siRNA-mediated depletion of endogenous protein phosphatase 2Acα markedly attenuates ceramide-activated protein phosphatase activity in insulin-secreting INS-832/13 cells

Jangati, Giridhar Rao; Veluthakal, Rajakrishnan; Kowluru, Anjaneyulu

doi:10.1016/j.bbrc.2006.07.100

Cited by 8 publications

(5 citation statements)

References 26 publications

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“…It is, therefore, likely that additional regulatory mechanisms might underlie β-cell demise seen under the duress of lipotoxic conditions including those involving progressive alterations in the mitochondrial membrane permeability transition pore as suggested by recent studies of Koshkin et al [37] in MIN6 and INS-1 cells. Furthermore, PA-induced CER-mediated effects might also include regulation of key target proteins such as the CER-activated protein phosphatase 2A that we have characterized in isolated β-cells [27, 38], leading to the inactivation of key cellular events including inhibition of extracellular-regulated kinase and inhibition of proinsulin gene expression [39]. …”

Section: Discussionmentioning

confidence: 99%

Tiam1/Rac1 signaling pathway mediates palmitate-induced, ceramide-sensitive generation of superoxides and lipid peroxides and the loss of mitochondrial membrane potential in pancreatic β-cells

Syed

Jayaram

Subasinghe

et al. 2010

Biochemical Pharmacology

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The phagocytic NADPH-oxidase [NOX] has been implicated in the generation of superoxides in the pancreatic β-cell. Herein, using normal rat islets and clonal INS 832/13 cells, we tested the hypothesis that activation of the small G-protein Rac1, which is a member of the NOX holoenzyme, is necessary for palmitate [PA]-induced generation of superoxides in pancreatic β-cells. Incubation of isolated β-cells with PA potently increased the NOX activity culminating in a significant increase in the generation of superoxides and lipid peroxides in these cells; such effects of PA were attenuated by diphenyleneiodonium [DPI], a known inhibitor of NOX. In addition, PA caused a transient, but significant activation [i.e., GTP-bound form] of Rac1 in these cells. NSC23766, a selective inhibitor of Rac1, but not Cdc42 or Rho activation, inhibited Rac1 activation and the generation of superoxides and lipid peroxides induced by PA. Fumonisin B-1 [FB-1], which inhibits de novo synthesis of ceramide [CER] from PA, also attenuated PA-induced superoxide and lipid peroxide generation and NOX activity implicating intracellularly generated CER in the metabolic effects of PA; such effects were also demonstrable in the presence of the cell-permeable C2-CER. Further, NSC23766 prevented C2-CER-induced Rac1 activation and production of superoxides and lipid peroxides. Lastly, C2-CER, but not its inactive analogue, significantly reduced the mitochondrial membrane potential, which was prevented to a large degree by NSC23766. Together, our findings suggest that Tiam1/Rac1 signaling pathway regulates PA-induced, CER-dependent superoxide generation and mitochondrial dysfunction in pancreatic β-cells.

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Section: Discussionmentioning

confidence: 99%

Tiam1/Rac1 signaling pathway mediates palmitate-induced, ceramide-sensitive generation of superoxides and lipid peroxides and the loss of mitochondrial membrane potential in pancreatic β-cells

Syed

Jayaram

Subasinghe

et al. 2010

Biochemical Pharmacology

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“…In other contexts, PP2A activation can be prosurvival by decreasing activity of proapoptotic substrates such as p38 (Choi et al, 2004;Ou et al, 2006;Ruano et al, 2006) and p53 (Yan et al, 1997;Messner et al, 2006). It is thus not surprising that broad inhibition of PP2A using okadaic acid, calyculin A, or siRNA can produce inconsistent effects on cell survival (Wolf and Eastman, 1999;Zeevalk et al, 2001;Strack et al, 2004;Jangati et al, 2006). In our culture models, inhibition of PP2A by okadaic acid, calyculin A, or PP2A siRNA did not protect BE(2)-M17 cells from 6-OHDA in contrast to G-substrate, which was protective (supplemental Fig.…”

Section: Mechanism Of G-substrate-mediated Protectionmentioning

confidence: 99%

An Endogenous Serine/Threonine Protein Phosphatase Inhibitor, G-Substrate, Reduces Vulnerability in Models of Parkinson's Disease

Chung

Koprich²,

Endo³

et al. 2007

J. Neurosci.

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Relative neuronal vulnerability is a universal yet poorly understood feature of neurodegenerative diseases. In Parkinson's disease, dopaminergic (DA) neurons in the substantia nigra (SN) (A9) are particularly vulnerable, whereas adjacent DA neurons within the ventral tegmental area (A10) are essentially spared. Our previous laser capture microdissection and microarray study (Chung et al., 2005) demonstrated that molecular differences between these DA neurons may underlie their differential vulnerability. Here we show that G-substrate, an endogenous inhibitor of Ser/Thr protein phosphatases, exhibits higher expression in A10 compared with A9 DA neurons in both rodent and human midbrain. Overexpression of G-substrate protected dopaminergic BE(2)-M17 cells against toxins, including 6-OHDA and MG-132 (carbobenzoxy-L-leucyl-L-leucyl-L-leucinal), whereas RNA interference (RNAi)-mediated knockdown of endogenous G-substrate increased their vulnerability to these toxins. G-substrate reduced 6-OHDA-mediated protein phosphatase 2A (PP2A) activation in vitro and increased phosphorylated levels of PP2A targets including Akt, glycogen synthase kinase 3␤, and extracellular signal-regulated kinase 2 but not p38. RNAi to Akt diminished the protective effect of G-substrate against 6-OHDA. In vivo, lentiviral delivery of G-substrate to the rat SN increased baseline levels of phosphorylated Akt and protected A9 DA neurons from 6-OHDA-induced toxicity. These results suggest that inherent differences in the levels of G-substrate contribute to the differential vulnerability of DA neurons and that enhancing G-substrate levels may be a neuroprotective strategy for the vulnerable A9 (SN) DA neurons in Parkinson's disease.

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“…There are at least 4 subfamilies of regulatory B subunits (including PPP2R2A) and it is thought that this diversity in regulatory subunits dictates substrate specificity and the subcellular localization of PP2A (13). PPP2R2A is expressed in a wide range of tissues including but not limited to; insulin sensitive tissues (liver, skeletal muscle and adipose tissue) and beta cells of the pancreatic islet (14–16). PP2A is a tumor suppressor and is inactivated or down regulated in colorectal cancer, myeloid leukemia, small cell lung carcinomas and luminal breast cancers (17–22).…”

Section: Discussionmentioning

confidence: 99%

Haploinsufficiency of the Insulin Receptor in the Presence of a Splice-Site Mutation inPpp2r2aResults in a Novel Digenic Mouse Model of Type 2 Diabetes

Goldsworthy

Bai

et al. 2016

Diabetes

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Insulin resistance in mice typically does not manifest as diabetes due to multiple compensatory mechanisms. Here, we present a novel digenic model of type 2 diabetes in mice heterozygous for a null allele of the insulin receptor and an N-ethyl-N-nitrosourea-induced alternative splice mutation in the regulatory protein phosphatase 2A (PP2A) subunit PPP2R2A. Inheritance of either allele independently results in insulin resistance but not overt diabetes. Doubly heterozygous mice exhibit progressive hyperglycemia, hyperinsulinemia, and impaired glucose tolerance from 12 weeks of age without significant increase in body weight. Alternative splicing of Ppp2r2a decreased PPP2R2A protein levels. This reduction in PPP2R2A containing PP2A phosphatase holoenzyme was associated with decreased serine/threonine protein kinase AKT protein levels. Ultimately, reduced insulin-stimulated phosphorylated AKT levels were observed, a result that was confirmed in Hepa1-6, C2C12, and differentiated 3T3-L1 cells knocked down using Ppp2r2a small interfering RNAs. Altered AKT signaling and expression of gluconeogenic genes in the fed state contributed to an insulin resistance and hyperglycemia phenotype. This model demonstrates how genetic changes with individually small phenotypic effects interact to cause diabetes and how differences in expression of hypomorphic alleles of PPP2R2A and potentially other regulatory proteins have deleterious effects and may therefore be relevant in determining diabetes risk.Type 2 diabetes is a complex disease where cellular resistance to insulin combined with a failure in b-cell compensation results in the development of the disease. Underlying this process are multiple genetic and environmental factors that interact to determine susceptibility risk. However, there are relatively few examples of patients with diabetes whose disease can be demonstrated to be due to the interaction of mutations in two or more genes. One of these is due to heterozygous mutations in two unlinked genes, peroxisome proliferator-activated receptor g (PPARG) and protein phosphatase 1, regulatory (inhibitor) subunit 3A (PPP1R3A), expressed in adipocytes and skeletal muscle, respectively, resulting in severe insulin resistance and lipodystrophy (1). A second example is haploinsufficiency for the insulin receptor (IR) in combination with chimerin 2 (CHN2), a GTPase-activating protein, that results in insulin resistance and deficiency in intrauterine growth (2). In this latter example, the CHN2 mutation implicates a novel gene in insulin signaling and its regulation of metabolism and growth (2). Although there are other examples of doubly heterozygous individuals with diabetes, e.g., in the maturity-onset diabetes of the young HNF1A and HNF4A genes, it is unclear how these impact the severity of disease (3). In a mouse model, a digenic insulin resistance phenotype has been described whereby 40% of mice heterozygous for both IR and insulin receptor substrate 1 (IRS-1) null alleles develop overt diabetes at 4-6 months of age, demonstra...

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siRNA-mediated depletion of endogenous protein phosphatase 2Acα markedly attenuates ceramide-activated protein phosphatase activity in insulin-secreting INS-832/13 cells

Cited by 8 publications

References 26 publications

Tiam1/Rac1 signaling pathway mediates palmitate-induced, ceramide-sensitive generation of superoxides and lipid peroxides and the loss of mitochondrial membrane potential in pancreatic β-cells

Tiam1/Rac1 signaling pathway mediates palmitate-induced, ceramide-sensitive generation of superoxides and lipid peroxides and the loss of mitochondrial membrane potential in pancreatic β-cells

An Endogenous Serine/Threonine Protein Phosphatase Inhibitor, G-Substrate, Reduces Vulnerability in Models of Parkinson's Disease

Haploinsufficiency of the Insulin Receptor in the Presence of a Splice-Site Mutation inPpp2r2aResults in a Novel Digenic Mouse Model of Type 2 Diabetes

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