Hyperglycemia Acutely Increases Cytosolic Reactive Oxygen Species via O-linked GlcNAcylation and CaMKII Activation in Mouse Ventricular Myocytes.

RATIONALE: Diabetes mellitus is a complex, multisystem disease, affecting large populations worldwide. Chronic CaMKII (Ca2+/calmodulin-dependent kinase II) activation may occur in diabetes mellitus and be arrhythmogenic. Diabetic hyperglycemia was shown to activate CaMKII by (1) O-linked attachment of N-acetylglucosamine (O-GlcNAc) at S280 leading to arrhythmia and (2) a reactive oxygen species (ROS)-mediated oxidation of CaMKII that can increase postinfarction mortality. OBJECTIVE: To test whether high extracellular glucose (Hi-Glu) promotes ventricular myocyte ROS generation and the role played by CaMKII. METHODS AND RESULTS: We tested how extracellular Hi-Glu influences ROS production in adult ventricular myocytes, using DCF (2',7'-dichlorodihydrofluorescein diacetate) and genetically targeted Grx-roGFP2 redox sensors. Hi-Glu (30 mmol/L) significantly increased the rate of ROS generation-an effect prevented in myocytes pretreated with CaMKII inhibitor KN-93 or from either global or cardiac-specific CaMKIIδ KO (knockout) mice. CaMKII KO or inhibition also prevented Hi-Glu-induced sarcoplasmic reticulum Ca2+ release events (Ca2+ sparks). Thus, CaMKII activation is required for Hi-Glu-induced ROS generation and sarcoplasmic reticulum Ca2+ leak in cardiomyocytes. To test the involvement of O-GlcNAc-CaMKII pathway, we inhibited GlcNAcylation removal by Thiamet G (ThmG), which mimicked the Hi-Glu-induced ROS production. Conversely, inhibition of GlcNAcylation (OSMI-1 [(αR)-α-[[(1,2-dihydro-2-oxo-6-quinolinyl)sulfonyl]amino]-N-(2-furanylmethyl)-2-methoxy-N-(2-thienylmethyl)-benzeneacetamide]) prevented ROS induction in response to either Hi-Glu or ThmG. Moreover, in a CRSPR-based knock-in mouse in which the functional GlcNAcylation site on CaMKIIδ was ablated (S280A), neither Hi-Glu nor ThmG induced myocyte ROS generation. So CaMKIIδ-S280 is required for the Hi-Glu-induced (and GlcNAc dependent) ROS production. To identify the ROS source(s), we used different inhibitors of NOX (NADPH oxidase) 2 (Gp91ds-tat peptide), NOX4 (GKT137831), mitochondrial ROS (MitoTempo), and NOS (NO synthase) pathway inhibitors (L-NAME, L-NIO, and L-NPA). Only NOX2 inhibition or KO prevented Hi-Glu/ThmG-induced ROS generation. CONCLUSIONS: Diabetic hyperglycemia induces acute cardiac myocyte ROS production by NOX2 that requires O-GlcNAcylation of CaMKIIδ at S280. This novel ROS induction may exacerbate pathological consequences of diabetic hyperglycemia.

Repetitive spikes of glucose and lipid induce senescence-like phenotypes of bone marrow stem cells through H3K27me3 demethylase-mediated epigenetic regulation.

Bone marrow-derived endothelial progenitor cells (EPCs) contribute to endothelial repair and angiogenesis. Reduced number of circulating EPCs is associated with future cardiovascular events. We tested whether dysregulated glucose and/or triglyceride (TG) metabolism has an impact on EPC homeostasis. The analysis of metabolic factors associated with circulating EPC number in humans revealed that postprandial hyperglycemia is negatively correlated with circulating EPC number, and this correlation appears to be further enhanced in the presence of postprandial hypertriglyceridemia (hTG). We therefore examined the effect of glucose/TG spikes on bone marrow lineage-sca-1+ c-kit+ (LSK) cells in mice, because primitive EPCs reside in bone marrow LSK fraction. Repetitive glucose + lipid (GL) spikes, but not glucose (G) or lipid (L) spikes alone, induced senescence-like phenotypes of LSK cells, and this phenomenon was reversible after cessation of GL spikes. G spikes and GL spikes differentially affected transcriptional program of LSK cell metabolism and differentiation. GL spikes upregulated a histone H3K27 demethylase JMJD3, and inhibition of JMJD3 eliminated GL spikes-induced LSK cell senescence-like phenotypes. These observations suggest that postprandial glucose/TG dysmetabolism modulate transcriptional regulation in LSK cells through H3K27 demethylase-mediated epigenetic regulation, leading to senescence-like phenotypes of LSK cells, reduced number of circulating EPCs, and development of atherosclerotic cardiovascular disease.NEW & NOTEWORTHY Combination of hyperglycemia and hypertriglyceridemia is associated with increased risk of atherosclerotic cardiovascular disease. We found that 1) hypertriglyceridemia may enhance the negative impact of hyperglycemia on circulating EPC number in humans and 2) metabolic stress induced by glucose + triglyceride spikes in mice results in senescence-like phenotypes of bone marrow stem/progenitor cells via H3K27me3 demethylase-mediated epigenetic regulation. These findings have important implications for understanding the pathogenesis of atherosclerotic cardiovascular disease in patients with T2DM.

Ameliorative effect of phosphodiesterase 4 and 5 inhibitors in deoxycorticosterone acetate-salt hypertensive uni-nephrectomized KKAy mice.

Diabetic nephropathy (DN) is a leading cause of end-stage renal disease (ESRD). Hypertension increases kidney stress, which deteriorates function, and leads to peripheral renal vascular resistance. Long-term hypoperfusion promotes interstitial fibrosis and glomerular sclerosis, resulting in nephrosclerosis. Although hypertension and DN are frequent ESRD complications, relevant animal models remain unavailable. We generated a deoxycorticosterone acetate (DOCA)-salt hypertensive uni-nephrectomized (UNx) KKAy mouse model demonstrating hypertension, hyperglycemia, cardiac hypertrophy, kidney failure, increased urinary albumin creatinine ratio (UACR), and increased renal PDE4D and cardiac PDE5A mRNA levels. We hypothesized that the novel PDE4 selective inhibitor, compound A, and PDE5 inhibitor, sildenafil, exhibit nephroprotective, and cardioprotective effects in this new model. Compound A, sildenafil, and the angiotensin II receptor blocker, irbesartan, significantly reduced ventricular hypertrophy and pleural effusion volume. Meanwhile, compound A and sildenafil significantly suppressed the UACR, urinary kidney injury molecule-1, and monocyte chemoattractant protein-1 levels, as well as that of renal pro-fibrotic marker mRNAs, including collagen 1A1, fibronectin, and transforming growth factor-beta (TGF-ß). Moreover, compound A significantly suppressed TGF-ß-induced pro-fibrotic mRNA expression in vitro in all major kidney lesions, including within the glomerular mesangial region, podocytes, and epithelial region. Hence, PDE4 and PDE5 inhibitors may be promising treatments, in combination with irbesartan, for DN with hypertension as they demonstrate complementary mechanisms.

Opuntia dillenii (Ker Gawl.) Haw., Seeds Oil Antidiabetic Potential Using In Vivo, In Vitro, In Situ, and Ex Vivo Approaches to Reveal Its Underlying Mechanism of Action.

Opuntia dillenii Ker Gawl. is one of the medicinal plants used for the prevention and treatment of diabetes mellitus (DM) in Morocco. This study aims to investigate the antihyperglycemic effect of Opuntia dillenii seed oil (ODSO), its mechanism of action, and any hypoglycemic risk and toxic effects. The antihyperglycemic effect was assessed using the OGTT test in normal and streptozotocin (STZ)-diabetic rats. The mechanisms of action were explored by studying the effect of ODSO on the intestinal absorption of d-glucose using the intestinal in situ single-pass perfusion technique. An Ussing chamber was used to explore the effects of ODSO on intestinal sodium-glucose cotransporter 1 (SGLT1). Additionally, ODSO's effect on carbohydrate degrading enzymes, pancreatic α-amylase, and intestinal α-glucosidase was evaluated in vitro and in vivo using STZ-diabetic rats. The acute toxicity test on mice was performed, along with a single-dose hypoglycemic effect test. The results showed that ODSO significantly attenuated the postprandial hyperglycemia in normal and STZ-diabetic rats. Indeed, ODSO significantly decreased the intestinal d-glucose absorption in situ. The ex vivo test (Ussing chamber) showed that the ODSO significantly blocks the SGLT1 (IC50 = 60.24 µg/mL). Moreover, ODSO indu\ced a significant inhibition of intestinal α-glucosidase (IC50 = 278 ± 0.01 µg/mL) and pancreatic α-amylase (IC50 = 0.81 ± 0.09 mg/mL) in vitro. A significant decrease of postprandial hyperglycemia was observed in sucrose/starch-loaded normal and STZ-diabetic ODSO-treated rats. On the other hand, ODSO had no risk of hypoglycemia on the basal glucose levels in normal rats. Therefore, no toxic effect was observed in ODSO-treated mice up to 7 mL/kg. The results of this study suggest that ODSO could be suitable as an antidiabetic functional food.

Selective extraction of galactomannan from fenugreek (Trigonella foenum-graecum L.) seed husk and its enzyme inhibitory potential associated with hyperglycaemia.

BACKGROUND: Fenugreek seeds host various bioactive compounds, and galactomannan (GM) is a significant soluble fibre. In this study, selective extraction is adapted to extract fenugreek seed GM to improvise the yield recovery. The seeds are fractionated, separated and classified as husk and cotyledons. Comparative studies have been performed to evaluate the crude and pure GM yield between different groups such as the whole seed, and the classified fractions. Characterization is done using Fourier transform infrared, differential scanning calorimetry, scanning electron microscopy, monosaccharide composition and optical density, and the structure is elucidated through nuclear magnetic resonance. The GM obtained through extraction is used to study its enzyme inhibitory property associated with hyperglycaemia. RESULTS: GM yield extracted from the husk is highly significant compared to other groups. Crude GM and pure GM yield was 2 and 3.25 times higher than that obtained through whole seed samples. The characterization of the pure GM is on a par with the existing reports. The purified GM inhibited α-amylase and α-glucosidase enzymes in vitro, with an IC50 of 21.08 ± 0.085 and 67.17 ± 5.15 µg mL-1 , respectively. CONCLUSION: Selective extraction prompts enhancement in the recovery of the bioactive compound, minimal use of resources, and promotes industrial viability. Characterization of the compound confirms the structure. Its enzyme inhibitory property makes GM a valuable compound in diabetic prevention/treatment. © 2021 Society of Chemical Industry.

Proinflammatory cytokines trigger biochemical and neurochemical changes in mouse retinal explants exposed to hyperglycemic conditions.

Purpose: Diabetic retinopathy (DR) is one of the most frequent complications of diabetes affecting the retina and eventually causing vision impairment. Emerging evidence suggests that inflammation plays a vital role in DR progression. In this study, we evaluated the early biochemical and neurochemical changes in mouse retinal explants to understand the contribution of proinflammatory cytokines to disease progression. Methods: DR was modeled in vitro by incubating mouse retinal explants in a physiological buffer supplemented with high glucose and the proinflammatory cytokines TNF-α and IL-1ß. Key metabolites of retinal energy metabolism, including glucose, lactate, ATP, glutamate, glutamine, and enzymes supporting retinal ATP levels were assessed 40 min after the application of high glucose and proinflammatory cytokines. As retinal energy metabolism is tightly coupled to retinal neurochemistry, we also determined the short-term effect on the amino acid distribution of glutamate, gamma aminobutyric acid (GABA), glutamine, and glycine. Results: The results indicated that the combined application of high glucose and proinflammatory cytokines increased retinal glucose, lactate, and ATP levels, and decreased retinal glutamate, without affecting glutamine levels or the enzymes supporting ATP levels. Moreover, we observed a statistically significant increase in ATP and glutamate release. Correspondingly, statistically significant alterations in amino acid distribution were observed in retinal explants coexposed to high glucose and proinflammatory cytokines. Conclusions: These data suggest that short-term exposure to proinflammatory cytokines contributes to the early biochemical and neurochemical changes caused by hyperglycemia, by affecting retinal energy metabolism and amino acid distribution. These data are consistent with the idea that early intervention to prevent inflammation-triggered loss of metabolic homeostasis in patients with diabetes is necessary to prevent DR progression.

Deletion of pancreatic ß-cell adenosine kinase improves glucose homeostasis in young mice and ameliorates streptozotocin-induced hyperglycaemia.

Severe reduction in the ß-cell number (collectively known as the ß-cell mass) contributes to the development of both type 1 and type 2 diabetes. Recent pharmacological studies have suggested that increased pancreatic ß-cell proliferation could be due to specific inhibition of adenosine kinase (ADK). However, genetic evidence for the function of pancreatic ß-cell ADK under physiological conditions or in a pathological context is still lacking. In this study, we crossed mice carrying LoxP-flanked Adk gene with Ins2-Cre mice to acquire pancreatic ß -cell ADK deficiency (Ins2-Cre± Adkfl/fl ) mice. Our results revealed that Ins2-Cre+/- Adkfl/fl mice showed improved glucose metabolism and ß-cell mass in younger mice, but showed normal activity in adult mice. Moreover, Ins2-Cre± Adkfl/fl mice were more resistant to streptozotocin (STZ) induced hyperglycaemia and pancreatic ß-cell damage in adult mice. In conclusion, we found that ADK negatively regulates ß-cell replication in young mice as well as under pathological conditions, such as STZ induced pancreatic ß-cell damage. Our study provided genetic evidence that specific inhibition of pancreatic ß-cell ADK has potential for anti-diabetic therapy.

Enhanced phosphorylation of AMPK by lutein and oxidised lutein that lead to mitochondrial biogenesis in hyperglycemic HepG2 cells.

The stimulation of adenosine monophosphate-activated protein kinase (AMPK) is a prime target to decrease the hyperglycemic condition, hence it is a lutein (L) and oxidised lutein (OXL) is a target molecule for the treatment of type II diabetes. In the current study, a plausible interaction of L and OXL with AMPK was investigated by molecular docking. In addition, the effect of L and OXL for the activation of AMPK that triggers the downstream regulator peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), TFAM expression, mitochondrial DNA (mtDNA), mitochondrial biogenesis and superoxide dismutase 2 (SOD2) in high glucose treated HepG2 cells were investigated by quantitative polymerase chain reaction and Western blot analysis. Molecular docking reveals higher binding affinity of L (ΔG = -6.3 kcal/mol) and OXL (ΔG = -15.5 kcal/mol) with AMPK, compared with metformin (ΔG = -5.0 kcal/mol). The phosphorylation of AMPK increased by 1.3- and 1.5-fold with L and OXL treatment, respectively, in high glucose induced HepG2 cells. The activation of PGC-1α is significant (P < 0.05) in OXL group than L. Similarly, TFAM expression is increased with L and OXL compared with the high glucose group. Further increase in SOD2 and mtDNA, confirms the efficacy of L and OXL in restoring the mitochondrial biogenesis in high glucose induced cells through AMPK, PGC-1α, and TFAM.

Comparison of diabetic nephropathy between male and female eNOS-/-db/db mice.

Sex is an important biological variable that impacts diverse physiological and pathological processes, including the progression of diabetic nephropathy. Diabetic nephropathy is one of the most common complications of diabetes mellitus and is the leading cause of end-stage renal disease. The endothelial nitric oxide synthase-deficient (eNOS-/-) db/db mouse is an appropriate and valuable model to study mechanisms in the development of diabetic nephropathy because of the similarities of the features of diabetic kidney disease in this model to those in humans. The aim of the present study was to determine whether there was a sex difference in renal injury in eNOS-/-db/db mice. Both male and female eNOS-/-db/db mice showed hyperglycemia, obesity, and renal hypertrophy. However, there was no significant difference in those variables between male and female mice. Furthermore, both male and female diabetic mice showed progressive albuminuria and significantly greater levels of serum creatinine and blood urea nitrogen compared with the same sex of wild-type mice (nondiabetic controls). Although all three variables in female eNOS-/-db/db mice had a tendency to be greater than those in male eNOS-/-db/db mice, those sex differences were not statistically significant. Moreover, both male and female eNOS-/-db/db mice showed significant mesangial expansion, higher glomerular injury scores, profound renal fibrosis, and substantial accumulation of fibronectin and collagen type IV proteins. However, sex differences in those structural changes were not observed. Similarly, survival rates of male and female eNOS-/-db/db mice were comparable. Taken together, the results from the present study suggest no sex difference in renal structural and functional damage in eNOS-/-db/db mice.

Sequential testicular atrophy involves changes in cellular proliferation and apoptosis associated with variations in aromatase P450 expression levels in Irs-2-deficient mice.

Insulin receptor substrate 2 (Irs-2) is an intracellular protein susceptible to phosphorylation after activation of the insulin receptor. Its suppression affects testis development and its absence induces peripheral resistance to insulin. The aim of this study was to identify changes induced by the deletion of Irs-2 in the testicular structure and by the altered expression of cytochrome P450 aromatase, a protein necessary for the development and maturation of germ cells. Adult knockout (KO) mice (Irs-2-/- , 6 and 12 weeks old) and age-matched wild-type (WT) mice were used in this study. Immunohistochemistry and Western blot analyses were performed to study proliferation (PCNA), apoptosis (active caspase-3) and P450 aromatase expression in testicular histological sections. Deletion of Irs-2 decreased the number of epithelial cells in the seminiferous tubule and rete testis. Aberrant cells were frequently detected in the epithelia of Irs-2-/- mice, accompanied by variations in spermatogonia, which were shown to exhibit small hyperchromatic nuclei as well as polynuclear and anuclear structures. The amount of cell proliferation was significantly lower in Irs-2-/- mice than in WT mice, whereas apoptotic processes were more common in Irs-2-/- mice. Aromatase P450 reactivity was higher in 6-week-old KO mice than in WT mice of the same age and was even higher at 12 weeks. Our results suggest that Irs-2 is a key element in spermatogenesis because silencing Irs-2 induces the sequential development of testicular atrophy. The effects are observed mainly in germ cells present in the seminiferous tubule, which may be due to changes in cytochrome P450 aromatase expression.

Diabetic hyperglycaemia activates CaMKII and arrhythmias by O-linked glycosylation.

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is an enzyme with important regulatory functions in the heart and brain, and its chronic activation can be pathological. CaMKII activation is seen in heart failure, and can directly induce pathological changes in ion channels, Ca(2+) handling and gene transcription. Here, in human, rat and mouse, we identify a novel mechanism linking CaMKII and hyperglycaemic signalling in diabetes mellitus, which is a key risk factor for heart and neurodegenerative diseases. Acute hyperglycaemia causes covalent modification of CaMKII by O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAc modification of CaMKII at Ser 279 activates CaMKII autonomously, creating molecular memory even after Ca(2+) concentration declines. O-GlcNAc-modified CaMKII is increased in the heart and brain of diabetic humans and rats. In cardiomyocytes, increased glucose concentration significantly enhances CaMKII-dependent activation of spontaneous sarcoplasmic reticulum Ca(2+) release events that can contribute to cardiac mechanical dysfunction and arrhythmias. These effects were prevented by pharmacological inhibition of O-GlcNAc signalling or genetic ablation of CaMKIIδ. In intact perfused hearts, arrhythmias were aggravated by increased glucose concentration through O-GlcNAc- and CaMKII-dependent pathways. In diabetic animals, acute blockade of O-GlcNAc inhibited arrhythmogenesis. Thus, O-GlcNAc modification of CaMKII is a novel signalling event in pathways that may contribute critically to cardiac and neuronal pathophysiology in diabetes and other diseases.

Antidiabetic effects of glucokinase regulatory protein small-molecule disruptors.

Glucose homeostasis is a vital and complex process, and its disruption can cause hyperglycaemia and type II diabetes mellitus. Glucokinase (GK), a key enzyme that regulates glucose homeostasis, converts glucose to glucose-6-phosphate in pancreatic ß-cells, liver hepatocytes, specific hypothalamic neurons, and gut enterocytes. In hepatocytes, GK regulates glucose uptake and glycogen synthesis, suppresses glucose production, and is subject to the endogenous inhibitor GK regulatory protein (GKRP). During fasting, GKRP binds, inactivates and sequesters GK in the nucleus, which removes GK from the gluconeogenic process and prevents a futile cycle of glucose phosphorylation. Compounds that directly hyperactivate GK (GK activators) lower blood glucose levels and are being evaluated clinically as potential therapeutics for the treatment of type II diabetes mellitus. However, initial reports indicate that an increased risk of hypoglycaemia is associated with some GK activators. To mitigate the risk of hypoglycaemia, we sought to increase GK activity by blocking GKRP. Here we describe the identification of two potent small-molecule GK-GKRP disruptors (AMG-1694 and AMG-3969) that normalized blood glucose levels in several rodent models of diabetes. These compounds potently reversed the inhibitory effect of GKRP on GK activity and promoted GK translocation both in vitro (isolated hepatocytes) and in vivo (liver). A co-crystal structure of full-length human GKRP in complex with AMG-1694 revealed a previously unknown binding pocket in GKRP distinct from that of the phosphofructose-binding site. Furthermore, with AMG-1694 and AMG-3969 (but not GK activators), blood glucose lowering was restricted to diabetic and not normoglycaemic animals. These findings exploit a new cellular mechanism for lowering blood glucose levels with reduced potential for hypoglycaemic risk in patients with type II diabetes mellitus.

Protein hydrolysates and ultrafiltered < 1 KDa fractions from Phaseolus lunatus, Phaseolus vulgaris and Mucuna pruriens exhibit antihyperglycemic activity, intestinal glucose absorption and α-glucosidase inhibition with no acute toxicity in rodents.

BACKGROUND: Protein hydrolysates from food plants, such as legumes, have emerged as a new alternative to treat hyperglycemia, an important risk factor contributing to the development of type 2 diabetes mellitus (T2DM) and its complications. The aim of this work was to assess the antihyperglycemic activity and inhibition of α-glucosidase, and intestinal glucose absorption, and acute toxicity of total hydrolysates and < 1 kDa fractions from Phaseolus lunatus L., Phaseolus vulgaris L., and Mucuna pruriens (L.) DC., obtained by hydrolysis with Alcalase®-Flavourzyme® or pepsine-pancreatin enzymatic systems. RESULTS: In vivo results showed that three of six total hydrolysates and four of six < 1 kDa fractions suppressed starch-induced postprandial hyperglycemia (ED50 range between 1.4 and 93 mg kg-1 ). In vitro, total hydrolysates and fractions, particularly from M. pruriens, inhibited carbohydrate intestinal absorption (from 19.2 to 40%), and α-glucosidase activity (IC50 from 0.86 to 75 mg mL-1 ). Finally, none of the hydrolysates and fractions tested did not show any signs of toxicity (LD50 > 5000 mg kg-1 ). CONCLUSION: These results suggest that hydrolysates and < 1 kDa fractions from P. lunatus, P. vulgaris and M. pruriens are suitable candidates to treat or prevent T2DM. © 2018 Society of Chemical Industry.

Podocyte-specific knockin of PTEN protects kidney from hyperglycemia.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has proven to be downregulated in podocytes challenged with high glucose (HG), and knockout of PTEN in podocytes aggravated the progression of diabetic kidney disease (DKD). However, whether podocyte-specific knockin of PTEN protects the kidney against hyperglycemia in vivo remains unknown. The inducible podocyte-specific PTEN knockin (PPKI) mice were generated by crossing newly created transgenic loxP-stop- loxP-PTEN mice with podocin-iCreERT2 mice. Diabetes mellitus was induced in mice by intraperitoneal injection of streptozotocin at a dose of 150 mg/kg. In vitro, small interfering RNA and adenovirus interference were used to observe the role of PTEN in HG-treated podocytes. Our data demonstrated that PTEN was markedly reduced in the podocytes of patients with DKD and focal segmental glomerulosclerosis, as well as in those of db/db mice. Interestingly, podocyte-specific knockin of PTEN significantly alleviated albuminuria, mesangial matrix expansion, effacement of podocyte foot processes, and incrassation of glomerular basement membrane in diabetic PPKI mice compared with wild-type diabetic mice, whereas no alteration was observed in the level of blood glucose. The potential renal protection of overexpressed PTEN in podocytes was partly attributed with an improvement in autophagy and motility and the inhibition of apoptosis. Our results showed that podocyte-specific knockin of PTEN protected the kidney against hyperglycemia in vivo , suggesting that targeting PTEN might be a novel and promising therapeutic strategy against DKD.

Demonstration of subcellular migration of CK2α localization from nucleus to sarco(endo)plasmic reticulum in mammalian cardiomyocytes under hyperglycemia.

The cellular control of glucose uptake and glycogen metabolism in mammalian tissues is in part mediated through the regulation of protein-serine/threonine kinases including CK2. Although it participates to several cellular signaling processes, however, its subcellular localization is not well-defined while some documents mentioned its localization change under pathological conditions. The activation/phosphorylation of some proteins including Zn2+-transporter ZIP7 in cardiomyocytes is controlled with CK2α, thereby, inducing changes in the level of intracellular free Zn2+ ([Zn2+] i ). In this regard, we aimed to examine cellular localization of CK2α in cardiomyocytes and its possible subcellular migration under hyperglycemia. Our confocal imaging together with biochemical analysis in isolated sarco(endo)plasmic reticulum [S(E)R] and nuclear fractions from hearts have shown that CK2α localized highly to S(E)R and Golgi and weakly to nuclear fractions in physiological condition. However, it can migrate from nuclear fractions to S(E)R under hyperglycemia. This migration can further underlie phosphorylation of a target protein ZIP7 as well as some endogenous kinases and phosphatases including PKA, CaMKII, and PP2A. We also have shown that CK2α activation is responsible for hyperglycemia-associated [Zn2+] i increase in diabetic heart. Therefore, our present data demonstrated, for the first time, the physiological relevance of CK2α in cellular control of Zn2+-distribution via inducing ZIP7 phosphorylation and activation of these above endogenous actors in hyperglycemia/diabetes-associated cardiac dysfunction. Moreover, our present data also emphasized the multi-subcellular compartmental localizations of CK2α and a tightly regulation of these localizations in cardiomyocytes. Therefore, taken into consideration of all data, one can emphasize the important role of the subcellular localization of CK2α as a novel target-pathway for understanding of diabetic cardiomyopathy.

Insulin promotes progression of colon cancer by upregulation of ACAT1.

BACKGROUND: Insulin resistant and the progression of cancer is closely related. The aim of this study was to  investigate the effect of insulin on the proliferation and migration of colon cancer cells and its underlying mechanism. METHODS: Colon carcinoma tissues from the 80 cases of colon cancer patients were collected. Immunohistochemistry was used to detect the expression of acyl coenzyme A: cholesterol acyltransferase1 (ACAT1), and we analyzed the correlation between hyperglycemia and ACAT1, hyperglycemia and metastasis. CCK8 assay and transwell assay were used to investigate the effect of different concentrations of insulin and ACAT1siRNA on human colon cancer cell line HT-29. ACAT1 mRNA expression and protein level in HT-29 cells were determined by real-time quantitative PCR and western blotting, respectively. RESULTS: Biopsies from patients with colon carcinoma showed hyperglycemia links ACAT1, lymph nodes metastasis and distant metastasis. Insulin markedly promoted cell proliferation and migration in human colon cancer HT-29 cells. Moreover, ACAT1mRNA expression and protein level were increased by insulin. ACAT1siRNA resulted in a complete inhibition of the ACAT1 mRNA expression. Consequently insulin-triggered cell proliferation and migration on colon cancer cells were inhibited. CONCLUSION: The progression of colon cancer has a positive correlation with hyperinsulinemia. Insulin-triggered cell proliferation and metastatic effects on colorectal cancer cells are mediated by ACAT1. Therefore, insulin could promote colon cancer progression by upregulation of ACAT1, which maybe is a potential therapeutic target for colon cancer.

Soluble epoxide hydrolase in podocytes is a significant contributor to renal function under hyperglycemia.

BACKGROUND: Diabetic nephropathy (DN) is the leading cause of renal failure, and podocyte dysfunction contributes to the pathogenesis of DN. Soluble epoxide hydrolase (sEH, encoded by Ephx2) is a conserved cytosolic enzyme whose inhibition has beneficial effects on renal function. The aim of this study is to investigate the contribution of sEH in podocytes to hyperglycemia-induced renal injury. MATERIALS AND METHODS: Mice with podocyte-specific sEH disruption (pod-sEHKO) were generated, and alterations in kidney function were determined under normoglycemia, and high-fat diet (HFD)- and streptozotocin (STZ)-induced hyperglycemia. RESULTS: sEH protein expression increased in murine kidneys under HFD- and STZ-induced hyperglycemia. sEH deficiency in podocytes preserved renal function and glucose control and mitigated hyperglycemia-induced renal injury. Also, podocyte sEH deficiency was associated with attenuated hyperglycemia-induced renal endoplasmic reticulum (ER) stress, inflammation and fibrosis, and enhanced autophagy. Moreover, these effects were recapitulated in immortalized murine podocytes treated with a selective sEH pharmacological inhibitor. Furthermore, pharmacological-induced elevation of ER stress or attenuation of autophagy in immortalized podocytes mitigated the protective effects of sEH inhibition. CONCLUSIONS: These findings establish sEH in podocytes as a significant contributor to renal function under hyperglycemia. GENERAL SIGNIFICANCE: These data suggest that sEH is a potential therapeutic target for podocytopathies.

Synthesis, biological evaluation and in silico studies of 5-(3-methoxybenzylidene)thiazolidine-2,4-dione analogues as PTP1B inhibitors.

PTP1B (protein tyrosine phosphatase 1B) dephosphorylates the insulin receptor substrate and thus acts as a negative regulator of the insulin and leptin signalling pathway. Recently, it has been considered as a new therapeutic target of intervention for the treatment of type2 diabetes. A series of aryl/alkylsulfonyloxy-5-(3-methoxybenzylidene)thiazolidine-2,4-dione derivatives were synthesized, screened in vitro for their PTP1B inhibitory activity and in vivo for anti-hyperglycaemic activity. Docking results further helped in understanding the nature of interactions governing the binding mode of ligands inside the active site of PTP1B. Among the synthesized compounds, 13 and 16 were found to be potent PTP1B inhibitors having IC50 of 7.31 and 8.73µM respectively. Significant lowering of blood glucose level was observed in some of the synthesized compounds in in vivo study.

Effect of solvents extraction on phytochemical components and biological activities of Tunisian date seeds (var. Korkobbi and Arechti).

BACKGROUND: The interest in natural antioxidants, especially polyphenols, is growing more and more thanks to their positive contribution to human health. Thus, the prevention from the harmful action of oxidative stress which has been involved in many diseases such as cancer, inflammation diabetes, and cardiovascular illness. Recent research proved the bioactive compounds richness of date seeds which could be a good biological matrix of natural antioxidants. Unfortunately, an important quantity of Tunisian dates seed is discarded yearly. METHODS: In this study, different solvents extraction (water, methanol, absolute acetone and aqueous acetone 80%) were used and the evaluation of its effect on phytochemical level, in vitro antioxidant activities, in vitro hyperglycemia key enzymes inhibition and in vivo anti-inflammatory proprieties were established for Tunisian date seeds. RESULTS: The result revealed that the polar solvent exhibited the highest amount of bioactive compounds. The correlation between polyphenol compounds and the antioxidant potentiality explains the powerful effect of used polar solvents on inflammation, TBARS and hyperglycemia inhibition. Furthermore, it showed its higher capacity to scavenge radicals. CONCLUSIONS: Therefore, this big waste of Tunisian seeds could be used as cheap source of natural antioxidant compounds which are considered as a health challenge for the poor countries.

Inhibitory Effects of Siegesbeckia orientalis Extracts on Advanced Glycation End Product Formation and Key Enzymes Related to Metabolic Syndrome.

Metabolic syndrome typically includes Type 2 diabetes associated with hyperglycemia, central obesity, dyslipidemia and hypertension. It is highly related to oxidative stress, formation of advanced glycated end products (AGEs) and key enzymes, such as carbohydrate digesting enzymes like pancreatic α-amylase and intestinal α-glucosidase, pancreatic lipase and angiotensin I-converting enzyme (ACE). This study used an in vitro approach to assess the potential of four extracts of Siegesbeckia orientalis linne on key enzymes relevant to metabolic syndrome. In this research, S. orientailis was firstly extracted by ethanol. The ethanol extract (SE) was then partitioned sequentially with hexane, ethyl acetate and methanol, and these extracts were named SE-Hex, SE-EA and SE-MeOH, respectively. The experimental results showed that SE-EA had the highest total phenolic content (TPC, 76.9 ± 1.8 mg/g) and the total flavonoids content (TFC, 5.3 ± 0.3 mg/g). This extract exhibited the most significant antioxidant activities, including DPPH radical-scavenging capacity (IC50 = 161.8 ± 2.4 µg/mL), ABTS radical-scavenging capacity (IC50 = 13.9 ± 1.5 µg/mL) and reducing power. For anti-glycation activities, SE-EA showed the best results in the inhibition of AGEs, as well as inhibitory activities against α-glucosidase (IC50 = 362.3 ± 9.2 µg/mL) and α-amylase (IC50 = 119.0 ± 17.7 µg/mL). For anti-obesity activities, SE-EA indicated the highest suppression effect on pancreatic lipase (IC50 = 3.67 ± 0.52 mg/mL). Finally, for anti-hypertension activity, SE-EA also demonstrated the strongest inhibitory activity on ACE (IC50 = 626.6 ± 15.0 µg/mL). Close relationships were observed among the parameters of TPC, antioxidant activities, inhibitory activities on α-amylase, α-glucosidase, lipase and ACE (R > 0.9). Moderate correlations were found among the parameters of TFC, antioxidant activities, and suppression of dicarbonyl compounds formation (R = 0.5-0.9). Taken together these in vitro studies reveal the therapeutic potential of SE-EA extract in the prevention and treatment of metabolic disorders.