GRK2 negatively regulates glycogen synthesis in mouse liver FL83B cells

G-protein-coupled receptor (GPCR) kinases (GRKs) are serine/threonine kinases that desensitize agonist-occupied classical GPCRs. Although the insulin receptor (IR) is a tyrosine kinase receptor, the IR also couples to G-proteins and utilizes G-protein signaling components. The present study was designed to test the hypothesis that GRK2 negatively regulates IR signaling. FL83B cells, derived from mouse liver, were treated with insulin and membrane translocation of GRK2 was determined using immunofluoresecence and Western blotting. Insulin caused an increase in the translocation of GRK-2 from cytosol to the plasma membrane. To determine the role of GRK2 in IR signaling, GRK2 was selectively down-regulated ( approximately by 90%) in FL83B cells using a small interfering RNA technique. Basal as well as insulin-induced glycogen synthesis (measured by d-[U-(14)C]glucose incorporation) was increased in GRK2-deficient cells compared with control cells. Similarly, GRK2 deficiency increased the basal and insulin-stimulated phosphorylation of Ser(21) in glycogen synthase kinase-3alpha. Insulin-induced tyrosine phosphorylation of the IR was similar in control and GRK2-deficient cells. Basal and insulin-stimulated phosphorylation of Tyr(612) in insulin receptor subunit 1 was significantly increased while phosphorylation of Ser(307) was decreased in GRK2-deficient FL83B cells compared with control cells. Chronic insulin treatment (24 h) in control cells caused an increase in GRK2 (56%) and a decrease in IR (50%) expression associated with the absence of an increase in glycogen synthesis, suggesting impairment of IR function. However, chronic insulin treatment (24 h) did not decrease IR expression or impair IR effects on glycogen synthesis in GRK2-deficient cells. We conclude that (i) GRK2 negatively regulates basal and insulin-stimulated glycogen synthesis via a post-IR signaling mechanism, and (ii) GRK2 may contribute to reduced IR expression and function during chronic insulin exposure.     

N-(2-hydroxyphenyl)acetamide and its gold nanoparticle conjugation prevent glycerol-induced acute kidney injury by attenuating inflammation and oxidative injury in mice

Molecular and Cellular Biochemistry - The protective activity of N-(2-hydroxyphenyl)acetamide (NA-2) and NA-2-coated gold nanoparticles (NA-2-AuNPs) in glycerol-treated model of acute kidney injury...     

Effects of Pro1266Leu mutation on structure and function of glycoprotein Ib binding domain of von Willebrand factor

Glycoprotein Ibα (GpIbα) binding ability of A1 domain of von Willebrand factor (vWF) facilitates platelet adhesion that plays a crucial role in maintaining hemostasis and thrombosis at the site of vascular damage. There are both “loss as well as gain of function” mutations observed in this domain. Naturally occurring “gain of function” mutations leave self-activating impacts on the A1 domain which turns the normal binding to characteristic constitutive binding with GPIbα. These “gain of function” mutations are associated with the von Willebrand disease type 2B. In recent years, studies focused on understanding the mechanism and conformational patterns attached to these phenomena have been conducted, but the conformational pathways leading to such binding patterns are poorly understood as of now. To obtain a microscopic picture of such events for the better understanding of pathways, we used molecular dynamics (MD) simulations along with principal component analysis and normal mode analysis to study the effects of Pro1266Leu (Pro503Leu in structural context) mutation on the structure and function of A1 domain of vWF. MD simulations have provided atomic-level details of intermolecular motions as a function of time to understand the dynamic behavior of A1 domain of vWF. Comparative analysis of the trajectories obtained from MD simulations of both the wild type and Pro503Leu mutant suggesting appreciable conformational changes in the structure of mutant which might provide a basis for assuming the “gain of function” effects of these mutations on the A1 domain of vWF, resulting in the constitutive binding with GpIbα.     

Niacin deficiency modulates genes involved in cancer: Are smokers at higher risk?

The role of niacin’s metabolite, nicotinamide adenine dinucleotide (NAD), in DNA repair via base-excision repair pathway is well documented. We evaluated if niacin deficiency results in genetic instability in normal human fetal lung fibroblasts (MRC-5), and further, does it leads to enhanced accumulation of cigarette smoke–induced genetic damage? MRC-5 cells were grown discretely in niacin-proficient/deficient media, and exposed to nicotine-derived nitrosamine ketone (NNK, a cigarette smoke carcinogen). Niacin deficiency abated the NAD polymerization, augmented the spontaneous induction of micronuclei (MN) and chromosomal aberrations (CA) and raised the expression of 10 genes and suppressed 12 genes involved in different biological functions. NNK exposure resulted in genetic damage as measured by the induction of MN and CA in cells grown in niacin-proficient medium, but the damage became practically marked when niacin-deficient cells were exposed to NNK. NNK exposure raised the expression of 16 genes and suppressed the expression of 56 genes in cells grown in niacin-proficient medium. NNK exposure to niacin-deficient cells raised the expression of eight genes including genes crucial in promoting cancer such as FGFR3 and DUSP1 and suppressed the expression of 33 genes, including genes crucial in preventing the onset and progression of cancer like RASSF2, JUP, and IL24, in comparison with the cells grown in niacin-proficient medium. Overall, niacin deficiency interferes with the DNA damage repair process induced by chemical carcinogens like NNK, and niacin-deficient population are at the higher risk of genetic instability caused by cigarette smoke carcinogen NNK.     

Investigation of deleterious effects of nsSNPs in the POT1 gene: a structural genomics-based approach to understand the mechanism of cancer development

Protection of telomere 1 (POT1) is one of the key components of shelterin complex, implicated in maintaining the telomere homeostasis, and thus stability of the eukaryotic genome. A large number of non-synonymous single nucleotide polymorphisms (nsSNPs) in the POT1 gene have been reported to cause varieties of human diseases, including cancer. In recent years, a number of mutations in POT1 has been markedly increased, and interpreting the effect of these large numbers of mutations to understand the mechanism of associated diseases seems impossible using experimental approaches. Herein, we employ varieties of computational methods such as PROVEAN, PolyPhen-2, SIFT, PoPMuSiC, SDM2, STRUM, and MAESTRO to identify the effects of 387 nsSNPs on the structure and function of POT1 protein. We have identified about 183 nsSNPs as deleterious and termed them as “high-confidence nsSNPs.” Distribution of these high-confidence nsSNPs demonstrates that the mutation in oligonucleotide binding domain 1 is highly deleterious (one in every three nsSNPs), and high-confidence nsSNPs show a strong correlation with residue conservation. The structure analysis provides a detailed insights into the structural changes occurred in consequence of conserved mutations which lead to the cancer progression. This study, for the first time, offers a newer prospective on the role of POT1 mutations on the structure, function, and their relation to associated diseases.     

Heterogeneity of the memory CD4 T cell response: persisting effectors and resting memory T cells

Defining the cellular composition of the memory T cell pool has been complicated by an inability to distinguish effector and memory T cells. We present here an activation profile assay, using anti-CD3 and antigenic stimuli, that clearly distinguishes effector and memory CD4 T cells and defines subsets of long-lived memory CD4 T cells based on CD62 ligand (CD62L) expression. The CD62L(low) memory subset functionally resembles effector cells, exhibiting hyper-responsiveness to antigenic and anti-CD3 mediated stimuli, high proliferative capacity, and rapid activation kinetics. The CD62L(high) memory subset functionally resembles resting memory cells, exhibiting hyporesponsiveness to anti-CD3 stimuli, lower proliferative capacity, and slower activation kinetics. Our results indicate that the memory CD4 T cell pool is heterogeneous, consisting of persisting effectors and resting memory T cells.     

Retinyl ester secretion by intestinal cells: a specific and regulated process dependent on assembly and secretion of chylomicrons

Retinyl esters (RE) have been used extensively as markers to study chylomicron (CM) catabolism because they are secreted in the postprandial state with CM and do not exchange with other lipoproteins in the plasma. To understand the mechanism of secretion of RE by the intestine under the fasting and postprandial states, differentiated Caco-2 cells were supplemented with radiolabeled retinol under conditions that support or do not support CM secretion. We observed that these cells assimilate vitamin A by a rapid uptake mechanism. After uptake, cells store retinol in both esterified and unesterified forms. Under fasting conditions, cells do not secrete RE but secrete free retinol unassociated with lipoproteins. Under postprandial conditions, cells secrete significant amounts of RE only with CM. The secretion of RE with CM was independent of the rate of uptake of retinol and intracellular free and esterified retinol levels, and was absolutely dependent on the assembly and secretion of CM. The secretion of RE was correlated with the secretion of CM and not with the secretion of total apolipoprotein B. Inhibition of CM secretion by Pluronic L81 decreased the secretion of RE and did not result in their increased secretion with smaller lipoproteins. These data strongly suggest that RE secretion by the intestinal cells is a specific and regulated process that occurs in the postprandial state and is dependent on the assembly and secretion of CM. We propose that RE are added to CM during final stages of lipoprotein assembly and may serve as signposts for these steps.