Insulin-induced de novo lipid synthesis occurs mainly via mTOR-dependent regulation of proteostasis of SREBP-1c.

Insulin stimulates de novo lipid synthesis in the liver and in cultured hepatocytes via its ability to activate sterol regulatory element-binding protein 1c (SREBP-1c). Although PI3K-AKT-mTORC1-p70S6K-signaling kinases are known to drive feed-forward expression of SREBP-1c, the identity of the phosphorylated amino acid residue(s) putatively involved in insulin-stimulated de novo lipogenesis remains elusive. We obtained in silico and mass spectrometry evidence, that was combined with siRNA strategies, to discover that insulin-induced phosphorylation of serine 418, serine 419, and serine 422 in rat SREBP-1c was most likely mediated by p70S6 kinase. Here, for the first time, we show that insulin-induced phosphorylation of these 3 serine residues mainly impinged on the mechanisms of proteostasis of both full-length and mature SREBP-1c in the McArdle-RH7777 hepatoma cells. Consistent with this conclusion, nascent SREBP-1c, substituted with phosphomimetic aspartic acid residues at these 3 sites, was resistant to proteasomal degradation. As a consequence, endoplasmic reticulum to Golgi migration and proteolytic maturation of pSREBP-1c was significantly enhanced which led to increased accumulation of mature nSREBP-1c, even in the absence of insulin. Remarkably, aspartic acid substitutions at S418, S419 and S422 also protected the nascent SREBP-1c from ubiquitin-mediated proteasome degradation thus increasing its steady-state levels and transactivation potential in the nucleus. These complementary effects of p70S6K-mediated phosphorylation on proteostasis of pSREBP-1c were necessary and sufficient to account for insulin's ability to enhance transcription of genes controlling de novo lipogenesis in hepatocytes.

Patients experiencing statin-induced myalgia exhibit a unique program of skeletal muscle gene expression following statin re-challenge.

Statins, the 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase inhibitors, are widely prescribed for treatment of hypercholesterolemia. Although statins are generally well tolerated, up to ten percent of statin-treated patients experience myalgia symptoms, defined as muscle pain without elevated creatinine phosphokinase (CPK) levels. Myalgia is the most frequent reason for discontinuation of statin therapy. The mechanisms underlying statin myalgia are not clearly understood. To elucidate changes in gene expression associated with statin myalgia, we compared profiles of gene expression in skeletal muscle biopsies from patients with statin myalgia who were undergoing statin re-challenge (cases) versus those of statin-tolerant controls. A robust separation of case and control cohorts was revealed by Principal Component Analysis of differentially expressed genes (DEGs). To identify putative gene expression and metabolic pathways that may be perturbed in skeletal muscles of patients with statin myalgia, we subjected DEGs to Ingenuity Pathways (IPA) and DAVID (Database for Annotation, Visualization and Integrated Discovery) analyses. The most prominent pathways altered by statins included cellular stress, apoptosis, cell senescence and DNA repair (TP53, BARD1, Mre11 and RAD51); activation of pro-inflammatory immune response (CXCL12, CST5, POU2F1); protein catabolism, cholesterol biosynthesis, protein prenylation and RAS-GTPase activation (FDFT1, LSS, TP53, UBD, ATF2, H-ras). Based on these data we tentatively conclude that persistent myalgia in response to statins may emanate from cellular stress underpinned by mechanisms of post-inflammatory repair and regeneration. We also posit that this subset of individuals is genetically predisposed to eliciting altered statin metabolism and/or increased end-organ susceptibility that lead to a range of statin-induced myopathies. This mechanistic scenario is further bolstered by the discovery that a number of single nucleotide polymorphisms (e.g., SLCO1B1, SLCO2B1 and RYR2) associated with statin myalgia and myositis were observed with increased frequency among patients with statin myalgia.

Trichostatin A induces a unique set of microRNAs including miR-129-5p that blocks cyclin-dependent kinase 6 expression and proliferation in H9c2 cardiac myocytes.

The pan-histone deacetylase inhibitor (HDACI), trichostatin A (TSA), was shown to normalize interleukin-18-induced cardiac hypertrophy in vivo and in vitro; evidently, this occurred via epigenetic mechanisms that profoundly altered cardiac gene expression (Majumdar et al. in, Physiol Genom, 43: 1392, 2011; BMC Genom, 13: 709, 2012). Here, we tested the hypothesis that TSA-induced changes in chromatin architecture also led to altered expression of microRNAs that in turn, contributed to the unique transcriptome of cardiac myocytes exposed to the HDACI. Using miRCURY LNA™ Universal microRNA PCR system, we demonstrate that H9c2 cells exposed to TSA for 6 and 24 h elicited differential expression of 19 and 16 microRNAs, respectively. H9c2 cells incubated in medium-containing 100 nM of TSA elicited a rapid and robust induction of miR-129-5p. Enhanced expression of miR-129-5p was also observed in the hearts of TSA-treated mice. Induction of miR-129-5p in H9c2 cells was accompanied by reduced expression of its direct target, cyclin-dependent kinase 6 (CDK6) that is a key regulator of cell cycle. Using cell division-dependent dilution of Cell Trace™ violet measurements we showed that concomitant induction of miR-129-5p and reduced CDK6 expression were mechanistically involved in TSA-induced inhibition of proliferation of H9c2 cells. Consistent with this scenario, cells expressing an antagomiR of miR-129-5p were resistant to the anti-proliferative actions of TSA. These data indicate that although TSA treatment led to altered expression of several microRNAs, the overarching action of TSA (i.e., inhibition of cell division) in H9c2 cells was achieved via miR-129-5p.

Role of PTEN in TNFα induced insulin resistance.

AIMS/HYPOTHESIS: PTEN may play a reversible role in TNFα induced insulin resistance, which has been linked to obesity-associated insulin resistance (IR). METHODS: Western blots for PTEN and p-Akt were performed on H-411E liver cells incubated with insulin, TNFα, and in selected experiments VO-OHpic vanadium complex in the presence and absence of PTEN siRNA. Total PTEN was compared to ß-actin loading control and p-Akt was compared to total Akt. RESULTS: Western blot and Real Time RT-PCR experiments showed increased PTEN after TNFα treatment (p = 0.04); slightly decreased PTEN after insulin treatment; and slightly increased PTEN after insulin + TNFα treatment. PTEN siRNA markedly inhibited the TNFα-induced increase in PTEN (p < 0.01) without significantly changing the p-Akt levels. The vanadium complex, exhibiting insulin-like effects, also significantly prevented the TNFα-induced increase in PTEN. Combining insulin and VO-OHpic was additive, providing both proof of concept and insight into mechanism. DISCUSSION: The PTEN increase due to TNFα treatment was reversible by both PTEN siRNA knockdown and VO-OHpic treatment. Thus, PTEN is identified as a potential new therapeutic target for reducing IR in Type 2 DM.

A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle.

We combined the TRIzol method of nucleic acid extraction with QIAamp columns to achieve coextraction of RNA and genomic DNA from peripheral blood mononuclear cells (PBMCs) and biopsied skeletal muscle, both stored at -80 °C for many months. Total RNA was recovered from the upper aqueous phase of TRIzol. The interphase and organic phases were precipitated with ethanol, digested with proteinase K, and filtered through QIAamp MinElute columns to recover DNA. The combined protocol yielded excellent quality and quantity of nucleic acids from archived human PBMCs and muscle and may be easily adapted for other tissues.

Pan-histone deacetylase inhibitors regulate signaling pathways involved in proliferative and pro-inflammatory mechanisms in H9c2 cells.

BACKGROUND: We have shown previously that pan-HDAC inhibitors (HDACIs) m-carboxycinnamic acid bis-hydroxamide (CBHA) and trichostatin A (TSA) attenuated cardiac hypertrophy in BALB/c mice by inducing hyper-acetylation of cardiac chromatin that was accompanied by suppression of pro-inflammatory gene networks. However, it was not feasible to determine the precise contribution of the myocytes- and non-myocytes to HDACI-induced gene expression in the intact heart. Therefore, the current study was undertaken with a primary goal of elucidating temporal changes in the transcriptomes of cardiac myocytes exposed to CBHA and TSA. RESULTS: We incubated H9c2 cardiac myocytes in growth medium containing either of the two HDACIs for 6h and 24h and analyzed changes in gene expression using Illumina microarrays. H9c2 cells exposed to TSA for 6h and 24h led to differential expression of 468 and 231 genes, respectively. In contrast, cardiac myocytes incubated with CBHA for 6h and 24h elicited differential expression of 768 and 999 genes, respectively. We analyzed CBHA- and TSA-induced differentially expressed genes by Ingenuity Pathway (IPA), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Core_TF programs and discovered that CBHA and TSA impinged on several common gene networks. Thus, both HDACIs induced a repertoire of signaling kinases (PTEN-PI3K-AKT and MAPK) and transcription factors (Myc, p53, NFkB and HNF4A) representing canonical TGFß, TNF-α, IFNγ and IL-6 specific networks. An overrepresentation of E2F, AP2, EGR1 and SP1 specific motifs was also found in the promoters of the differentially expressed genes. Apparently, TSA elicited predominantly TGFß- and TNF-α-intensive gene networks regardless of the duration of treatment. In contrast, CBHA elicited TNF-α and IFNγ specific networks at 6 h, followed by elicitation of IL-6 and IFNγ-centered gene networks at 24h. CONCLUSIONS: Our data show that both CBHA and TSA induced similar, but not identical, time-dependent, gene networks in H9c2 cardiac myocytes. Initially, both HDACIs impinged on numerous genes associated with adipokine signaling, intracellular metabolism and energetics, and cell cycle. A continued exposure to either CBHA or TSA led to the emergence of a number of apoptosis- and inflammation-specific gene networks that were apparently suppressed by both HDACIs. Based on these data we posit that the anti-inflammatory and anti-proliferative actions of HDACIs are myocyte-intrinsic. These findings advance our understanding of the mechanisms of actions of HDACIs on cardiac myocytes and reveal potential signaling pathways that may be targeted therapeutically.

Panhistone deacetylase inhibitors inhibit proinflammatory signaling pathways to ameliorate interleukin-18-induced cardiac hypertrophy.

We investigated the genome-wide consequences of pan-histone deacetylase inhibitors (HDACIs) trichostatin A (TSA) and m-carboxycinnamic acid bis-hydroxamide (CBHA) in the hearts of BALB/c mice eliciting hypertrophy in response to interleukin-18 (IL-18). Both TSA and CBHA profoundly altered cardiac chromatin structure that occurred concomitantly with normalization of IL-18-induced gene expression and amelioration of cardiac hypertrophy. The hearts of mice exposed to IL-18+/-TSA or CBHA elicited distinct gene expression profiles. Of 184 genes that were differentially regulated by IL-18 and TSA, 33 were regulated in an opposite manner. The hearts of mice treated with IL-18 and/or CBHA elicited 147 differentially expressed genes (DEGs), a third of which were oppositely regulated by IL-18 and CBHA. Ingenuity Pathways and Kyoto Encyclopedia of Genes and Genomes analyses of DEGs showed that IL-18 impinged on TNF-α- and IFNγ-specific gene networks relegated to controlling immunity and inflammation, cardiac metabolism and energetics, and cell proliferation and apoptosis. These TNF-α- and IFNγ-specific gene networks, extensively connected with PI3K, MAPK, and NF-κB signaling pathways, were oppositely regulated by IL-18 and pan-HDACIs. Evidently, both TSA and CBHA caused a two- to fourfold induction of phosphatase and tensin homolog expression to counteract IL-18-induced proinflammatory signaling and cardiac hypertrophy.

A critical role of Sp1 transcription factor in regulating gene expression in response to insulin and other hormones.

Specificity protein 1 (Sp1) belongs to a family of ubiquitously expressed, C(2)H(2)-type zinc finger-containing DNA binding proteins that activate or repress transcription of many genes in response to physiological and pathological stimuli. There is emerging evidence to indicate that in addition to functioning as 'housekeeping' transcription factors, members of Sp family may be key mediators of gene expression induced by insulin and other hormones. The founding member of the family, Sp1, by virtue of its multi-domain organization, potential for posttranslational modifications and interactions with numerous transcription factors, represents an ideal mediator of nuclear signaling in response to hormones. Insulin regulates the sub-cellular localization, stability and trans-activation potential of Sp1 by dynamically modulating its post-translational modification by O-linked beta-N-acetylglucosamine (O-GlcNAc) or phosphate residues. We briefly review the recent literature demonstrating that an involvement of Sp-family of transcription factors in the regulation of differential gene expression in response to hormones is more common than previously appreciated and may represent a key regulatory mechanism.

Epigenetic regulation of cardiac muscle-specific genes in H9c2 cells by Interleukin-18 and histone deacetylase inhibitor m-carboxycinnamic acid bis-hydroxamide.

Interleukin-18 (IL-18) elicited a robust hypertrophy response in H9c2 cardiomyocytes as judged by their accelerated rates of protein synthesis and increased cell size. Evidently, IL-18 treatment also induced a cardiac hypertrophy-specific program of gene expression in H9c2 cardiomyocytes since they elicited enhanced expression of atrial naturetic factor (ANF), desmin, and skeletal alpha-actin genes accompanied by a canonical switch in the transcription of alpha- and beta-myosin heavy chain (MyHC) genes. Co-treatment of H9c2 cells with m-carboxycinnamic acid bis-hydroxamide (CBHA), an inhibitor of histone deacetylases, significantly blocked both morphological and molecular manifestations of IL-18-induced cardiac hypertrophy in vitro. IL-18 treatment led to activation of phosphoinositide-3-kinase and phosphorylated Akt/protein kinase B, while CBHA blunted this pathway via inducing the expression of its upstream regulator, PTEN (phosphatase and tensin homolog). The architecture of bulk chromatin of H9c2 cells exposed to IL-18 and/or CBHA was significantly altered as judged by the extent of covalent modifications of its constituent histones. The chromatin immuno-precipitation (ChIP) assays revealed that IL-18-induced specific epigenetic changes in the chromatin of ANF, desmin, skeletal alpha-actin, and MyHC genes that were largely neutralized by CBHA. We demonstrate for the first time that 'histone code' of the entire approximately 50 kb genomic DNA encoding the alpha- and beta-MyHC genes and the intergenic DNA that generates anti-beta-MyHC RNA was uniquely modulated by pro- and anti-hypertrophy signals of IL-18 and CBHA, respectively.

Insulin dynamically regulates calmodulin gene expression by sequential o-glycosylation and phosphorylation of sp1 and its subcellular compartmentalization in liver cells.

O-glycosylation and phosphorylation of Sp1 are thought to modulate the expression of a number of genes in normal and diabetic state. Sp1 is an obligatory transcription factor for constitutive and insulin-responsive expression of the calmodulin gene (Majumdar, G., Harmon, A., Candelaria, R., Martinez-Hernandez, A., Raghow, R., and Solomon, S. S. (2003) Am. J. Physiol. 285, E584-E591). Here we report the temporal dynamics of accumulation of total, O-GlcNAc-modified, and phosphorylated Sp1 in H-411E hepatoma cells by immunohistochemistry with monospecific antibodies, confocal microscopy, and matrix-assisted laser desorption and ionization-time of flight mass spectrometry. Insulin elicited sequential and reciprocal post-translational modifications of Sp1. The O-glycosylation of Sp1 and its nuclear accumulation induced by insulin peaked early (approximately 30 min), followed by a steady decline of O-GlcNAc-modified Sp1 to negligible levels by 240 min. The accumulation of phosphorylated Sp1 in the nuclei of insulin-treated cells showed an opposite pattern, increasing steadily until reaching a maximum around 240 min after treatment. Analyses of the total, O-GlcNAc-modified, or phosphorylated Sp1 by Western blot and mass spectrometry corroborated the sequential and reciprocal control of post-translational modifications of Sp1 in response to insulin. Treatment of cells with streptozotocin (a potent inhibitor of O-GlcNAcase) led to hyperglycosylation of Sp1 that failed to be significantly phosphorylated. The mass spectrometry data indicated that a number of common serine residues of Sp1 undergo time-dependent, reciprocal O-glycosylation and phosphorylation, paralleling its rapid translocation from cytoplasm to the nucleus. Later, changes in the steady state levels of phosphorylated Sp1 mimicked the enhanced steady state levels of calmodulin mRNA seen after insulin treatment. Thus, O-glycosylation of Sp1 appears to be critical for its localization into the nucleus, where it undergoes obligatory phosphorylation that is needed for Sp1 to activate calmodulin gene expression.

Proteome of H-411E (liver) cells exposed to insulin and tumor necrosis factor-alpha: analysis of proteins involved in insulin resistance.

Insulin resistance may be modeled in H-411E liver cells in tissue culture with the use of the cytokine tumor necrosis factor-alpha (TNF-alpha) and insulin. This tissue-culture model nicely mimics IR in human type 2 diabetes mellitus. After incubation of liver cells in tissue culture with INS alone, TNF-alpha alone, and TNF-alpha plus insulin, as well as a control sample, liver-cell extracts were separated on 2D polyacrylamide-gel electrophoresis on the basis of isoelectric point and molecular weight. We analyzed the gel images with the use of PD Quest software (Bio-Rad Laboratories, Hercules, Calif) to identify differentially expressed protein spots (ie, up or down with insulin vs down or up with TNF-alpha plus insulin). In separate experiments, phosphorus-32 incorporation/autoradiography and phosphoprotein staining were used to characterize treatment-induced phosphorylations. Affected protein spots were identified with the use of peptide fingerprinting and matrix-assisted laser desorption ionization time of flight mass spectrometry. The first series of experiments identified 6 differentially expressed proteins: eukaryotic translation initiation factor-3, subunit 2, regulator of G-protein signaling-5, superoxide dismutase, protein disulfide isomerase A6, proteasome subunit-alpha type 3, and regucalcin. In addition, we observed changes in the phosphorylation of protein disulfide isomerase A6. A second series of experiments identified 7 additional proteins with significantly altered differential expression: cell-division protein kinase-4, kinogen heavy chain, carbonic anhydrase-7, E 3 ubiquitin protein ligase, URE-B1; Rab GDP dissociation inhibitor-beta, Rab GDP dissociation inhibitor-beta2, and MAWDBP. It can be seen that differentially expressed proteins, affected by treatment with insulin or with TNF-alpha plus insulin, include regulators of translation, protein degradation, cellular Ca ++ , G-proteins, and free-radical production. Although one cannot detail the mechanism or mechanisms of TNF-alpha induced IR from this data alone, it is easy to relate all of these proteins to a role in insulin signal transduction and, hence, insulin resistance.

Insulin stimulates and diabetes inhibits O-linked N-acetylglucosamine transferase and O-glycosylation of Sp1.

Insulin stimulates both the biosynthesis of transcription factor Sp1 and its O-linked N-acetylglucosaminylation (O-GlcNAcylation), which promotes nuclear localization of Sp1 and its ability to transactivate calmodulin (CaM) gene transcription. To investigate this further, we incubated H-411E liver cells with insulin (10,000 microU/ml) and quantified the subcellular distribution of O-GlcNAc transferase (OGT) and O-GlcNAc-modified Sp1. We also examined the phosphorylation of Sp1 using both Western blot and incorporation of 32P into Sp1. The results demonstrate that insulin, but not glucagon, stimulates OGT synthesis and enhances cytosolic staining of OGT (histochemical). Insulin increases O-GlcNAc-Sp1, which peaks at 30 min, followed by decline at 4 h. In contrast, insulin initiates phosphorylation of Sp1 early, followed by a continued increase in phosphorylated Sp1 (PO4-Sp1) at 4 h. A reciprocal relationship between O-GlcNAc-Sp1 and PO4-Sp1 was observed. To explore the pathophysiological relevance, we localized OGT in liver sections from streptozotocin (STZ)-induced diabetic rats. We observed that staining of OGT in STZ-induced diabetic rat liver is clearly diminished, but it was substantially restored after 6 days of insulin treatment. We conclude that insulin stimulates CaM gene transcription via a dynamic interplay between O-glycosylation and phosphorylation of Sp1 that modulates stability, mobility, subcellular compartmentalization, and activity.

O-glycosylation of Sp1 and transcriptional regulation of the calmodulin gene by insulin and glucagon.

Both insulin and glucagon stimulate steady-state levels of Sp1 transcription factor, but only insulin stimulates transcription of the calmodulin (CaM) gene in liver. Because O-glycosylation of Sp1 by O-linked N-acetylglucosamine (O-GlcNAc) is thought to regulate its ability to activate transcription, we assayed the levels of Sp1 with anti-Sp1 and anti-O-GlcNAc antibodies in Western blots by use of extracts of H-411E liver cells treated with insulin (10,000 microU/ml) or glucagon (1.5 x 10(-5) M). We also assessed subcellular localization of the native and glycosylated Sp1 in H411E cells treated with either hormone in the presence of deoxynorleucine (DON, an indirect inhibitor of O-glycosylation) or streptozotocin (STZ, an indirect stimulator of O-glycosylation). Insulin stimulated both total and O-GlcNAc-modified Sp1 primarily in the nucleus and induced CaM gene transcription (P < 0.0001). In contrast, glucagon promoted accumulation of Sp1 in the cytoplasm but not the nucleus, without significantly stimulating (P = not significant) either its O-glycosylation or transcription of the CaM gene. DON inhibited O-glycosylation of Sp1 and its ability to migrate to the nucleus and transactivate CaM gene transcription. In contrast, cotreatment of cells with STZ and glucagon enhanced O-glycosylation of Sp1, promoting its migration to the nucleus and resulting in increased CaM gene transcription. Thus O-glycosylation of Sp1 by insulin, but not glucagon, apparently enhances its (Sp1) nuclear recruitment and results in activation of CaM gene transcription.

Paradoxical regulation of Sp1 transcription factor by glucagon.

Insulin is a potent regulator of Sp1 transcription factor. To examine if glucagon, which usually antagonizes insulin, regulates Sp1, we assessed the levels of Sp1 by Western blotting from H-411E cells exposed to glucagon with or without insulin. Glucagon alone (1.5 x 10(-9) to 1.5 x 10(-5) M) stimulated Sp1 accumulation but inhibited insulin's (10,000 microU/ml) stimulatory effect on Sp1. We also assessed the effect of TNF-alpha, wortmannin, a PI3K inhibitor, and cAMP-dependent protein kinase inhibitor on Sp1 accumulation. While TNF-alpha (5 ng/ml) blocked insulin-stimulated Sp1, it failed to block stimulation of Sp1 by glucagon (1.5 x 10(-5) M). Similarly, wortmannin inhibited insulin- but not glucagon-stimulated Sp1, whereas protein kinase inhibitor had an opposite effect. Thus, insulin acts primarily via PI3K, and glucagon apparently stimulates through a cAMP-dependent pathway. Insulin increased the staining intensity of Sp1 seen exclusively in the nuclei of H-411E cells. Sp1 was demonstrable in both nucleus and cytoplasm after glucagon treatment. Finally, as judged by immunoblotting to specific antibody, insulin but not glucagon, stimulated O-glycosylation of Sp1. Thus, unique signaling mechanisms mediate the response of Sp1 to glucagon in the presence or absence of insulin.