Regulatory Actions of Glucocorticoid Hormones: From Organisms to Mechanisms.

The history of glucocorticoid hormone research is an excellent example of "bedside to bench" investigation. It started with two very insightful clinical observations. Thomas Addison described the syndrome of what came to be known as adrenal hormone insufficiency and Harvey Cushing the syndrome of glucocorticoid hormone excess. These dramatic and life-threatening conditions spawned 150 years of active research that has involved many disciplines; indeed some of the fundamental observations of molecular biology are the result of this work. We have a fundamental knowledge of how glucocorticoids regulate gene transcription, their major effect. The challenge facing current and future investigators is to discern how to use this information to make these powerful therapeutic agents safer and more effective.

Insulin represses phosphoenolpyruvate carboxykinase gene transcription by causing the rapid disruption of an active transcription complex: a potential epigenetic effect.

Insulin represses gluconeogenesis, in part, by inhibiting the transcription of genes that encode rate-determining enzymes, such as phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase). Glucocorticoids stimulate expression of the PEPCK gene but the repressive action of insulin is dominant. Here, we show that treatment of H4IIE hepatoma cells with the synthetic glucocorticoid, dexamethasone (dex), induces the accumulation of glucocorticoid receptor, as well as many transcription factors, coregulators, and RNA polymerase II, on the PEPCK gene promoter. The addition of insulin to dex-treated cells causes the rapid dissociation of glucocorticoid receptor, polymerase II, and several key transcriptional regulators from the PEPCK gene promoter. These changes are temporally related to the reduced rate of PEPCK gene transcription. A similar disruption of the G-6-Pase gene transcription complex was observed. Additionally, insulin causes the rapid demethylation of arginine-17 on histone H3 of both genes. This rapid, insulin-induced, histone demethylation is temporally related to the disruption of the PEPCK and G-6-Pase gene transcription complex, and may be causally related to the mechanism by which insulin represses transcription of these genes.

Compartmentalization of transport and phosphorylation of glucose in a hepatoma cell line.

The first steps of glucose metabolism are carried out by members of the families of GLUTs (glucose transporters) and HKs (hexokinases). Previous experiments using the inhibitor of glucose transport, CB (cytochalasin B), revealed that compartmentalization of GLUTs and HKs is a major factor in the control of glucose uptake in L6 myotubes [Whitesell, Ardehali, Printz, Beechem, Knobel, Piston, Granner, Van Der Meer, Perriott and May (2003) Biochem. J. 370, 47-56]. In the present paper, we evaluate compartmentalization of GLUTs and HKs in a hepatoma cell line, H4IIE, which is characterized by excess GLUT activity, HKI in a particulate and a cytosolic fraction, and insignificant G6Pase (glucose-6-phosphatase) activity. The measured activity of glucose transport exceeded the rate of phosphorylation approx. 30-fold. Treatment with 25 microM CB (K(i) approximately 3 microM in H4IIE cells) paradoxically increased the excess of GLUTs over phosphorylation (GLUTs are inhibited 80%, while phosphorylation is inhibited 98%). The global relationships of the data could be reconciled most simply by a two-compartment model. In this model, phosphorylation of glucose is carried out by a subset of HK molecules supplied by a subset of GLUTs that are more sensitive to CB than the other GLUTs. The agent, DCC (dicyclohexylcarbodi-imide) caused HKI to translocate from the particulate compartment to the cytosolic compartment and potently inhibited glucose phosphorylation. The particulate compartment may represent the mitochondria, to which the more CB-sensitive GLUTs may control the transport of glucose.

Identification of the insulin-regulated interaction of phosphodiesterase 3B with 14-3-3 beta protein.

Phosphodiesterase (PDE)-3B, a major PDE isoform in adipocytes, plays a pivotal role in the antilipolytic action of insulin. Insulin-induced phosphorylation and activation of PDE3B is phosphatidylinositol 3-kinase (PI3-K) and Akt dependent, but the precise mechanism of PDE3B activation is not fully understood. We have identified 14-3-3 beta, a critical scaffolding molecule in signal transduction, as a protein that interacts with PDE3B using the yeast two-hybrid system. The interaction between PDE3B and 14-3-3 beta was then confirmed in vitro. The glutathione S-transferase (GST)-tagged 14-3-3 beta interacts with endogenous PDE3B of rat adipocytes, and this interaction is enhanced when adipocytes are treated with insulin. Coimmunoprecipitation experiments reveal that endogenous PDE3B also associates with endogenous 14-3-3 beta in rat adipocytes, and this interaction is enhanced by insulin. Two different PI3-K inhibitors, wortmannin and Ly294002, block this induction, suggesting that PI3-K is required. Synthetic 15 amino acid peptides of rat PDE3B containing phosphorylated Ser-279 or -302 inhibit this interaction, indicating that the insulin-regulated phosphorylation of these serine residues is involved. Because insulin receptor substrate-1 also associates with 14-3-3, the dimeric 14-3-3 beta could function as a scaffolding protein in the activation of PDE3B by insulin.

Hexokinase II overexpression improves exercise-stimulated but not insulin-stimulated muscle glucose uptake in high-fat-fed C57BL/6J mice.

The aim of the present study was to determine the specific sites of impairment to muscle glucose uptake (MGU) in the insulin-resistant high-fat-fed, conscious C57BL/6J mouse. Wild type (WT) and hexokinase II overexpressing (HK(Tg)) mice were fed either a standard diet or high-fat diet and studied at 4 months of age. A carotid artery and jugular veins had catheters chronically implanted for sampling and infusions, respectively, and mice were allowed to recovery for at least 5 days. Mice were fasted for 5 h and underwent a hyperinsulinemic-euglycemic clamp or saline infusion for 120 min. Separate groups of mice were studied during 30-min sedentary or treadmill exercise periods. A bolus of 2-deoxy[(3)H]glucose was administered 25 min before the end of each study for determination of R(g), an index of tissue-specific glucose uptake. Fasting blood glucose was increased in high-fat compared with standard diet-fed WT (194 +/- 4 vs. 171 +/- 4 mg/dl) but not HK(Tg) (179 +/- 5 vs. 171 +/- 3 mg/dl) mice. High-fat feeding created hyperinsulinemia in both WT and HK(Tg) mice (58 +/- 8 and 77 +/- 15 micro U/ml) compared with standard diet-fed mice (21 +/- 2 and 20 +/- 1 micro U/ml). R(g) was not affected by genotype or diet during either saline infusion or sedentary conditions. HK II overexpression augmented insulin-stimulated R(g) in standard diet-fed but not high-fat-fed mice. Exercise-stimulated R(g) was impaired by high-fat feeding in WT mice, but this impairment was largely rectified in HK(Tg) mice. In conclusion, high-fat feeding impairs both insulin- and exercise-stimulated MGU, but only exercise-stimulated MGU was corrected by HK II overexpression.

Peroxisome proliferator-activated receptor gamma coactivator-1alpha, as a transcription amplifier, is not essential for basal and hormone-induced phosphoenolpyruvate carboxykinase gene expression.

Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the initial step in hepatic gluconeogenesis. In the fasted state, PEPCK gene expression is activated by glucagon (via cAMP) and glucocorticoids. Peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha) plays an important role in energy homeostasis and is considered to be a key regulator of hepatic gluconeogenesis in response to fasting. It is not clear whether PGC-1alpha is obligatory for the activation of the transcription program of gluconeogenic genes, or whether it amplifies an existing process. H4IIE hepatoma cells were used to address this key point. These cells respond appropriately to all of the hormones involved in the regulation of gluconeogenic genes, yet they are devoid of PGC-1alpha. Also, these hormone responses occur in the absence of ongoing protein synthesis, so the necessary complement of transcription factors exists in untreated cells. However, exogenous expression of PGC-1alpha in these cells does enhance basal and hormone-induced expression of the PEPCK and glucose-6-phosphatase genes. Mutational analyses of the PEPCK gene promoter reveal that one element in the PEPCK gene promoter, glucocorticoid accessory factor 3, which binds chicken ovalbumin upstream promoter-transcription factor, is of particular importance. Taken together, these data suggest that, under chronic fasting conditions, i.e. when high levels of cAMP and glucocorticoids induce PGC-1alpha expression, this coactivator markedly amplifies PEPCK gene expression and gluconeogenesis.

Estrogen-related receptor alpha is a repressor of phosphoenolpyruvate carboxykinase gene transcription.

The orphan nuclear receptor estrogen-related receptor (ERR) alpha is a downstream effector of the transcriptional coactivator PGC-1alpha in the regulation of genes important for mitochondrial oxidative capacity. PGC-1alpha is also a potent activator of the transcriptional program required for hepatic gluconeogenesis, and in particular of the key gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK). We report here that the regulatory sequences of the PEPCK gene harbor a functional ERRalpha binding site. However, in contrast to the co-stimulating effects of ERRalpha and PGC-1alpha on mitochondrial gene expression, ERRalpha acts as a transcriptional repressor of the PEPCK gene. Suppression of ERRalpha expression by small interfering RNA leads to reduced binding of ERRalpha to the endogenous PEPCK gene, and an increase in promoter occupancy by PGC-1alpha, suggesting that part of the ERRalpha function at this gene is to antagonize the action of PGC-1alpha. In agreement with the in vitro studies, animals that lack ERRalpha show increased expression of gluconeogenic genes, including PEPCK and glycerol kinase, but decreased expression of mitochondrial genes, such as ATP synthase subunit beta and cytochrome c-1. Our findings suggest that ERRalpha has opposing effects on genes important for mitochondrial oxidative capacity and gluconeogenesis. The different functions of ERRalpha in the regulation of these pathways suggest that enhancing ERRalpha activity could have beneficial effects on glucose metabolism in diabetic subjects by two distinct mechanisms: increasing mitochondrial oxidative capacity in peripheral tissues and liver, and suppressing hepatic glucose production.

SRC-1 and GRIP1 coactivate transcription with hepatocyte nuclear factor 4.

Hepatocyte nuclear factor-4 (HNF4), a member of the nuclear receptor superfamily, plays an important role in tissue-specific gene expression, including genes involved in hepatic glucose metabolism. In this study, we show that SRC-1 and GRIP1, which act as coactivators for various nuclear receptors, associate with HNF4 in vivo and enhance its transactivation potential. The AF-2 domain of HNF4 is required for this interaction and for the potentiation of transcriptional activity by these coactivators. p300 can also serve as a coactivator with HNF4, and it synergizes with SRC-1 to further augment the activity of HNF4. HNF4 is also a key regulator of the expression of hepatocyte nuclear factor-1 (HNF1). The overexpression of SRC-1 or GRIP1 enhances expression from a HNF1 gene promoter-reporter in HepG2 hepatoma cells, and this requires an intact HNF4-binding site in the HNF1 gene promoter. Type 1 maturity onset diabetes of young (MODY), which is characterized by abnormal glucose-mediated insulin secretion, is caused by mutations of the HNF4 gene. A mutation of the HNF4-binding site in the HNF1 gene promoter has also been associated with MODY. Thus, HNF4 is involved in the regulation of glucose homeostasis at several levels and along with the SRC-1, GRIP1, and p300 may play an important role in the pathophysiology of non-insulin-dependent diabetes mellitus.

A point mutation of the AF2 transactivation domain of the glucocorticoid receptor disrupts its interaction with steroid receptor coactivator 1.

Glucocorticoids cause a 10-fold increase in hepatic phosphoenolpyruvate carboxykinase (PEPCK) gene transcription through two low affinity glucocorticoid receptor (GR) binding sites and a complex array of accessory factor DNA elements and associated proteins. To analyze how co-activators interact with the GR in this context, we took advantage of the C656G GR mutant that binds ligand with very high affinity. This GR activates PEPCK gene transcription at a 500-fold lower dexamethasone concentration than does wild type GR. Transfected C656G GR containing additional mutations or deletions was tested on PEPCK gene expression in H4IIE hepatoma cells. We found that the AF2 domain is the only one of the three defined transactivation domains in GR that is required for PEPCK gene expression and that mutation of this domain disrupts the direct interaction of GR with steroid receptor coactivator 1 (SRC-1). These data help define the functional interaction between GR and SRC-1 and further define the role of the GR in glucocorticoid-mediated expression of the PEPCK gene.

Insulin inhibits hepatocellular glucose production by utilizing liver-enriched transcriptional inhibitory protein to disrupt the association of CREB-binding protein and RNA polymerase II with the phosphoenolpyruvate carboxykinase gene promoter.

Hormones regulate glucose homeostasis, in part, by controlling the expression of gluconeogenic enzymes, such as phosphoenolpyruvate carboxykinase (PEPCK). Insulin and glucocorticoids reciprocally regulate PEPCK expression primarily at the level of gene transcription. We demonstrate here that glucocorticoids promote, whereas insulin disrupts, the association of CREB-binding protein (CBP) and RNA polymerase II with the hepatic PEPCK gene promoter in vivo. We also show that accessory factors, such as CCAAT/enhancer-binding protein beta (C/EBP beta), can recruit CBP to drive transcription. Insulin increases protein levels of liver-enriched transcriptional inhibitory protein (LIP), an inhibitory form of C/EBP beta, in a phosphatidylinositol 3-kinase-dependent manner. LIP concomitantly replaces liver-enriched transcriptional activator protein on the PEPCK gene promoter, which can abrogate the recruitment of CBP and polymerase II, culminating in the repression of PEPCK expression and the attenuation of hepatocellular glucose production.

Epigallocatechin gallate, a constituent of green tea, represses hepatic glucose production.

Herbs have been used for medicinal purposes, including the treatment of diabetes, for centuries. Plants containing flavonoids are used to treat diabetes in Indian medicine and the green tea flavonoid, epigallocatechin gallate (EGCG), is reported to have glucose-lowering effects in animals. We show here that the regulation of hepatic glucose production is decreased by EGCG. Furthermore, like insulin, EGCG increases tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 (IRS-1), and it reduces phosphoenolpyruvate carboxykinase gene expression in a phosphoinositide 3-kinase-dependent manner. EGCG also mimics insulin by increasing phosphoinositide 3-kinase, mitogen-activated protein kinase, and p70(s6k) activity. EGCG differs from insulin, however, in that it affects several insulin-activated kinases with slower kinetics. Furthermore, EGCG regulates genes that encode gluconeogenic enzymes and protein-tyrosine phosphorylation by modulating the redox state of the cell. These results demonstrate that changes in the redox state may have beneficial effects for the treatment of diabetes and suggest a potential role for EGCG, or derivatives, as an antidiabetic agent.

The synergistic effect of dexamethasone and all-trans-retinoic acid on hepatic phosphoenolpyruvate carboxykinase gene expression involves the coactivator p300.

Activation of phosphoenolpyruvate carboxykinase (PEPCK) gene transcription in response to all-trans-retinoic acid (RA) or a glucocorticoid such as dexamethasone (Dex) requires a distinct arrangement of DNA-response elements and their cognate transcription activators on the gene promoter. Two of the accessory factor-binding elements involved in the Dex response (gAF1 and gAF3) coincide with the DNA-response elements involved in the RA response. We demonstrate here that the combination of Dex/RA has a synergistic effect on endogenous PEPCK gene expression in rat hepatocytes and H4IIE hepatoma cells. Reporter gene studies show that the gAF3 element and one of the two glucocorticoid receptor-binding elements (GR1) are most important for this effect. Chromatin immunoprecipitation assays revealed that when H4IIE cells were treated with Dex/RA, ligand-activated retinoic acid receptors (retinoic acid receptor/retinoid X receptor) and glucocorticoid receptors are recruited to this gene promoter, as are the transcription coregulators p300, CREB-binding protein, p/CIP, and SRC-1. Notably, the recruitment of p300 and RNA polymerase II to the PEPCK promoter is increased by the combined Dex/RA treatment compared with Dex or RA treatment alone. The functional importance of p300 in the Dex/RA response is illustrated by the observation that selective reduction of this coactivator, but not that of CREB-binding protein, abolishes the synergistic effect in H4IIE cells.

Elements of the glucocorticoid and retinoic acid response units are involved in cAMP-mediated expression of the PEPCK gene.

Although many genes are regulated by the concerted action of several hormones, hormonal signaling to gene promoters has generally been studied one hormone at a time. The phosphoenolpyruvate carboxykinase (PEPCK) gene is a case in point. Transcription of this gene is induced by glucagon (acting by the second messenger, cAMP), glucocorticoids, and retinoic acid, and it is dominantly repressed by insulin. These hormonal responses require the presence of different hormone response units (HRUs), which consist of constellations of DNA elements and associated transcription factors. These include the glucocorticoid response unit (GRU), cAMP response unit (CRU), retinoic acid response unit (RARU), and the insulin response unit. HRUs are known to have functional overlap. In particular, the cAMP response element of the CRU is also a component of the GRU. The purpose of this study was to determine whether known GRU or RARU elements or transcription factors function as components of the CRU. We show here that the glucocorticoid accessory factor binding site 1 and glucocorticoid accessory factor binding site 3 elements, which are components of both the GRU and RARU, are an important part of the CRU. Furthermore, we find that the transcription factor, chicken ovalbumin upstream promoter-transcription factor, and two coactivators, cAMP response element-binding protein-binding protein and steroid receptor coactivator-1, participate in both the cAMP and glucocorticoid responses. This provides a further illustration of how the PEPCK gene promoter integrates different hormone responses through overlapping HRUs that utilize some of the same transcription factors and coactivators.

Control of glucose phosphorylation in L6 myotubes by compartmentalization, hexokinase, and glucose transport.

In muscle, insulin enhances influx of glucose and its conversion to glucose 6-phosphate (G6P) by hexokinase (HK). While effects of insulin on glucose transport have been demonstrated, its effect on the activity of HK of cells has not. In L6 myotubes treated for 24 h with insulin there was increased expression of the HK isoform, HKII, and increased glucose phosphorylation without a concomitant increase in glucose transport, indirectly suggesting that phosphorylation of glucose was a target of insulin action [Osawa, Printz, Whitesell and Granner (1995) Diabetes 44, 1426-1432]. In the present work the same treatment led to a 2-fold rise in G6P, suggesting that transport and/or HK were important targets of insulin action. We used a method to identify the site of rate control involving the specificity of phosphorylation towards 2-deoxy-[1-14C]glucose and D-[2-3H]glucose. Glucose transport does not greatly discriminate between these two tracers while HK shows increased specificity for glucose. Specificity of the glucose phosphorylation of the cells increased with addition of insulin and when extracellular glucose was raised. Specificity was reduced at low glucose concentrations or when the inhibitor of transport, cytochalasin B, was added. We conclude that transport and HK share nearly equal control over glucose phosphorylation in these cells. A computer program was used to test models for compatibility with the different types of experiments. The predicted intracellular glucose and transport rates associated with phosphorylation activity were lower than their measured values for the whole cell. In the most likely model, 15+/-4% of the glucose transporters serve a proportionate volume of the cytoplasm. Insulin activation of glucose phosphorylation might then result from stimulation of these transporters together with HK recruitment or relief from inhibition by G6P.