Gluconeogenetic Enzymes Research Articles

Simultaneous activation of genes encoding urea cycle enzymes and gluconeogenetic enzymes coincides with a corticosterone surge period before metamorphosis in Xenopus laevis.

Amphibian tadpoles are postulated to excrete ammonia as nitrogen metabolites but to shift from ammonotelism to ureotelism during metamorphosis. However, it is unknown whether ureagenesis occurs or plays a functional role before metamorphosis. Here, the mRNA-expression levels of two urea cycle enzymes (carbamoyl phosphate synthetase I [CPSI] and ornithine transcarbamylase [OTC]) were measured beginning with stage-47 Xenopus tadpoles at 5 days post-fertilization (dpf), between the onset of feeding (stage 45, 4 dpf) and metamorphosis (stage 55, 32 dpf). CPSI and OTC expression levels increased significantly from stage 49 (12 dpf). Urea excretion was also detected at stage 47. A transient corticosterone surge peaking at stage 48 was previously reported, supporting the hypothesis that corticosterone can induce CPSI expression in tadpoles, as found in adult frogs and mammals. Stage-46 tadpoles were exposed to a synthetic glucocorticoid, dexamethasone (Dex, 10-500 nM) for 3 days. CPSI mRNA expression was significantly higher in tadpoles exposed to Dex than in tadpoles exposed to the vehicle control. Furthermore, glucocorticoid receptor mRNA expression increased during the pre-metamorphic period. In addition to CPSI and OTC mRNA upregulation, the expression levels of three gluconeogenic enzyme genes (glucose 6-phosphatase, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase 1) increased with the onset of urea synthesis and excretion. These results suggest that simultaneous induction of the urea cycle and gluconeogenic enzymes coincided with a corticosterone surge occurring prior to metamorphosis. These metabolic changes preceding metamorphosis may be closely related to the onset of feeding and nutrient accumulation required for metamorphosis.

Dynamic Metabolic Zonation of the Hepatic Glucose Metabolism Is Accomplished by Sinusoidal Plasma Gradients of Nutrients and Hormones.

Being the central metabolic organ of vertebrates, the liver possesses the largest repertoire of metabolic enzymes among all tissues and organs. Almost all metabolic pathways are resident in the parenchymal cell, hepatocyte, but the pathway capacities may largely differ depending on the localization of hepatocytes within the liver acinus-a phenomenon that is commonly referred to as metabolic zonation. Metabolic zonation is rather dynamic since gene expression patterns of metabolic enzymes may change in response to nutrition, drugs, hormones and pathological states of the liver (e.g., fibrosis and inflammation). This fact has to be ultimately taken into account in mathematical models aiming at the prediction of metabolic liver functions in different physiological and pathological settings. Here we present a spatially resolved kinetic tissue model of hepatic glucose metabolism which includes zone-specific temporal changes of enzyme abundances which are driven by concentration gradients of nutrients, hormones and oxygen along the hepatic sinusoids. As key modulators of enzyme expression we included oxygen, glucose and the hormones insulin and glucagon which also control enzyme activities by cAMP-dependent reversible phosphorylation. Starting with an initially non-zonated model using plasma profiles under fed, fasted and diabetic conditions, zonal patterns of glycolytic and gluconeogenetic enzymes as well as glucose uptake and release rates are created as an emergent property. We show that mechanisms controlling the adaptation of enzyme abundances to varying external conditions necessarily lead to the zonation of hepatic carbohydrate metabolism. To the best of our knowledge, this is the first kinetic tissue model which takes into account in a semi-mechanistic way all relevant levels of enzyme regulation.

Stability of Proteins Out of Service: the GapB Case of Bacillus subtilis.

Bacillus subtilis possesses two glyceraldehyde-3-phosphate dehydrogenases with opposite roles, the glycolytic NAD-dependent GapA and the NADP-dependent GapB enzyme, which is exclusively required during gluconeogenesis but not active under conditions promoting glycolysis. We propose that proteins that are no longer needed will be recognized and proteolyzed by Clp proteases and thereby recycled. To test this postulation, we analyzed the stability of the glycolytic enzyme GapA and the gluconeogenetic enzyme GapB in the presence and absence of glucose. It turned out that GapA remained rather stable under both glycolytic and gluconeogenetic conditions. In contrast, the gluconeogenetic enzyme GapB was degraded after a shift from malate to glucose (i.e., from gluconeogenesis to glycolysis), displaying an estimated half-life of approximately 3 h. Comparative in vivo pulse-chase labeling and immunoprecipitation experiments of the wild-type strain and isogenic mutants identified the ATP-dependent ClpCP protease as the enzyme responsible for the degradation of GapB. However, arginine protein phosphorylation, which was recently described as a general tagging mechanism for protein degradation, did not seem to play a role in GapB proteolysis, because GapB was also degraded in a mcsB mutant, lacking arginine kinase, in the same manner as in the wild type.IMPORTANCE GapB, the NADP-dependent glyceraldehyde-3-phosphosphate dehydrogenase, is essential for B. subtilis under gluconeogenetic conditions. However, after a shift to glycolytic conditions, GapB loses its physiological function within the cell and becomes susceptible to degradation, in contrast to GapA, the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, which remains stable under glycolytic and gluconeogenetic conditions. Subsequently, GapB is proteolyzed in a ClpCP-dependent manner. According to our data, the arginine kinase McsB is not involved as adaptor protein in this process. ClpCP appears to be in charge in the removal of inoperable enzymes in B. subtilis, which is a strictly regulated process in which the precise recognition mechanism(s) remains to be identified.

3-(2-amino-ethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-dione (K145) ameliorated dexamethasone induced hepatic gluconeogenesis through activation of Akt/FoxO1 pathway

3-(2-amino-ethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-dione (K145) is identified as a selective SphK2 inhibitor. It was previously reported as an anti-tumor agent, in this study we demonstrated that K145 was able to regulate hepatic gluconeogenesis and improve glucose intolerance in mice. C57BL/6 mice treated with dexamethasone injection were used as experimental animals, which exhibited impaired glucose tolerance and increased gluconeogenetic enzymes. After K145 treatment, we found that the impairment of glucose tolerance and gluconeogenetic genes mRNA expression were improved. Besides, both in vivo and in votro studies suggested that K145 stimulated insulin dependent Akt phosphorylation and subsequently activates FoxO1 phosphorylation therefore inhibited gluconeogenetic genes expression including PEPCK and G6pase. Our study figures out a potential extent increase the value of developing K145 as therapeutic candidate for diabetes.

Organophosphorus insecticide, monocrotophos, possesses the propensity to induce insulin resistance in rats on chronic exposure 大鼠长期暴露于有机磷杀虫剂—久效磷后可能诱发胰岛素抵抗

Our earlier studies had shown that monocrotophos (MCP), an organophosphorus insecticide (OPI), has the propensity to augment the secondary complications associated with type-1 diabetes. The present study investigates whether rats exposed for prolonged periods to monocrotophos would develop insulin resistance mediated by alteration in glucose homeostasis. Male rats were administered sublethal doses of monocrotophos daily for 180 days. Interim blood samples were collected to measure alteration in blood glucose and lipid profile. Rats were also subjected to glucose and insulin tolerance test and fasting blood glucose and insulin levels were measured to calculate insulin resistance by HOMA-IR method. After 180 days, the rats were also evaluated for pancreatic histology and activities of hepatic gluconeogenetic enzymes. Monocrotophos elicited a gradual and sustained increase in blood glucose and insulin resistance in rats with concomitant glucose intolerance and reduced insulin sensitivity. MCP exposure was also associated with increase in weights of key white adipose pads, activities of gluconeogenesis enzymes and increase in pancreatic islet diameter, all of which led to hyperglycemia, hyperinsulinemia and dyslipidaemia. Long-term exposure of rats to MCP resulted in glucose intolerance with hyperinsulinemia, a hallmark of insulin resistance. Our data suggest that chronic exposure to low doses of monocrotophos, might lead to development of insulin resistance by altering lipid profile and glucose homeostasis.

The alpha-glucosidase inhibitor miglitol affects bile acid metabolism and ameliorates obesity and insulin resistance in diabetic mice

ObjectiveAlpha-glucosidase inhibitors (α-GIs) show various anti-diabetic or anti-obesity effects in addition to the suppression of postprandial hyperglycemia. Based on recent observations that bile acids (BAs) are involved in glucose and energy homeostasis, we examined the ability of miglitol, an α-GI, to influence BA metabolism and ameliorate insulin resistance and obesity. Materials/methodsNSY mice, representing an obese type 2 diabetic model, were fed with a high-fat diet and treated with miglitol for 4 or 12weeks. BAs were quantified in feces, blood from the portal vein or the vena cava and in the liver. The gene expression of type 2 iodothyronine deiodinase (D2) in brown adipose tissues, gluconeogenetic enzymes in the liver and adipokines in epididymal fat was measured, and portal blood glucagon-like peptide-1 (GLP-1) levels, body weight changes, glucose tolerance along with insulin sensitivity were evaluated. ResultsMiglitol significantly increased BAs in both feces and portal blood while the hepatic BA level was reduced. The drug clearly enhanced active GLP-1 secretion into the portal blood and there was a good positive correlation between the active GLP-1 levels and portal blood BA concentrations. D2 expression in brown adipose tended to increase in association with the elevated BA concentrations. Miglitol ameliorated body weight gain, glucose intolerance, insulin resistance and inflammatory adipokine upregulation that were induced by a high-fat diet. ConclusionsCollectively, miglitol substantially affects BA regulation in mice and this novel finding may explain in part the known favourable effects of the drug on diabetes and obesity.

Glucocorticoids induce insulin resistance independent of GR in the liver

Glucocorticoids (GC) are potent anti-inflammatory drugs which are widely used in steroid therapy for autoimmune diseases. But besides their beneficial effects, long term treatment induces insulin resistance leading to type 2 diabetes. Until now, the precise molecular mechanisms how GCs induce insulin resistance are ill-defined. GCs act via the glucocorticoid receptor (GR) that can alter gene expression via two different modes of action: either by protein-protein interaction of the monomeric GR with transcription factors such as AP-1 and NF-κB or by direct binding as a homodimer to glucocorticoid response elements (GREs) in the promoter region of target genes. Traditionally GRE induced expression of gluconeogenetic enzymes in the liver by the GR was assumed to be responsible for insulin resistance due to excessive gluconeogenesis.

Augmentation of hepatic and renal oxidative stress and disrupted glucose homeostasis by monocrotophos in streptozotocin-induced diabetic rats

Several recent studies have demonstrated that organophosphorus insecticides (OPI) possess the potential to disrupt glucose homeostasis leading to hyperglycemia in experimental animals. The propensity of OPI to induce hyperglycemia along with oxidative stress may have far-reaching consequences on diabetic outcomes and associated complications. The primary objective of this study was to assess the potential of monocrotophos (MCP), an extensively used OPI, on hepatic and renal oxidative stress markers and dysregulation of hepatic glucose homeostasis in experimentally induced diabetic rats. Rats rendered diabetic by a single dose of streptozotocin (60 mg/kg b.w) were orally administered MCP (0.9 mg/kg b.w/d for 5 d). Monocrotophos per se caused only a marginal increase in blood glucose levels but significantly elevated the blood glucose levels and also disrupted glucose homeostasis by depleting liver glycogen content and increasing the gluconeogenetic enzyme activities in diabetic rats. Experimentally induced diabetes was also associated with alterations in antioxidant enzymes in liver and kidney. MCP markedly enhanced lipid peroxidation in kidney and altered the enzymatic antioxidant defense mechanisms in both liver and kidney of diabetic rats. Collectively our data provides evidence that MCP has the propensity to augment the oxidative stress and further disrupt glucose homeostasis in diabetic rats.

Waterborne fluoxetine disrupts feeding and energy metabolism in the goldfish Carassius auratus

Fluoxetine (FLX) is one of the most commonly detected pharmaceuticals in wastewater and bioaccumulates in wild-caught fish, especially in brain, liver and muscle tissues. Previous studies indicated that FLX is pharmacologically active in fish species exerting anorexigenic effects, but it is not clear whether waterborne FLX has any potential effects on regulating food intake and energy metabolism. In this study, we investigated the effect of two doses of FLX, an environmental concentration of 540 ng/L, and 100-times this concentration (54 μg/L), on feeding and key metabolic parameters in goldfish. Fish were exposed for a period of 28 days and changes in food intake and body mass were assessed. Pair-fed groups were maintained to discern primary FLX-induced effects from secondary metabolic responses induced by the decreased food intake. Additionally, an untreated control group and a fasted group were used to further compare physiological changes in the context of nutritional status of the animals. Significant decreases in food intake and weight gain were recorded in goldfish exposed to 54 μg/L FLX. Furthermore a significant decrease occurred in circulating glucose levels in the group exposed to 540 ng/L FLX. To elucidate potential mechanisms, we investigated gene expression of feeding neuropeptides in the neuroendocrine brain of goldfish as well as gene expression and enzymatic activity of glycolytic and gluconeogenetic enzymes in liver and muscle tissues. The results confirm brain gene expression patterns in line with potential anorexigenic effects in the hypothalamus, with increased expression in corticotropin-releasing factor (CRF) and decreased expression of neuropeptide Y (NPY). With respect to glucose metabolism, liver gene expression of the gluconeogenic enzyme fructose-1,6-bisphosphatase decreased and muscle hexokinase activity increased in fish exposed to 540 ng/L FLX. Overall, this study demonstrated anorectic properties of FLX at a dose of 54 μg/L FLX and moderate but significant effects on glucose metabolism in goldfish exposed to 540 ng/L FLX. Future studies investigating the importance of these changes in fish are warranted.

Mitochondrial respiration and membrane potential are regulated by the allosteric ATP-inhibition of cytochrome c oxidase

This paper describes the problems of measuring the allosteric ATP-inhibition of cytochrome c oxidase (CcO) in isolated mitochondria. Only by using the ATP-regenerating system phosphoenolpyruvate and pyruvate kinase full ATP-inhibition of CcO could be demonstrated by kinetic measurements. The mechanism was proposed to keep the mitochondrial membrane potential (∆Ψ m) in living cells and tissues at low values (100–140 mV), when the matrix ATP/ADP ratios are high. In contrast, high ∆Ψ m values (180–220 mV) are generally measured in isolated mitochondria. By using a tetraphenyl phosphonium electrode we observed in isolated rat liver mitochondria with glutamate plus malate as substrates a reversible decrease of ∆Ψ m from 233 to 123 mV after addition of phosphoenolpyruvate and pyruvate kinase. The decrease of ∆Ψ m is explained by reversal of the gluconeogenetic enzymes pyruvate carboxylase and phosphoenolpyruvate carboxykinase yielding ATP and GTP, thus increasing the matrix ATP/ADP ratio. With rat heart mitochondria, which lack these enzymes, no decrease of ∆Ψ m was found. From the data we conclude that high matrix ATP/ADP ratios keep ∆Ψ m at low values by the allosteric ATP-inhibition of CcO, thus preventing the generation of reactive oxygen species which could generate degenerative diseases. It is proposed that respiration in living eukaryotic organisms is normally controlled by the ∆Ψ m-independent “allosteric ATP-inhibition of CcO.” Only when the allosteric ATP-inhibition is switched off under stress, respiration is regulated by “respiratory control,” based on ∆Ψ m according to the Mitchell Theory.

TrmB an archael transcriptional regulator

In Pyrococcus furiosus two binding protein–dependent, substrate–specific ABC transporters could be identified, one of it being a trehalose/maltose (TM)–transporter, which is responsible for the uptake of trehalose and maltose. By binding to the respective operator sites TrmB prevents the further binding of transcription factors and subsequent RNA polymerase recruitment, which leads to an inhibition of transcription. With the aid of in vitro transcription assays using purified components of P. furiosus it could be shown that TrmB–mediated inhibition of transcription on both promoters is released by specific carbohydrates with maltose and trehalose being inducers for the TM operon, and maltodextrins as well as sucrose releasing inhibition on the MD promoter.TrmBL1 is homologue to TrmB and is involved in the regulation of genes encoding glycolytic and gluconeogenetic enzymes, respectively, where it recognizes a conserved sequence motif called TGM. It inhibits transcription of glycolytic genes with maltose, maltotriose and fructose as inducers (3). On the other hand TrmBL1 activates the expression of gluconeogenetic genes as well. Since the TGM is not present in the promoter region of the TrmBL1 gene, the TGM is not essential for TrmBL1 target recognition (3).Both TrmB and TrmBL1 share the unique property of recognizing different DNA sequences on different promoters and showing distinctive, promoter–dependent sugar specificity.In vitro transcription assays, EMSA and DNaseI footprinting experiments proved that TrmB and TrmBL1 have at least two different HTH motifs and recognize their respective target promoters with different helices within their N–terminal DNA binding domains.

Curcumin inhibits glucose production in isolated mice hepatocytes

Curcumin is a compound derived from the spice turmeric, and is a potent anti-oxidant, anti-carcinogenic, and anti-hepatotoxic agent. We have investigated the acute effects of curcumin on hepatic glucose production. Gluconeogenesis and glycogenolysis in isolated hepatocytes, and gluconeogenetic enzyme activity after 120 min exposure to curcumin were measured. Hepatic gluconeogenesis from 1 mM pyruvate was inhibited in a concentration-dependent manner, with a maximal decrease of 45% at the concentration of 25 μM. After 120 min exposure to 25 μM curcumin, hepatic gluconeogenesis from 2 mM dihydroxyacetone phosphate and hepatic glycogenolysis were inhibited by 35% and 20%, respectively. Insulin also inhibited hepatic gluconeogenesis from 1 mM pyruvate and inhibited hepatic glycogenolysis in a concentration-dependent manner. Curcumin (25 μM) showed an additive inhibitory effect with insulin on both hepatic gluconeogenesis and glycogenolysis, indicating that curcumin inhibits hepatic glucose production in an insulin-independent manner. After 120 min exposure to 25 μM curcumin, hepatic glucose-6-phosphatase (G6Pase) activity and phosphoenolpyruvate carboxykinase (PEPCK) activity both were inhibited by 30%, but fructose-1,6-bisphosphatase (FBPase) was not reduced. After 120 min exposure to 25 μM curcumin, phosphorylation of AMP kinase α-Thr 172 was increased. Thus, the anti-diabetic effects of curcumin are partly due to a reduction in hepatic glucose production caused by activation of AMP kinase and inhibition of G6Pase activity and PEPCK activity.

Characterization of Hepatic Glucose Metabolism Disorder with the Progress of Diabetes in Male Spontaneously Diabetic Torii Rats

The Spontaneously Diabetic Torii (SDT) rat has recently been established as a new model of non-obese type 2 diabetes. In this study, we examined changes in hepatic glucose metabolism in prediabetic and diabetic SDT rats compared with age-matched control rats. The prediabetic state was confirmed at 16 weeks of age, and the diabetic state was confirmed at 24 and 32 weeks of age. Decreases in glucokinase mRNA levels and activity were observed in the prediabetic state. In this state, glycogen synthase activity and glycogen content were also decreased in the SDT rat. In addition to the above changes, glycogen phosphorylase mRNA and activity were decreased and gluconeogenetic enzyme mRNA levels were significantly increased in the diabetic state. These results indicate there is a great potential that abnormalities in hepatic glucose metabolism play a role in the progression to onset of diabetes. We suggest that the SDT rat is a valuable diabetic model for investigations into mechanisms or causes of progression to diabetes.

Chloroplast phosphoglycerate kinase, a gluconeogenetic enzyme, is required for efficient accumulation of Bamboo mosaic virus

The tertiary structure in the 3′-untranslated region (3′-UTR) of Bamboo mosaic virus (BaMV) RNA is known to be involved in minus-strand RNA synthesis. Proteins found in the RNA-dependent RNA polymerase (RdRp) fraction of BaMV-infected leaves interact with the radio labeled 3′-UTR probe in electrophoretic mobility shift assays (EMSA). Results derived from the ultraviolet (UV) cross-linking competition assays suggested that two cellular factors, p43 and p51, interact specifically with the 3′-UTR of BaMV RNA. p43 and p51 associate with the poly(A) tail and the pseudoknot of the BaMV 3′-UTR, respectively. p51-containing extracts specifically down-regulated minus-strand RNA synthesis when added to in vitro RdRp assays. LC/MS/MS sequencing indicates that p43 is a chloroplast phosphoglycerate kinase (PGK). When the chloroplast PKG levels were knocked down in plants, using virus-induced gene silencing system, the accumulation level of BaMV coat protein was also reduced.

Hyporesponsiveness to Glucocorticoids in Mice Genetically Deficient for the Corticosteroid Binding Globulin

Corticosteroid binding globulin (CBG) is the carrier for glucocorticoids in plasma. The protein is believed to keep the steroids inactive and to regulate the amount of free hormone acting on target tissues (free hormone hypothesis). Here, we generated a mouse model genetically deficient for CBG to test the contribution of the carrier to glucocorticoid action and adrenocortical stress response. The absence of CBG resulted in a lack of corticosterone binding activity in serum and in an approximately 10-fold increase in free corticosterone levels in CBG-null mice, consistent with its role in regulation of circulating free hormone levels. Surprisingly, cbg(-/-) animals did not exhibit features seen in organisms with enhanced glucocorticoid signaling. Rather, the mice exhibited increased activity of the pituitary axis of hormonal control, normal levels of gluconeogenetic enzymes, and fatigue, as well as an aggravated response to septic shock, indicating an inability to appropriately respond to the excess free corticosterone in the absence of CBG. Thus, our data suggest an active role for CBG in bioavailability, local delivery, and/or cellular signal transduction of glucocorticoids that extends beyond a function as a mere cargo transporter.

Ontogenetic development of mRNA levels and binding sites of hepatic β-adrenergic receptors in cattle

Catecholamines regulate glucose metabolism and affect hepatic glucose production mainly through β 2-adrenergic receptors. The hypothesis was tested that gene expression and numbers of hepatic β-adrenergic receptors in calves are influenced by age. Examined developmental stages included pre-term (P0) and full-term (F0) calves immediately after birth, full-term calves on day 5 of life (F5), and veal calves (VC) at the age of 159 days. Expression of β 1-, β 2-, and β 3-adrenergic receptor mRNA was measured by real-time PCR. Receptor binding was quantified by saturation binding assays using ( 3H)-CGP-12177 as a ligand. Abundance of mRNA differed among β-adrenergic subtypes (β 2 > β 1 > β 3; P < 0.01). β 3-Adrenergic receptor mRNA was undetectable in VC. mRNA abundance for β 2-adrenergic receptors was higher ( P < 0.05) in VC than P0 and for β 3-adrenergic receptors was higher ( P < 0.001) in F5 than P0. Binding studies revealed most binding of ( 3H)-CGP-12177 to β 2-adrenergic receptors, which were highest in VC ( P < 0.001) and higher ( P < 0.05) in F5 than P0. Binding sites correlated positively with mRNA levels of β 2-adrenergic receptors ( r = 0.67; P < 0.001), with hepatic activities of phosphoenolpyruvate kinase ( r = 0.73; P < 0.001) and with pyruvate kinase ( r = 0.4; P < 0.05), and with plasma glucose concentrations ( r = 0.5; P < 0.01). In conclusion, mRNA of all three β-adrenergic receptor subtypes were found in liver, with β 2-adrenergic receptors being the dominant subtype. Numbers of β 2-adrenergic receptors increased with age and were mainly regulated at the transcriptional level. Numbers of β-adrenergic receptors were positively associated with hepatic activities of gluconeogenetic enzymes and with plasma glucose levels, suggesting functional importance.

Metabolic Flux Analysis of Candida tropicalis Growing on Xylose in an Oxygen-Limited Chemostat

We have studied the metabolism of xylose by Candida tropicalis in oxygen-limited chemostat. In vitro enzyme assays indicated that glycolytic and gluconeogenetic enzymes are expressed simultaneously facilitating substrate cycling. Enhancing the redox imbalance by cofeeding of formate increased xylose and oxygen consumption rates and ethanol, xylitol, glycerol and CO 2 production rates at steady state. Metabolic flux analysis (MFA) indicated that fructose 6-phosphate is replenished from the pentose phosphate pathway in sufficient amounts without contribution of the gluconeogenetic pathway. Substrate cycling between pyruvate kinase, pyruvate carboxylase and phospho- enol-pyruvate kinase increased ATP turnover. Cofeeding of formate increased the ATP yield. The ATP yields of xylose and xylose–formate cultivation were 6.9 and 8.7 mol ATP/C-mol CDW, respectively, as calculated from the MFA.

Analysis of glucocorticoid signalling by gene targeting

Glucocorticoids are involved in the regulation of numerous physiological processes. The majority of these effects are thought to be mediated by the glucocorticoid receptor (GR) via activation and repression of gene expression. In most cases activation requires binding of a receptor-dimer to DNA while repression is mediated by protein–protein-interaction of GR-monomers with other transcription factors. To analyse the molecular mechanisms that underlie glucocorticoid effects, mouse mutations in the GR gene were generated and analysed. In order to address the role of glucocorticoid receptor signalling during development and in physiology, the gene was disrupted by gene targeting. Most of the mice homozygous for the mutation die shortly after birth due to severe lung atelectasis. Additional defects were found in the adrenals, liver, brain, bone marrow and thymus as well as in the feedback-regulation of the HPA-axis. To approach the question which functions of the GR are regulated by DNA-binding and which by protein–protein-interaction, a point mutation was introduced into the dimerization domain of the GR which is located in the DNA-binding domain. By homologous recombination in ES-cells using the Cre/loxP-system, mice carrying this mutation were generated [GR(dim) mice]. The mice are fully viable although they show impaired inducibility of gluconeogenetic enzymes in liver, defects in longterm renewal of erythroid progenitors and increased expression of POMC and ACTH in the pituitary. However neither in the lung nor the adrenals were any histological abnormalties found. In conclusion GR(dim)-mice represent a valuable tool to further analyse mechanisms of physiological effects of the GR.

Hypoglycemic effect of insulin-like growth factor-1 in mice lacking insulin receptors.

We have investigated the metabolic actions of recombinant human IGF-1 in mice genetically deficient of insulin receptors (IR-/-). After intraperitoneal administration, IGF-1 caused a prompt and sustained decrease of plasma glucose levels in IR-/- mice. Plasma free fatty acid concentrations were unaffected. Interestingly, the effects of IGF-1 were identical in normal mice (IR+/+) and in IR-/- mice. Despite decreased glucose levels, IR-/- mice treated with IGF-1 died within 2-3 d of birth, like sham-treated IR-/- controls. In skeletal muscle, IGF-1 treatment caused phosphorylation of IGF-1 receptors and increased the levels of the phosphatidylinositol-3-kinase p85 subunit detected in antiphosphotyrosine immunoprecipitates, consistent with the possibility that IGF-1 stimulates glucose uptake in a phosphatidylinositol-3-kinase-dependent manner. IGF-1 receptor phosphorylation and coimmunoprecipitation of phosphatidylinositol3-kinase by antiphosphotyrosine antibodies was also observed in liver, and was associated with a decrease in mRNA levels of the key gluconeogenetic enzyme phosphoenolpyruvate carboxykinase. Thus, the effect of IGF-1 on plasma glucose levels may be accounted for by increased peripheral glucose use and by inhibition of hepatic gluconeogenesis. These data indicate that IGF-1 can mimic insulin's effects on glucose metabolism by acting through its own receptor. The failure of IGF-1 to rescue the lethal phenotype due to lack of insulin receptors suggests that IGF-1 receptors cannot effectively mediate all the metabolic actions of insulin receptors.

Differences in binding of epidermal growth factor to liver membranes of TCDD-resistant and TCDD-sensitive rats after a single dose of TCDD

Epidermal growth factor (EGF) receptor has been implied as having a role in certain actions of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). After a single dose of TCDD, the receptor has been shown to be downregulated in several tissues including the liver. Two rat substrains, the Han/Wistar (Kuopio; H/W) rat and the Long-Evans (Turku AB; L-E) rat exhibit over a 1000-fold difference in their sensitivity to the lethal effect of TCDD. This large sensitivity difference was utilized in the current study to investigate whether or not a correlation exists between TCDD lethality and biochemical endpoints related to the hepatic EGF receptor. In the TCDD-sensitive L-E strain both the B max of the EGF receptor and the receptor protein as measured by Western blots, decreased dose and time dependently. Ten days after a lethal dose of TCDD (50 μg/kg), the downregulation was 80%. In the resistant H/W strain, two non-lethal doses were used (50 and 500 μg/kg), since the lethal dose is not known. These doses caused a downregulation already at 4 days after dosing, but no further decrease by day 10. The activity of phosphoenolpyruvate carboxykinase (PEPCK, the main gluconeogenetic enzyme in the liver and a proposed target of TCDD) decreased in H/W rats at least to the same extent as in L-E rats at both 4 and 10 days. It is concluded that EGF receptor downregulation is different in the two rat strains studied, despite the fact that a classical Ah receptor-regulated response (CYP1A1 induction) is similar. The results demonstrate that downregulation of the EGF receptor by TCDD is strain-dependent as well as dose- and time-dependent.