprine. METHODS: One hundred and forty-one IBD patients were followed for azathioprine Adverse Drug Reaction (ADR). Patients were genotyped for TPMT*2, TPMT*3A, TPMT*3B, TPMT*3C. RESULTS: The frequency of Azathioprine adverse effect was about 35.5%. An association between TPMT genotypes 1/3A and 3B/3B and azathioprine related bone marrow suppression was found (P value ≤ 0.05). CONCLUSION: The findings suggest that there was a significant association between TPMT genotypes 1/3A and 3B/3B and azathioprine related bone marrow suppression.

Effect of obstructive sleep apnea on the response to hypertension therapy.

Obstructive sleep apnea (OSA) often precedes cardiovascular disease, partly due to treatment resistant hypertension. The nocturnal apneas of OSA trigger increased sympathetic nervous discharge during both sleep and wakefulness. Apneas also trigger cardiac release of the endogenous diuretic atrial natriuretic peptide. We hypothesized that treatment of the excess sympathetic nervous activity of OSA with a ß1 blocker would lower 24 h blood pressure (BP) more than diuretic therapy. Subjects with OSA associated hypertension received 2 weeks of placebo followed by the ß1 blocker nebivolol or hydrochlorothiazide (HCTZ) for 6 weeks in a blinded crossover study. BP, baroreflex sensitivity (BRS), heart rate variability (HRV), arterial reactivity, and stiffness were measured after placebo and each treatment. The ß1 blocker lowered clinic BP by -11/-8 mmHg, more than the -3/-1 effect of HCTZ (P < 0.01). The ß1 blocker lowered 24 h diastolic blood pressure (DBP) more than HCTZ. Although given at bedtime, neither drug increased BP dipping. Nebivolol increased HRV in the high-frequency band. Nebivolol did not alter BRS while HCTZ significantly diminished BRS compared to nebivolol (P < 0.01). Nebivolol increased flow-mediated brachial artery dilation when compared to HCTZ and slowed pulse wave velocity, indicating a decrease in arterial stiffness. Diuretic therapy failed to lower BP in OSA subjects and this might account for the frequent association of OSA with treatment resistant hypertension. However, blockade of the excess sympathetic nervous activity of OSA with a ß1 blocker lowered both clinic and 24 h DBP.

Chronic ß2 adrenergic agonist, but not exercise, improves glucose handling in older type 2 diabetic mice.

Insulin resistant type 2 diabetes mellitus in the obese elderly has become a worldwide epidemic. While exercise can prevent the onset of diabetes in young subjects its role in older diabetic people is less clear. Exercise stimulates the release of the ß(2)-agonist epinephrine more in the young. Although epinephrine and ß(2)-agonist drugs cause acute insulin resistance, their chronic effect on insulin sensitivity is unclear. We fed C57BL/6 mice a high fat diet to induce diabetes. These overweight animals became very insulin resistant. Exhaustive treadmill exercise 5 days a week for 8 weeks had no effect on their diabetes, nor did the ß(2)-blocking drug ICI 118551. In contrast, exercise combined with the ß(2)-agonist salbutamol (albuterol) had a beneficial effect on both glucose tolerance and insulin sensitivity after 4 and 8 weeks of exercise. The effect was durable and persisted 5 weeks after exercise and ß(2)-agonist had stopped. To test whether ß(2)-agonist alone was effective, the animals that had received ß(2)-blockade were then given ß(2)-agonist. Their response to a glucose challenge improved but their response to insulin was not significantly altered. The ß(2)-agonists are commonly used to treat asthma and asthmatics have an increased incidence of obesity and type 2 diabetes. Although ß(2)-agonists cause acute hyperglycemia, chronic treatment improves insulin sensitivity, probably by improving muscle glucose uptake.

Epinephrine and the metabolic syndrome.

Epinephrine is the prototypical stress hormone. Its stimulation of all α and ß adrenergic receptors elicits short-term systolic hypertension, hyperglycemia, and other aspects of the metabolic syndrome. Acute epinephrine infusion increases cardiac output and induces insulin resistance, but removal of the adrenal medulla has no consistent effect on blood pressure. Epinephrine is the most effective endogenous agonist at the ß2 receptor. Transgenic mice that cannot make epinephrine and mice that lack the ß2 receptor become hypertensive during exercise, presumably owing to the absence of ß2-mediated vasodilatation. Epinephrine-deficient mice also have cardiac remodeling and poor cardiac responses to stress, but do not develop resting hypertension. Mice that cannot make epinephrine have a normal metabolism on a regular 14% fat diet but become hyperglycemic and insulin resistant when they eat a high fat diet. Vigorous exercise prevents diabetes in young mice and humans that overeat. However, exercise is a less effective treatment in older type 2 human diabetics and had no effect on glucose or insulin responses in older, diabetic mice. Sensitivity of the ß2 receptor falls sharply with advancing age, and adrenal epinephrine release also decreases. However, treatment of older diabetic mice with a ß2 adrenergic agonist improved insulin sensitivity, indicating that ß2 subsensitivity can be overcome pharmacologically. Recent studies show that over the long term, epinephrine prevents hypertension during stress and improves glucose tolerance. The hyperglycemic influence of epinephrine is short-lived. Chronic administration of epinephrine and other ß2 agonists improves cellular glucose uptake and metabolism. Overall, epinephrine counteracts the metabolic syndrome.

The effect of ethyl acetate extract of pomelo mix on systemic exposure of verapamil in rabbits.

The effect of ethyl acetate extract of pomelo fruit on systemic exposure of verapamil was explored in rabbits. Two groups each of 8 locally-inbred New Zealand male rabbits were used. The first group was used for single-dose treatment (both verapamil and pomelo extract). The second group was used for multiple-dose treatment, pomelo extract (once daily for 14 days) and verapamil single doses (at days 7 and 14). A verapamil dose of 30 mg/kg and a pomelo extract dose of 45 mg/kg were used. Single-dose treatment with pomelo extract resulted in a minor change in mean C(max) of verapamil in plasma, while a decrease of 37.8% in AUC(0-24) and 28.3% in AUC(0-∞) was observed but did not reach statistical significance. After the first period of multiple dose treatment (pomelo extract for 7 days), the combination increased the concentration of verapamil in plasma with a significant increase in mean C(max), AUC(0-24) and AUC(0-∞) by 461.9%, 299.7%, and 261.1%, respectively (p values were 0.005, 0.002, and 0.006, respectively). In contrast, after the second period (day 14 of pomelo extract use), the combination decreased the concentration of verapamil in the plasma with a substantial decrease in mean C(max), AUC(0-24), and AUC(0-∞), by 68.2%, 69.7% and 58.3%, respectively. This decrease did not reach statistical significance (p values were 0.073, 0.081 and 0.083, respectively). The T(max) was not affected significantly in both studies. The study illustrates a complex time-dependent interaction between verapamil and the ethyl acetate extract of pomelo mix. More intensive studies are needed to further understand the nature of the interaction.