Separation of insulin signaling into distinct GLUT4 translocation and activation steps

GLUT4 (glucose transporter 4) plays a pivotal role in insulin-induced glucose uptake to maintain normal blood glucose levels. Here, we report that a cell-permeable phosphoinositide-binding peptide induced GLUT4 translocation to the plasma membrane without inhibiting IRAP (insulin-responsive aminopeptidase) endocytosis. However, unlike insulin treatment, the peptide treatment did not increase glucose uptake in 3T3-L1 adipocytes, indicating that GLUT4 translocation and activation are separate events. GLUT4 activation can occur at the plasma membrane, since insulin was able to increase glucose uptake with a shorter time lag when inactive GLUT4 was first translocated to the plasma membrane by pretreating the cells with this peptide. Inhibition of phosphatidylinositol (PI) 3-kinase activity failed to inhibit GLUT4 translocation by the peptide but did inhibit glucose uptake when insulin was added following peptide treatment. Insulin, but not the peptide, stimulated GLUT1 translocation. Surprisingly, the peptide pretreatment inhibited insulin-induced GLUT1 translocation, suggesting that the peptide treatment has both a stimulatory effect on GLUT4 translocation and an inhibitory effect on insulin-induced GLUT1 translocation. These results suggest that GLUT4 requires translocation to the plasma membrane, as well as activation at the plasma membrane, to initiate glucose uptake, and both of these steps normally require PI 3-kinase activation.     

Kaempferitrin inhibits GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes

Insulin stimulated GLUT4 (glucose transporter 4) translocation and glucose uptake in muscles and adipocytes is important for the maintenance of blood glucose homeostasis in our body. In this paper, we report the identification of kaempferitrin (kaempferol 3,7-dirhamnoside), a glycosylated flavonoid, as a compound that inhibits insulin stimulated GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes. In the absence of insulin, we observed that addition of kaempferitrin did not affect GLUT4 translocation or glucose uptake. On the other hand, kaempferitrin acted as an inhibitor of insulin-stimulated GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes by inhibiting Akt activation. Molecular docking studies using a homology model of GLUT4 showed that kaempferitrin binds directly to GLUT4 at the glucose transportation channel, suggesting the possibility of a competition between kaempferitrin and glucose during the transport. Taken together, our data demonstrates that kaempferitrin inhibits GLUT4 mediated glucose uptake at least by two different mechanisms, one by interfering with the insulin signaling pathway and the other by a possible competition with glucose during the transport.     

Enhanced Glucose Uptake in Phenylbutyric Acid-Treated 3T3-L1 Adipocytes

Diabetes Mellitus is a chronic metabolic disease marked by altered glucose homeostasis and insulin resistance. The phosphatase PTEN antagonizes the insulin-induced-PI3K-driven cascade that normally leads to GLUT4 membrane translocation. This study investigates the effect of Phenylbutyric Acid (PBA), a chemical chaperone and a potential mediator of PTEN activity, on glucose uptake in differentiated 3T3-L1 adipocytes. Adipocyte differentiation status was quantified by Oil Red O staining and the expression of AP2. Baseline and insulin-induced adipocyte glucose uptake were assayed with and without PBA treatment. Expression of GLUT1, GLUT4, PIP3, pAkt, pPTEN, and PARK-7 was examined by western blot. Plasma membrane expression of GLUT4 was determined using immunofluorescence. Leptin and adiponectin secretion was measure by enzyme-linked immunosorbent assay. PBA treatment, alone or with insulin induction, significantly increased glucose uptake in 3T3-L1 adipocytes. PBA significantly increased GLUT1 but not GLUT4 total protein expression. However, a significant increase in membrane GLUT4 protein translocation was observed. The expression of PIP3 and pAkt increased indicating enhanced PI3k pathway activity. There was a significant decrease in PTEN activity as evident by a rise in the phosphorylated form of this protein. PARK7 protein expression increased with PBA. Treating differentiated adipocytes with PBA did not alter their differentiation status, but decreased the leptin to adiponectin ratio. Conclusion: this study showed that PBA enhances adipocyte glucose uptake potentially through its effect on glucose transporter expression and/or trafficking via the PI3K signaling pathway; suggesting PBA as a possible candidate for the ancillary management of diabetes.     

The interrelation between aPKC and glucose uptake in the skeletal muscle during contraction and insulin stimulation

Contraction and insulin increase glucose uptake in skeletal muscle. While the insulin pathway, better characterized, requires activation of phosphoinositide 3‐kinase (PI3K) and atypical protein kinase (aPKC), muscle contraction seems to share insulin‐activated components to increase glucose uptake. This study aimed to investigate the interrelation between the pathway involved in glucose uptake evoked by insulin and muscle contraction. Isolated muscle of rats was treated with solvent (control), insulin, wortmannin (PI3K inhibitor) and the combination of insulin plus wortmannin. After treatment, muscles were electrically stimulated (contracted) or remained at rest. Glucose transporter 4 (GLUT4) localization, glucose uptake and phospho‐aPKC (aPKC activated form) were assessed. Muscle contraction and insulin increased glucose uptake in all conditions when compared with controls not stimulating an effect that was accompanied by an increase in GLUT4 and of phospho‐aPKC at the muscle membrane. Contracted muscles treated with insulin did not show additive effects on glucose uptake or aPKC activity compared with the response when these stimuli were applied alone. Inhibition of PI3K blocked insulin effect on glucose uptake and aPKC but not in the contractile response. Thus, muscle contraction seems to stimulate aPKC and glucose uptake independently of PI3K. Therefore, aPKC may be a convergence point and a rate limit step in the pathway by which, insulin and contraction, increase glucose uptake in skeletal muscle. Copyright © 2014 John Wiley & Sons, Ltd.     

Akt mediates insulin induction of glucose uptake and up-regulation of GLUT4 gene expression in brown adipocytes

Insulin acutely stimulated glucose uptake in rat primary brown adipocytes in a PI3-kinase-dependent but p70S6-kinase-independent manner. Since Akt represents an intermediate step between these kinases, this study investigated the contribution of Akt to insulin-induced glucose uptake by the use of a chemical compound, ML-9, as well as by transfection with a dominant-negative form of Akt (DeltaAkt). Pretreatment with ML-9 for 10 min completely inhibited insulin stimulation of (1) Akt kinase activity, (2) Akt phosphorylation on the regulatory residue Ser473 but not on Thr308, and (3) mobility shift in Akt1 and Akt2. However, ML-9 did not affect insulin-stimulated PI3-kinase nor PKCzeta activities. In consequence, ML-9 precluded insulin stimulation of glucose uptake and GLUT4 translocation to plasma membrane (determined by Western blot), without any effect on the basal glucose uptake. Moreover, DeltaAkt impaired insulin stimulation of glucose uptake and GFP-tagged GLUT4 translocation to plasma membrane in transiently transfected immortalised brown adipocytes and HeLa cells, respectively. Furthermore, ML-9 treatment for 6 h down-regulated insulin-induced GLUT4 mRNA accumulation, without affecting GLUT1 expression, in a similar fashion as LY294002. Indeed, co-transfection of brown adipocytes with DeltaAkt precluded the transactivation of GLUT4-CAT promoter by insulin in a similar fashion as a dominant-negative form of PI3-kinase. Our results indicate that activation of Akt may be an essential requirement for insulin regulation of glucose uptake and GLUT4 gene expression in brown adipocytes.     

A novel role of neuregulin in skeletal muscle. Neuregulin stimulates glucose uptake, glucose transporter translocation, and transporter expression in muscle cells

Neuregulins regulate the expression of acetylcholine receptor genes and induce development of the neuromuscular junction in muscle. In studying whether neuregulins regulate glucose uptake in muscle, we analyzed the effect of a recombinant neuregulin, (r)heregulin-beta1-(177-244) (HRG), on L6E9 muscle cells, which express the neuregulin receptors ErbB2 and ErbB3. L6E9 responded acutely to HRG by a time- and concentration-dependent stimulation of 2-deoxyglucose uptake. HRG-induced stimulation of glucose transport was additive to the effect of insulin. The acute stimulation of the glucose transport induced by HRG was a consequence of the translocation of GLUT4, GLUT1, and GLUT3 glucose carriers to the cell surface. The effect of HRG on glucose transport was dependent on phosphatidylinositol 3-kinase activity. HRG also stimulated glucose transport in the incubated soleus muscle and was additive to the effect of insulin. Chronic exposure of L6E9 cells to HRG potentiated myogenic differentiation, and under these conditions, glucose transport was also stimulated. The activation of glucose transport after chronic HRG exposure was due to enhanced cell content of GLUT1 and GLUT3 and to increased abundance of these carriers at the plasma membrane. However, under these conditions, GLUT4 expression was markedly down-regulated. Muscle denervation is associated with GLUT1 induction and GLUT4 repression. In this connection, muscle denervation caused a marked increase in the content of ErbB2 and ErbB3 receptors, which occurred in the absence of alterations in neuregulin mRNA levels. This fact suggests that neuregulins regulate glucose transporter expression in denervated muscle. We conclude that neuregulins regulate glucose uptake in L6E9 muscle cells by mechanisms involving the recruitment of glucose transporters to the cell surface and modulation of their expression. Neuregulins may also participate in the adaptations in glucose transport that take place in the muscle fiber after denervation.     

Quercetin suppresses insulin receptor signaling through inhibition of the insulin ligand–receptor binding and therefore impairs cancer cell proliferation

Although the flavonoid quercetin is known to inhibit activation of insulin receptor signaling, the inhibitory mechanism is largely unknown. In this study, we demonstrate that quercetin suppresses insulin induced dimerization of the insulin receptor (IR) through interfering with ligand–receptor interactions, which reduces the phosphorylation of IR and Akt. This inhibitory effect further inhibits insulin stimulated glucose uptake due to decreased cell membrane translocation of glucose transporter 4 (GLUT4), resulting in impaired cancer cell proliferation. The effect of quercetin in inhibiting tumor growth was also evident in an in vivo model, indicating a potential future application for quercetin in the treatment of cancers.     

Design and synthesis of novel histone deacetylase inhibitor derived from nuclear localization signal peptide

We describe herein the synthesis and characterization of a new class of histone deacetylase (HDAC) inhibitors derived from conjugation of a suberoylanilide hydroxamic acid-like aliphatic-hydroxamate pharmacophore to a nuclear localization signal peptide. We found that these conjugates inhibited the histone deacetylase activities of HDACs 1, 2, 6, and 8 in a manner similar to suberoylanilide hydroxamic acid (SAHA). Notably, compound 7b showed a threefold improvement in HDAC 1/2 inhibition, a threefold increase in HDAC 6 selectivity and a twofold increase in HDAC 8 selectivity when compared to SAHA.     

Transient effect of platelet-derived growth factor on GLUT4 translocation in 3T3-L1 adipocytes.

We earlier developed a novel method to detect translocation of the glucose transporter (GLUT) directly and simply using c-MYC epitope-tagged GLUT (GLUTMYC). To define the effect of platelet-derived growth factor (PDGF) on glucose transport in 3T3-L1 adipocytes, we investigated the PDGF- and insulin-induced glucose uptake, translocation of glucose transporters, and phosphatidylinositol (PI) 3-kinase activity in 3T3-L1, 3T3-L1GLUT4MYC, and 3T3-L1GLUT1MYC adipocytes. Insulin and PDGF stimulated glucose uptake by 9-10- and 5.5-6.5-fold, respectively, in both 3T3-L1 and 3T3-L1GLUT4MYC adipocytes. Exogenous GLUT4MYC expression led to enhanced PDGF-induced glucose transport. In 3T3-L1GLUT4MYC adipocytes, insulin and PDGF induced an 8- and 5-fold increase in GLUT4MYC translocation, respectively, determined in a cell-surface anti-c-MYC antibody binding assay. This PDGF-induced GLUT4MYC translocation was further demonstrated with fluorescent detection. In contrast, PDGF stimulated a 2-fold increase of GLUT1MYC translocation and 2.5-fold increase of glucose uptake in 3T3-L1GLUT1MYC adipocytes. The PDGF-induced GLUT4MYC translocation, glucose uptake, and PI 3-kinase activity were maximal (100%) at 5-10 min and thereafter rapidly declined to 40, 30, and 12%, respectively, within 60 min, a time when effects of insulin were maximal. Wortmannin (0.1 microM) abolished PDGF-induced GLUT4MYC translocation and glucose uptake in 3T3-L1GLUT4MYC adipocytes. These results suggest that PDGF can transiently trigger the translocation of GLUT4 and stimulate glucose uptake by translocation of both GLUT4 and GLUT1 in a PI 3-kinase-dependent signaling pathway in 3T3-L1 adipocytes.     

Arsenite stimulated glucose transport in 3T3-L1 adipocytes involves both Glut4 translocation and p38 MAPK activity.

The protein-modifying agent arsenite stimulates glucose uptake in 3T3-L1 adipocytes. In the current study we have analysed the signalling pathways that contribute to this response. By subcellular fractionation we observed that arsenite, like insulin, induces translocation of the GLUT1 and GLUT4 glucose transporters from the low-density membrane fraction to the plasma membrane. Arsenite did not activate early steps of the insulin receptor (IR)-signalling pathway and the response was insensitive to inhibition of phosphatidylinositol-3'-kinase (PI-3') kinase by wortmannin. These findings indicate that the 'classical' IR-IR substrate-PI-3' kinase pathway, that is essential for insulin-induced GLUT4 translocation, is not activated by arsenite. However, arsenite-treatment did induce tyrosine-phosphorylation of c-Cbl. Furthermore, treatment of the cells with the tyrosine kinase inhibitor, tyrphostin A25, abolished arsenite-induced glucose uptake, suggesting that the induction of a tyrosine kinase by arsenite is essential for glucose uptake. Both arsenite and insulin-induced glucose uptake were inhibited partially by the p38 MAP kinase inhibitor, SB203580. This compound had no effect on the magnitude of translocation of glucose transporters indicating that the level of glucose transport is determined by additional factors. Arsenite- and insulin-induced glucose uptake responded in a remarkably similar dose-dependent fashion to a range of pharmacological- and peptide-inhibitors for atypical PKC-lambda, a downstream target of PI-3' kinase signalling in insulin-induced glucose uptake. These data show that in 3T3-L1 adipocytes both arsenite- and insulin-induced signalling pathways project towards a similar cellular response, namely GLUT1 and GLUT4 translocation and glucose uptake. This response to arsenite is not functionally linked to early steps of the IR-IRS-PI-3' kinase pathway, but does coincide with c-Cbl phosphorylation, basal levels of PKC-lambda activity and p38 MAPK activation.     

The Cdk5 inhibitor roscovitine strongly inhibits glucose uptake in 3T3‐L1 adipocytes without altering GLUT4 translocation from internal pools to the cell surface

Glucose entry into mammalian cells is facilitated by a family of glucose transport proteins known as GLUTs. Treatment of 3T3‐L1 adipocytes with the Cdk5 inhibitor roscovitine strongly inhibits insulin‐stimulated/GLUT4‐mediated glucose transport. Inhibition of glucose uptake occurs within 2–6 min of the addition of roscovitine and is slowly reversed. The roscovitine treatment interferes with neither the translocation nor the insertion of GLUT4 into the plasma membrane. These studies support recent evidence showing that insulin‐stimulated Cdk5 is implicated in the regulation of GLUT4‐mediated glucose uptake in 3T3‐L1 adipocytes. J. Cell. Physiol. 220: 238–244, 2009. © 2009 Wiley‐Liss, Inc.     

Depletion of mitochondrial DNA causes impaired glucose utilization and insulin resistance in L6 GLUT4myc myocytes

Mitochondrial dysfunction contributes to a number of human diseases, such as hyperlipidemia, obesity, and diabetes. The mutation and reduction of mitochondrial DNA (mtDNA) have been suggested as factors in the pathogenesis of diabetes. To elucidate the association of cellular mtDNA content and insulin resistance, we produced L6 GLUT4myc myocytes depleted of mtDNA by long term treatment with ethidium bromide. L6 GLUT4myc cells cultured with 0.2 mug/ml ethidium bromide (termed depleted cells) revealed a marked decrease in cellular mtDNA and ATP content, concomitant with a lack of mRNAs encoded by mtDNA. Interestingly, the mtDNA-depleted cells showed a drastic decrease in basal and insulin-stimulated glucose uptake, indicating that L6 GLUT4myc cells develop impaired glucose utilization and insulin resistance. The repletion of mtDNA normalized basal and insulin-stimulated glucose uptake. The mRNA level and expression of insulin receptor substrate (IRS)-1 associated with insulin signaling were decreased by 76 and 90% in the depleted cells, respectively. The plasma membrane (PM) GLUT4 in the basal state was decreased, and the insulin-stimulated GLUT4 translocation to the PM was drastically reduced by mtDNA depletion. Moreover, insulin-stimulated phosphorylation of IRS-1 and Akt2/protein kinase B were drastically reduced in the depleted cells. Those changes returned to control levels after mtDNA repletion. Taken together, our data suggest that PM GLUT4 content and insulin signal pathway intermediates are modulated by the alteration of cellular mtDNA content, and the reductions in the expression of IRS-1 and insulin-stimulated phosphorylation of IRS-1 and Akt2/protein kinase B are associated with insulin resistance in the mtDNA-depleted L6 GLUT4myc myocytes.     

An amino acid mixture is essential to optimize insulin-stimulated glucose uptake and GLUT4 translocation in perfused rodent hindlimb muscle

The purpose of this study was to investigate whether an amino acid mixture increases glucose uptake across perfused rodent hindlimb muscle in the presence and absence of a submaximal insulin concentration, and if the increase in glucose uptake is related to an increase in GLUT4 plasma membrane density. Sprague-Dawley rats were separated into one of four treatment groups: basal, amino acid mixture, submaximal insulin, or amino acid mixture with submaximal insulin. Glucose uptake was greater for both insulin-stimulated treatments compared with the non-insulin-stimulated treatment groups but amino acids only increased glucose uptake in the presence of insulin. Phosphatidylinositol 3-kinase (PI 3-kinase) activity was greater for both insulin-stimulated treatments with amino acids having no additional impact. Akt substrate of 160 kDa (AS160) phosphorylation, however, was increased by the amino acids in the presence of insulin, but not in the absence of insulin. AMPK was unaffected by insulin or amino acids. Plasma membrane GLUT4 protein concentration was greater in the rats treated with insulin compared with no insulin in the perfusate. In the presence of insulin, amino acids increased GLUT4 density in the plasma membrane but had no effect in the absence of insulin. AS160 phosphorylation and plasma membrane GLUT4 density accounted for 76% of the variability in muscle glucose uptake. Collectively, these findings suggest that the beneficial effects of an amino acid mixture on skeletal muscle glucose uptake, in the presence of a submaximal insulin concentration, are due to an increase in AS160 phosphorylation and plasma membrane-associated GLUT4, but independent of PI 3-kinase and AMPK activation.     

Shikonin increases glucose uptake in skeletal muscle cells and improves plasma glucose levels in diabetic Goto-Kakizaki rats

Background

There is considerable interest in identifying compounds that can improve glucose homeostasis. Skeletal muscle, due to its large mass, is the principal organ for glucose disposal in the body and we have investigated here if shikonin, a naphthoquinone derived from the Chinese plant Lithospermum erythrorhizon, increases glucose uptake in skeletal muscle cells.

Methodology/Principal Findings

Shikonin increases glucose uptake in L6 skeletal muscle myotubes, but does not phosphorylate Akt, indicating that in skeletal muscle cells its effect is medaited via a pathway distinct from that used for insulin-stimulated uptake. Furthermore we find no evidence for the involvement of AMP-activated protein kinase in shikonin induced glucose uptake. Shikonin increases the intracellular levels of calcium in these cells and this increase is necessary for shikonin-mediated glucose uptake. Furthermore, we found that shikonin stimulated the translocation of GLUT4 from intracellular vesicles to the cell surface in L6 myoblasts. The beneficial effect of shikonin on glucose uptake was investigated in vivo by measuring plasma glucose levels and insulin sensitivity in spontaneously diabetic Goto-Kakizaki rats. Treatment with shikonin (10 mg/kg intraperitoneally) once daily for 4 days significantly decreased plasma glucose levels. In an insulin sensitivity test (s.c. injection of 0.5 U/kg insulin), plasma glucose levels were significantly lower in the shikonin-treated rats. In conclusion, shikonin increases glucose uptake in muscle cells via an insulin-independent pathway dependent on calcium.

Conclusions/Significance

Shikonin increases glucose uptake in skeletal muscle cells via an insulin-independent pathway dependent on calcium. The beneficial effects of shikonin on glucose metabolism, both in vitro and in vivo, show that the compound possesses properties that make it of considerable interest for developing novel treatment of type 2 diabetes.     

Enhancement of Glucose Uptake in Mouse Skeletal Muscle Cells and Adipocytes by P2Y6 Receptor Agonists

Glucose uptake by peripheral tissues such as skeletal muscles and adipocytes is important in the maintenance of glucose homeostasis. We previously demonstrated that P2Y₆ receptor (P2Y₆R) agonists protect pancreatic islet cells from apoptosis and stimulate glucose-dependent insulin release. Here, we investigated the effects of P2Y₆R activation on glucose uptake in insulin target tissues. An agonist of the P2Y₆R, P¹-(5′-uridine)-P³-(5′-N⁴-methoxycytidine)-triphosphate (MRS2957), significantly increased the uptake of [³H]2-deoxyglucose in mouse C2C12 myotubes and 3T3-L1 adipocytes, and this stimulation was significantly decreased by a selective P2Y₆R antagonist N,N″-1,4-butanediyl-bis[N′-(3-isothiocyanatophenyl)thiourea] (MRS2578). Pre-incubation with Compound C (an inhibitor of 5′-AMP-activated protein kinase, AMPK), or AMPK siRNA abolished the stimulatory effect of MRS2957 on glucose uptake. Also, MRS2957 (60 min incubation) increased recruitment of the facilitated glucose transporter-4 (GLUT4) to the cell membrane, which was blocked by MRS2578. Treatment of C2C12 myotubes with MRS2957 induced significant phosphorylation of AMPK, which increase GLUT4 expression through histone deacetylase (HDAC)5 signaling. Glucose uptake in primary mouse adipocytes from wild-type mice was stimulated upon P2Y₆R activation by either MRS2957 or native agonist UDP, and the P2Y₆R effect was antagonized by MRS2578. However, in adipocytes from P2Y₆R-knockout mice P2Y₆R agonists had no effect on glucose uptake, and there was no change in the glucose uptake by insulin. Our results indicate that the P2Y₆R promotes glucose metabolism in peripheral tissues, which may be mediated through AMPK signaling.     

Maturation of the regulation of GLUT4 activity by p38 MAPK during L6 cell myogenesis

Insulin stimulates glucose uptake in skeletal muscle cells and fat cells by promoting the rapid translocation of GLUT4 glucose transporters to the plasma membrane. Recent work from our laboratory supports the concept that insulin also stimulates the intrinsic activity of GLUT4 through a signaling pathway that includes p38 MAPK. Here we show that regulation of GLUT4 activity by insulin develops during maturation of skeletal muscle cells into myotubes in concert with the ability of insulin to stimulate p38 MAPK. In L6 myotubes expressing GLUT4 that carries an exofacial myc-epitope (L6-GLUT4myc), insulin-stimulated GLUT4myc translocation equals in magnitude the glucose uptake response. Inhibition of p38 MAPK with SB203580 reduces insulin-stimulated glucose uptake without affecting GLUT4myc translocation. In contrast, in myoblasts, the magnitude of insulin-stimulated glucose uptake is significantly lower than that of GLUT4myc translocation and is insensitive to SB203580. Activation of p38 MAPK by insulin is considerably higher in myotubes than in myoblasts, as is the activation of upstream kinases MKK3/MKK6. In contrast, the activation of all three Akt isoforms and GLUT4 translocation are similar in myoblasts and myotubes. Furthermore, GLUT4myc translocation and phosphorylation of regulatory sites on Akt in L6-GLUT4myc myotubes are equally sensitive to insulin, whereas glucose uptake and phosphorylation of regulatory sites on p38 MAPK show lower sensitivity to the hormone. These observations draw additional parallels between Akt and GLUT4 translocation and between p38 MAPK and GLUT4 activation. Regulation of GLUT4 activity by insulin develops upon muscle cell differentiation and correlates with p38 MAPK activation by insulin.     

Thiol-based SAHA analogues as potent histone deacetylase inhibitors

In order to find novel nonhydroxamate histone deacetylase (HDAC) inhibitors, a series of thiol-based compounds modeled after suberoylanilide hydroxamic acid (SAHA) was synthesized, and their inhibitory effect on HDACs was evaluated. Compound 6, in which the hydroxamic acid of SAHA was replaced by a thiol, was found to be as potent as SAHA, and optimization of this series led to the identification of HDAC inhibitors more potent than SAHA.