Rizwana Sanaullah Waraich

Phosphorylation of Ser357 of Rat Insulin Receptor Substrate-1 Mediates Adverse Effects of Protein Kinase C-δ on Insulin Action in Skeletal Muscle Cells

The activation of the protein kinase C (PKC) family of serine/threonine kinases contributes to the modulation of insulin signaling, and the PKC-dependent phosphorylation of insulin receptor substrate (IRS)-1 has been implicated in the development of insulin resistance. Here we demonstrate Ser357 of rat IRS-1 as a novel PKC-δ-dependent phosphorylation site in skeletal muscle cells upon stimulation with insulin and phorbol ester using Ser(P)357 antibodies and active and kinase dead mutants of PKC-δ. Phosphorylation of this site was simulated using IRS-1 Glu357 and shown to reduce insulin-induced tyrosine phosphorylation of IRS-1, to decrease activation of Akt, and to subsequently diminish phosphorylation of glycogen synthase kinase-3. When the phosphorylation was prevented by mutation of Ser357 to alanine, these effects of insulin were enhanced. When the adjacent Ser358, present in mouse and rat IRS-1, was mutated to alanine, which is homologous to the human sequence, the insulin-induced phosphorylation of glycogen synthase kinase-3 or tyrosine phosphorylation of IRS-1 was not increased. Moreover, both active PKC-δ and phosphorylation of Ser357 were shown to be necessary for the attenuation of insulin-stimulated Akt phosphorylation. The phosphorylation of Ser357 could lead to increased association of PKC-δ to IRS-1 upon insulin stimulation, which was demonstrated with IRS-1 Glu357. Together, these data suggest that phosphorylation of Ser357 mediates at least in part the adverse effects of PKC-δ activation on insulin action.

Development and precise characterization of phospho-site-specific antibody of Ser357 of IRS-1: Elimination of cross reactivity with adjacent Ser358

Antibodies that recognize specifically phosphorylated sites on proteins are widely utilized for studying the regulation and biological function of phosphoproteins. The proposed strategy is a powerful, analytical tool allowing the generation of phospho-site specific antibodies albeit adjacent phosphorylation sites are present. Here, we demonstrate the assessment and elimination of cross reactivity of phospho-site-specific-Ser(357) IRS-1 antibody. While determining the specificity of p-Ser(357) antiserum we came across the cross reactivity of the antiserum with adjacent Ser(358) which was successfully abolished by an improved immuno-purification method. The specificity of the purified antiserum was then verified by indirect ELISA, results of ELISA were also mirrored in the experiments carried out in BHK-IR cells using different mutants of IRS-1 carrying mutations at either Ser(357)/Ser(358)/Ser(357/358). Immuno-purified-p-Ser(357) did not react with IRS-1 Ala(357) and IRS-1 Ala(357/358). In conclusion, the present study describes generation and characterization of p-Ser(357) IRS-1 antibody, which reacts with IRS-1 in site specific and phosphorylation state-dependent manner without showing cross reactivity to adjacent Ser(358). This antibody can be effectively used to further clarify the inhibitory role of Ser(357) in insulin signal transduction.

A new glycotoxins inhibitor attenuates insulin resistance in liver and fat cells.

Glycotoxins/Advanced glycation end products (AGEs) have implications in development of diabetes and related diseases. In the present study we deciphered the mechanisms of action of URM-II-81, a new derivative of isatin, in alleviation of insulin resistance in human hepatocytes and murine adipocytes. URM-II-81 reduced AGEs formation and receptor for advanced glycation end products (RAGE) expression in both cell types. We also observed suppression of methylglyoxal (MGO) mediated ROS production and deactivation of PKC-α. URM-II-81 restored proximal insulin signaling by modulating IRS-1 phosphorylation. URM-II-81 also alleviated MGO mediated diminished distal insulin signaling by increasing protein kinase B (PKB) and glycogen synthase kinase 3-beta (GSK-3-beta) phosphorylation. Glycogen synthesis was also increased in hepatocytes after treatment with URM-II-81. In adipocytes URM-II-81 prevented MGO induced reduced glucose uptake. We conclude that URM-II-81 can be a possible treatment target to address glycotoxins induced insulin resistance.

Insulin releasing effect of some pure compounds from Moringa oleifera on mice islets

The anti-diabetic activity of extracts, fractions and compounds of Moringa oleifera have been reported; however, several constituents from this well known medicinal plant are not yet screened for bio-perspecting role for diabetes. Current studies demonstrated the anti-diabetic properties of five chemical constituents of the plant viz, 4-hydroxyphenylacetonitrite (1), fluoropyrazine (3), methyl-4-hydroxybenzoate (4), vanillin (5), and 4-α-L-rhamnopyranosyloxybenzyl isothiocyanate (6) along with one related compound 3,4-dihydroxy benzonitrile (2) for the first time in vitro and in vivo. Furthermore, the mechanism of action of compounds was predicted by utilizing molecular docking with protein kinase A (PKA) and exchange protein activated by cAMP (Epac2A). The structure of compounds was elucidated by UV, IR, MS, and 1H NMR. The compounds 1, 3–5 induced significant insulin secretion at stimulatory (16.7 mM) glucose, but not at basal (3 mM) glucose concentration, and compound 3 seems to be the most active. Compounds 1, 3–5 showed dose-dependent insulin secretory activity with optimum response at 200 μM. In silico studies revealed that compound 3 has a noticeable electrostatic and hydrophobic interaction with protein kinase A (PKA). In vitro studies also showed that there was significant reduction of compounds 1–3 mediated insulin secretion in the presence of PKA inhibitor suggesting that there is a possible role of PKA signaling pathway on insulin secretion. Upon oral administration of 1, 3–5 to diabetic rats, compounds 1 and 3 significantly reduced blood glucose level in diabetic rats in a dose- and time-dependent manner. The oral glucose tolerance test in diabetic rats showed that compound 3 significantly enhanced plasma insulin and improved beta-cell function. In cytotoxicity assay, compounds 1, 3–5 did not show any toxic effect upto 200 μM. The insulin releasing characteristic of different constituents from M. oleifera conceivably correlate the lowering of blood glucose in in vivo diabetic rats by triggering glucose-induced insulin secretion from pancreatic islets possibly by PKA-mediated insulin secretory pathway.

Cucurbitacin E reduces obesity and related metabolic dysfunction in mice by targeting JAK-STAT5 signaling pathway

Several members of cucurbitaceae family have been reported to regulate growth of cancer by interfering with STAT3 signaling. In the present study, we investigated the unique role and molecular mechanism of cucurbitacins (Cucs) in reducing symptoms of metabolic syndrome in mice. Cucurbitacin E (CuE) was found to reduce adipogenesis in murine adipocytes. CuE treatment diminished hypertrophy of adipocytes, visceral obesity and lipogenesis gene expression in diet induced mice model of metabolic syndrome (MetS). CuE also ameliorated adipose tissue dysfunction by reducing hyperleptinemia and TNF-alpha levels and enhancing hypoadiponectinemia. Results show that CuE mediated these effects by attenuating Jenus kinase- Signal transducer and activator of transcription 5 (JAK- STAT5) signaling in visceral fat tissue. As a result, CuE treatment also reduced PPAR gamma expression. Glucose uptake enhanced in adipocytes after stimulation with CuE and insulin resistance diminished in mice treated with CuE, as reflected by reduced glucose intolerance and glucose stimulated insulin secretion. CuE restored insulin sensitivity indirectly by inhibiting JAK phosphorylation and improving AMPK activity. Consequently, insulin signaling was up-regulated in mice muscle. As CuE positively regulated adipose tissue function and suppressed visceral obesity, dyslipedemia, hyperglycemia and insulin resistance in mice model of MetS, we suggest that CuE can be used as novel approach to treat metabolic diseases.

Modulation of Pancreatic β-Cells and Antioxidant Status by Cinnamon in Type 2 Diabetic Rats

The effect of Cinnamomum cassia was investigated in a non-obese type 2 diabetic rat model to explore possible cellular mechanism(s) of its antidiabetic activity. Non-obese type 2 diabetes was developed in rats by injecting 60 mg/kg streptozotocin (STZ) along with 120 mg/kg nicotinamide (NA) in adult Wistar rats. Type 2 diabetes was confirmed after 14 days of STZ-NA induction. Diabetic rats were treated with cinnamon extract at 250 mg/kg (Cn250) or 500 mg/kg dose (Cn500) and the positive control, glibenclamide (5 mg/kg) for 28 days. After treatment, blood glucose, serum insulin, HbA1c and antioxidant status were measured. Additionally, insulin and glucagon immunostaining was performed in pancreatic sections. Moderate hyperglycemia and β-cells dysfunction was found in this diabetic model rats. Interestingly, cinnamon extract treatment lowered the elevated blood glucose (Cn250: 269.8 ± 18.5 mg/dl vs. 322.5 ± 12.5 mg/dl, p<0.01; Cn500: 195.2 ± 22.5 mg/dl vs. 322.5 ± 12.5 mg/dl, p<0.001) and enhanced serum insulin levels (Cn250: 0.28 ± 0.032 ng/ml vs. 0.195 ± 0.03 ng/ml; Cn500: 0.45 ± 0.035 ng/ml vs. 0.195 ± 0.03 ng/ml, p<0.001) in a dose-dependent manner. In diabetic rats, a drastic decrease of β-cell function was observed while cinnamon extract treatment elevated the β-cell function significantly (p<0.05) in Cn500 treated group. Qualitative and quantitative improvement of pancreatic β-cell morphology was found in cinnamon-treated rats. Total antioxidant status was improved by cinnamon extract suggesting its antioxidant potential in Cn500 dose significantly (1.83 ± 0.05 mmol/L vs. 1.49 ± 0.09 mmol/L, p<0.05). Glibenclamide showed similar action to that of 500 mg/kg cinnamon in all the assays. Collectively, the data suggest that cinnamon exerts antidiabetic activity by increasing insulin secretion, modulating β-cell function, and improving antioxidant status in non-obese type 2 diabetic model rats.

New isatin derivative inhibits neurodegeneration by restoring insulin signaling in brain

Diabetes is associated with neurodegeneration. Glycation ensues in diabetes and glycated proteins cause insulin resistance in brain resulting in amyloid plaques and NFTs. Also glycation enhances gliosis by promoting neuroinflammation. Currently there is no therapy available to target neurodegenration in brain therefore, development of new therapy that offers neuroprotection is critical. The objective of this study was to evaluate mechanistic effect of isatin derivative URM-II-81, an anti-glycation agent for improvement of insulin action in brain and inhibition of neurodegenration. Methylglyoxal induced stress was inhibited by treatment with URM-II-81. Also, Ser473 and Ser9 phosphorylation of Akt and GSK-3β respectively were restored by URM-II-81. Effect of URM-II-81 on axonal integrity was studied by differentiating Neuro2A using retinoic acid. URM-II-81 restored axonal length in MGO treated cells. Its effects were also studied in high fat and low dose streptozotocin induced diabetic mice where it reduced RBG levels and inhibited glycative stress by reducing HbA1c. URM-II-81 treatment also showed inhibition of gliosis in hippocampus. Histological analysis showed reduced NFTs in CA3 hippocampal region and restoration of insulin signaling in hippocampii of diabetic mice. Our findings suggest that URM-II-81 can be developed as a new therapeutic agent for treatment of neurodegenration.