Glucose Induction Research Articles

Effect of recombinant growth hormone and chromium picolinate on cytokine production and growth performance in swine.

The effect of dietary chromium picolinate (CrP) and recombinant porcine growth hormone, somatotropin (rPST) administration on growth performance and cytokine production in Landrace-Poland China gilts was determined using a 2 by 2 treatment array. Treatments were: (1) control (basal diet), (2) CrP-supplemented diet (basal diet + 300 micrograms Cr3+/kg diet as CrP), (3) rPST (100 pg/kg body weight/day), and (4) rPST+CrP. CrP-supplemented diets were fed beginning at 20 kg body weight through 90 kg. Administration of rPST was begun at 60 kg weight and continued through 90 kg. All rPST treated pigs demonstrated improvements in growth performance versus controls. Pigs given CrP-supplemented diets showed no differences in growth performance. At 90 kg, pigs were challenged with endotoxin (lipopolysaccharide, 0.2 microgram/kg i.v.). Blood samples were collected at 0, 1, and 3 h postchallenge. Plasma IL-6 levels increased from 23 U/ml at time 0 to 1,927 U/ml at 3 h for control swine. Swine from the CrP treatment group had IL-6 levels of 8,130 U/ml at 3 h post-LPS. There were no differences in plasma IL-6 from pigs in the rPST and rPST+CrP treatment groups compared to the controls. Endotoxin challenge had no effect on either blood glucose levels or induction of TNF-alpha in any treatment group. PBMC from CrP-treated animals produced more IL-2 than peripheral blood mononuclear cells from all other groups.

Four additional genes in the sigB operon of Bacillus subtilis that control activity of the general stress factor sigma B in response to environmental signals

sigma B of the gram-positive bacterium Bacillus subtilis is an alternative transcription factor activated by a variety of environmental stresses, including the stress imposed upon entry into the stationary growth phase. Previous reports have shown that this stationary-phase activation is enhanced when cells are grown in rich medium containing glucose and glutamine. The sigma B structural gene, sigB, lies in an operon with three other genes whose products have been shown to control sigma B activity in response to environmental stress. However, none of these is sufficient to explain the enhanced stationary-phase activation of sigma B in response to glucose. We show here that the four genes previously identified in the sigB operon constitute the downstream half of an eight-gene operon. The complete sigB operon is preceded by a sigma A-like promoter (PA) and has the order PA-orfR-orfS-orfT-orfU-PB-rsbV-rsbW-sig B-rsbX, where rsb stands for regulator of sigma-B and the previously identified sigma B-dependent promoter (PB) is an internal promoter preceding the downstream four-gene cluster. Although the genes downstream of PB were also transcribed by polymerase activity originating at PA, this transcription into the downstream cluster was not essential for normal induction of a sigma B-dependent ctc-lacZ fusion. However, deletion of all four upstream open reading frames was found to interfere with induction of the ctc-lacZ fusion in response to glucose. Additional deletion analysis and complementation studies showed that orfU was required for full glucose induction of sigma B-dependent genes. orfU encodes a trans-acting, positive factor with significant sequence identity to the RsbX negative regulator of sigma B. On the basis of these results, we rename orfU as rsbU to symbolize the regulatory role of its product.

The pancreatic β-cell glucose sensor

Pancreatic β cells secrete insulin in response to an increase in the level of blood glucose above 5m m, which is characteristic of the fasting state. Glucose metabolism is essential for glucose sensing, and both the high- K m glucose transporter GLUT2 and the high- K m glucose phosphorylating enzyme glucokinase have been implicated in coupling insulin secretion to extracellular glucose levels. Experiments in isolated islets, immortalized β-cell lines and transgenic animals, together with findings in humans with maturity-onset diabetes of the young, indicate that the primary β-cell glucose sensor is glucokinase. Although the level of GLUT2 is frequently reduced in animal models of type II diabetes, GLUT2 does not limit glucose metabolism in β cells and does not appear to regulate glucose induction of insulin secretion.

Carbon catabolite repression in Kluyveromyces lactis: isolation and characterization of the KIDLD gene encoding the mitochondrial enzyme D-lactate ferricytochrome c oxidoreductase.

In the "petite-negative" yeast Kluyveromyces lactis carbon catabolite repression of some cytoplasmic enzymes has been observed. However, with respect to mitochondrial enzymes, in K. lactis, unlike the case in the "petite-positive" yeast Saccharomyces cerevisiae, growth on fermentable carbon sources does not cause repression of respiratory enzymes. In this paper data are reported on carbon catabolite repression of mitochondrial enzymes in K. lactis, in particular on L- and D-lactate ferricytochrome c oxidoreductase (LCR). The L- and D-LCR (E.C. 1123, E.C. 1124) in yeast catalyze the stereospecific oxidation of D and L isomers of lactate to pyruvate. This pathway is linked to the respiratory chain, cytochrome c being the electron acceptor of the redox reaction. We demonstrate that the level of mitochondrial D- and L-LCR is controlled by the carbon source, being induced by the substrate lactate and catabolite-repressed by glucose. We cloned the structural gene for D-LCR of K. lactis (KlDLD), by complementation of growth on D,L-lactate in the S. cerevisiae strain WWF18-3D, carrying both a CYB2 disruption and the dld mutation. From the sequence analysis an open reading frame was identified that could encode a polypeptide of 579 amino acids, corresponding to a calculated molecular weight of 63,484 Da. Analysis of mRNA expression indicated that glucose repression and induction by lactate are exerted at the transcriptional level.

Glucose induces oscillatory Ca2+ signalling and insulin release in human pancreatic beta cells.

Mechanisms of pulsatile insulin release in man were explored by studying the induction of oscillatory Ca2+ signals in individual beta cells and islets isolated from the human pancreas. Evidence was provided for a glucose-induced closure of ATP-regulated K+ channels, resulting in voltage-dependent entry of Ca2+. The observation of step-wise increases of capacitance in response to depolarizing pulses suggests that an enhanced influx of Ca2+ is an effective means of stimulating the secretory activity of the isolated human beta cell. Activation of muscarinic receptors (1-10 mumol/l carbachol) and of purinergic P2 receptors (0.01-1 mumol/l ATP) resulted in repetitive transients followed by sustained elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i). Periodic mobilisation of intracellular calcium was seen also when injecting 100 mumol/l GTP-gamma-S into beta cells hyperpolarized to -70 mV. Individual beta cells responded to glucose and tolbutamide with increases of [Ca2+]i, manifested either as large amplitude oscillations (frequency 0.1-0.5/min) or as a sustained elevation. Glucose regulation was based on sudden transitions between the basal and the two alternative states of raised [Ca2+]i at threshold concentrations of the sugar characteristic for the individual beta cells. The oscillatory characteristics of coupled cells were determined collectively rather than by particular pacemaker cells. In intact pancreatic islets the glucose induction of well-synchronized [Ca2+]i oscillations had its counterpart in 2-5 min pulses of insulin. Each of these pulses could be resolved into regularly occurring short insulin transients. It is concluded that glucose stimulation of insulin release in man is determined by the number of beta cells entering into a state with Ca(2+)-induced secretory pulses.

Expression of high-affinity glucose transport protein Hxt2p of Saccharomyces cerevisiae is both repressed and induced by glucose and appears to be regulated posttranslationally

Expression of putative high-affinity glucose transport protein Hxt2p of Saccharomyces cerevisiae was repressed 15- to 20-fold in high concentrations of glucose or fructose. S. cerevisiae with either the ssn6-delta 9 or the hxk2-delta 1::URA3 mutation, each of which relieves glucose repression, exhibited high Hxt2p expression in both 2.0% glucose (normally repressing) and 0.05% glucose (normally derepressing) while S. cerevisiae with the snf1-delta 10 mutation, which causes constitutive repression, did not detectably express Hxt2p in either glucose concentration. In addition to repressing at high concentrations, glucose or fructose is required for induction of Hxt2p expression. Hxt2p was not expressed by wild-type S. cerevisiae in media containing only ethanol or galactose as carbon and energy source but was expressed if glucose was added. An hxk2-delta 1::URA3 mutant did not detectably express Hxt2p in ethanol or galactose, but an ssn6-delta9 mutant did highly express Hxt2p in both carbon sources. Thus, simple relief of glucose repression as occurs with hxk2 null mutants is insufficient for high-level Hxt2p expression. Mutation of ssn6, a general transcriptional repressor, does lead to Hxt2p expression in the absence of glucose induction, suggesting relief of an additional negative regulatory system. High expression of Hxt2p does not always result in HXT2-dependent high-affinity transport, implying that Hxt2p activity is regulated posttranslationally. In the high glucose condition for the ssn6 mutant, high-affinity glucose transport is derepressed. Deletion of the HXT2 locus does not diminish this level of transport. However, high-affinity glucose transport is diminished in the ssn6-delta9 hxt2 delta1 double mutant compared with ssn6-delta9 alone in low glucose. Thus, while constitutively expressed in ssn6 mutants, Hxt2p only appears to be active as a transporter under low-glucose conditions. Similarly, Hxt2p was found to be expressed under low-glucose conditions in an snf3 mutant which does not display high-affinity uptake. This finding suggests that SNF3 may be involved in the posttranslational regulation of Hxt2p.

Two glucose-signaling pathways in S14 gene transcription in primary hepatocytes: a common role of protein phosphorylation.

Transcription of the rat S14 gene is induced in response to increased carbohydrate metabolism in the liver. Because carbohydrate-induced changes in lipogenesis are mediated in part by changes in phosphorylation of multiple proteins, we investigated the role of protein phosphorylation on transcriptional regulation of the two carbohydrate response elements, a thyroid hormone receptor-independent carbohydrate response element and a thyroid receptor-dependent glucose response element located up-stream of the S14 gene. S14 reporter constructs were transiently transfected into rat primary hepatocytes and incubated with the protein or phosphatase inhibitor okadaic acid calyculin-A, or one of several protein kinase activators. Low dose okadaic acid blocked glucose induction from both elements without inhibiting glucose metabolism. Calyculin-A, a preferential phosphatase-1 inhibitor, only blocked the glucose response when glucose metabolism was inhibited. The protein kinase-C activator, 12-myristate 13-acetate, did not change the glucose responses, whereas the protein kinase-A activator, 8-(4-chlorophenylthio)cAMP, inhibited S14 transcription by inhibiting glucose metabolism. In contrast, the calcium ionophore A23187, a calmodulin kinase activator, mimicked the effect of low dose okadaic acid, but had no effect on glucose metabolism. We conclude that protein phosphatase-2A and calmodulin kinases may be involved in the glucose signaling pathway of the S14 gene. A similar phosphorylation step may be involved in the two distinct glucose response pathways.

Definition of the carbohydrate response element of the rat S14 gene. Context of the CACGTG motif determines the specificity of carbohydrate regulation.

Transcription of the S14 gene in primary hepatocytes is stimulated in response to increased carbohydrate metabolism. We have demonstrated previously that a 30-base pair (bp) segment of the S14 gene from -1457 to -1428 is a carbohydrate response element (ChoRE). This element contains a (5')CACGTG motif that is essential for control. DNase I footprinting experiments with liver nuclear extract revealed two factors binding within the S14 ChoRE. In transient transfection experiments, mutation of the upstream site between -1457 and -1450 did not affect the response to elevated glucose, whereas the downstream 21-bp site between -1448 and -1428 was sufficient to mediate the glucose induction. Electrophoretic mobility shift assays indicated that the hepatic factor binding to this site in vitro is closely related or identical to the major late transcription factor (MLTF). However, replacement of the 21-bp S14 ChoRE with the authentic MLTF binding site from the adenovirus major late promoter failed to elicit the glucose response. By systematically exchanging bases between the functional S14 and nonresponsive adenovirus sites, the sequence (5')CACGTGNNNGCC was found to be essential for carbohydrate regulation. A segment containing this specific motif from the rat fatty acid synthase gene, another carbohydrate-responsive gene in hepatocytes, conferred a carbohydrate response when linked to the S14 promoter. Thus, the context of the CACGTG motif provides the specificity for regulation by carbohydrate metabolism.

Characterisation of PDC2, a gene necessary for high level expression of pyruvate decarboxylase structural genes in Saccharomyces cerevlsiae

The regulatory gene PDC2 was identified in a screen for mutations affecting pyruvate decarboxylase activity in yeast. I have cloned and sequenced this gene. The predicted protein of 925 amino acids has no homology to any sequence in the databases. However, the protein sequence is rich in asparagine and serine residues, as is often found for transcriptional regulators. The PDC2 deletion mutant exhibits a phenotype very similar to, but more severe than that of the point mutant: a strongly reduced pyruvate decarboxylase specific activity, slow, respiration-dependent growth on glucose, and accumulation of pyruvate. The activity of other glycolytic enzymes seems to be unaffected by the pdc2 delta mutation. Synthesis of pyruvate decarboxylase is regulated by PDC2 at the transcriptional level. Expression of the major structural gene for pyruvate decarboxylase, PDC1, is strongly reduced in pdc2 delta mutants. Transcription of the generally more weakly expressed PDC5 gene appears to be entirely abolished. However, glucose induction of pyruvate decarboxylase synthesis is unaffected. Thus, PDC2 is either important for a high basal level of PDC gene expression or it plays a positive role in the autoregulation that controls expression of PDC1 and PDC5.

The thyroid hormone receptor can unmask a glucose response in the S14 gene.

The rat S14 gene is synergistically regulated by thyroid hormone and carbohydrates. The 5'-flanking region of this gene contains both carbohydrate and thyroid hormone response elements (TREs). We recently located the carbohydrate response element (CHORE) between -1583 and -1069 bases from the start site of transcription that is required for glucose induction of this gene. We have now studied the relationship between the effects of CHORE, TREs, and thyroid hormone receptor on the carbohydrate regulation of transcription. We used a transient cotransfection assay with a plasmid containing 5 kilobases (kb) of the up-stream region from the S14 gene ligated to a luciferase reporter and a thyroid hormone receptor expression vector. Unliganded thyroid hormone receptor reduced the response of the reporter to glucose. Ligand-bound thyroid hormone receptor significantly enhanced the glucose response from the 5-kb promoter. While deletion of the CHORE (5-kb delta CHORE) from the 5-kb S14 promoter eliminated the glucose response without cotransfection of the thyroid receptor, deletion of the CHORE did not affect the glucose response when both thyroid hormone receptor was cotransfected and thyroid hormone was present. However, deletion of the TREs (-3261 to -2110) from the 5-kb delta CHORE construct abolished the effect. These data suggest that the thyroid hormone receptor functions as or in association with a trans-acting glucose response factor. The site of interaction between thyroid hormone and glucose is localized to an area of the gene that is different from the previously described CHORE.

Carbohydrate regulation of the rat L-type pyruvate kinase gene requires two nuclear factors: LF-A1 and a member of the c-myc family

Transcription of the L-type pyruvate kinase (L-PK) gene is induced in response to increased carbohydrate metabolism in the liver. We have demonstrated previously that a segment of the 5'-flanking region of the L-PK gene between -183 and -96 is necessary and sufficient for the glucose response in primary hepatocytes. To explore the protein factors that are involved in carbohydrate regulation, we have performed mutational analyses and in vitro binding studies of this segment. Sequences critical for the glucose response were mapped from -171 to -124. This segment contains the consensus binding sites for two nuclear transcription factors: LF-A1 and MLTF. Both factors are capable of binding to the corresponding L-PK sites in vitro. Mutational and functional analyses indicated that LF-A1 is indeed involved in glucose induction of the L-PK gene. The PK MLTF-like site consists of two imperfect CACGTG motifs, the core binding site for a family of transcription factors related to c-myc. Unexpectedly, mutations in either motif that resulted in defective glucose stimulation retained in vitro binding to MLTF. Furthermore, an authentic MLTF binding site from the adenovirus major late promoter was not functionally interchangeable with the natural sequence. These data indicate that binding of MLTF, in presence of LF-A1, is not capable of supporting the glucose response. Conversion of either imperfect motif to CACGTG within the context of the mutations disrupting the opposite site restored the response to elevated glucose. Thus, the factor that recognizes the PK MLTF-like site and participates in mediating the carbohydrate response of the L-PK gene appears to be a member of the c-myc family distinct from MLTF.

The growth and signalling defects of the ggs1 (fdp1/byp1) deletion mutant on glucose are suppressed by a deletion of the gene encoding hexokinase PII.

Yeast cells defective in the GGS1 (FDP1/BYP1) gene are unable to adapt to fermentative metabolism. When glucose is added to derepressed ggs1 cells, growth is arrested due to an overloading of glycolysis with sugar phosphates which eventually leads to a depletion of phosphate in the cytosol. Ggs1 mutants lack all glucose-induced regulatory effects investigated so far. We reduced hexokinase activity in ggs1 strains by deleting the gene HXK2 encoding hexokinase PII. The double mutant ggs1 delta, hxk2 delta grew on glucose. This is in agreement with the idea that an inability of the ggs1 mutants to regulate the initiation of glycolysis causes the growth deficiency. However, the ggs1 delta, hxk2 delta double mutant still displayed a high level of glucose-6-phosphate as well as the rapid appearance of free intracellular glucose. This is consistent with our previous model suggesting an involvement of GGS1 in transport-associated sugar phosphorylation. Glucose induction of pyruvate decarboxylase, glucose-induced cAMP-signalling, glucose-induced inactivation of fructose-1,6-bisphosphatase, and glucose-induced activation of the potassium transport system, all deficient in ggs1 mutants, were restored by the deletion of HXK2. However, both the ggs1 delta and the ggs1 delta, hk2 delta mutant lack detectable trehalose and trehalose-6-phosphate synthase activity. Trehalose is undetectable even in ggs1 delta strains with strongly reduced activity of protein kinase A which normally causes a very high trehalose content. These data fit with the recent cloning of GGS1 as a subunit of the trehalose-6-phosphate synthase/phosphatase complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Physiologic concentrations of glucose regulate fatty acid synthase activity in HepG2 cells by mediating fatty acid synthase mRNA stability.

Carbohydrate feeding of animals results in striking increases in hepatic fatty acid synthesis but much of this induction is presumed to be hormone mediated. To clarify the mechanisms responsible for the specific effect of carbohydrate on fatty acid synthesis, the effects of D-glucose on the expression of human fatty acid synthase (FAS) in HepG2 cells cultured in serum-free medium were studied. Northern blots of total RNA from these cells showed a single FAS mRNA band of 9.3 kilobases that was increased 2.7-5.4-fold in the presence of D-glucose. Lactate and citrate but not L-glucose mimicked this effect. Glucose induction of FAS mRNA was time- and concentration-dependent and most of the increase in FAS message was detected within the range of physiologic glucose concentrations. Glucose induction of FAS mRNA, protein synthetic rate, and enzyme activity were similar suggesting that glucose regulates FAS expression at a pretranslational level in this system. Transcription run-off experiments showed that glucose feeding was associated with no change in the FAS transcription initiation rate despite a 5-fold increase in FAS mRNA. FAS mRNA stability as determined by actinomycin D chase was 7-fold greater in the presence of glucose. Differences between FAS mRNA levels in cells grown in the presence versus the absence of glucose were inhibited by cycloheximide but not puromycin, suggesting that glucose regulation of FAS mRNA stability is not dependent on translation. Glucose, at physiologic concentrations and in the absence of hormones, appears to regulate FAS gene expression in HepG2 cells predominantly by mediating FAS mRNA stability.

Expression and function of GLUT-1 and GLUT-2 glucose transporter isoforms in cells of cultured rat pancreatic islets.

We have previously investigated glucose induction of glucokinase, glucose usage and insulin release in isolated cultured rat pancreatic islets (Liang, Y., Najafit, H., Smith, R. M., Zimmerman, E. C., Magnuson, M. A., Tal, M., and Mastchinsky, F. M. (1992) Diabetes (1992) 41, 792-806). Here we studied the expression and function of GLUT-1 and GLUT-2 glucose transporter isoforms, using the same system, i.e. isolated pancreatic rat islets immediately after isolation or cultured in the presence of 3 or 30 mM glucose for as long as 10 days. We found by immunofluorescence microscopy and Western and Northern blot analysis of islet extracts that GLUT-1 expression was induced in islet beta-cells in tissue culture both with low or high glucose present. The induction of GLUT-1 was specific to beta-cells but was not present in all beta-cells and was not detected in alpha-cells. GLUT-2 expression was also specific for beta-cells and was not observed in all beta-cells. Some beta-cells in culture coexpressed GLUT-1 and GLUT-2. The expression of the two glucose transporters was regulated in the opposite direction in response to glucose concentration in the culture medium. GLUT-1 was more effectively induced when glucose was low, and GLUT-2 expression was more pronounced when glucose was high in the culture media. Another difference between the two glucose transporters was that GLUT-2 expression was increased while GLUT-1 expression was decreased as culturing continued as long as 7 days. Thus, after 7 days of culture GLUT-2 expression in beta-cells was nearly the same at low and high glucose, whereas GLUT-1 was practically absent no matter what the glucose level was. In attempts to correlate GLUT-1 and GLUT-2 expression to beta-cell function glucose uptake and glucose-stimulated insulin release in fresh and cultured islets were measured. In freshly isolated islet glucose uptake was estimated to be 100-fold in excess of actual glucose use. Glucose uptake was reduced by 7-day culture to about one-third of that observed in freshly isolated islets no matter what the glucose concentration of the culture media. We conclude that in the present experimental system GLUT-1 and GLUT-2 expression and function are not closely associated with glucose usage rates or the secretory function of beta-cells.

Concordant glucose induction of glucokinase, glucose usage, and glucose-stimulated insulin release in pancreatic islets maintained in organ culture.

Using cultured islets as the experimental system, this study established dosage-response and time-dependency curves of the inductive glucose effect on glucose-stimulated insulin release, glucose usage, and glucokinase activity. Glucose-stimulated insulin release in islets cultured for 1, 2, or 7 days was increased as a function of glucose concentration in the culture medium and as a function of time. Glucose usage in the cultured islets showed a close relationship with glucose concentration in the culture medium at both 2 and 7 days of culture. Glucokinase activity increased in islets cultured for 1, 2, or 7 days as a function of increasing glucose concentrations in the culture medium and as a function of time. The V(max) of glucokinase in islets cultured for 7 days in medium containing 30 mM glucose was twice the value of freshly isolated islets and was almost fivefold higher than that in islets cultured for 7 days in 3 mM glucose. The glucose induction of glucose-stimulated insulin release, of glucose usage, and of glucokinase activity were tightly correlated. The biochemical mechanisms of glucose induction of islet glucokinase were further studied. Immunoblotting with an antibody against C-terminal peptide of glucokinase showed that densities of a 52,000-kD protein band from tissue extracts of islets cultured for 7 days in 3, 12, and 30 mM glucose were 25, 44, and 270% compared with that of extract from freshly isolated islets (100%). RNA blot analysis of glucokinase mRNA demonstrated virtually the same levels in fresh islets and islets after 7 days of culture in 3 or 30 mM glucose. The adaptive response of glucokinase to glucose appears therefore to be occurring at a translational or posttranslational site in cultured islets. These data greatly strengthen the concept that glucose is the regulator that induces the activity of glucokinase, which in turn determines the rate change of glucose usage as well as glucose-stimulated insulin release from beta-cells. Thus, the hypothesis that glucokinase is the glucose sensor of beta-cells is strengthened further.

Concordant glucose induction of glucokinase, glucose usage, and glucose-stimulated insulin release in pancreatic islets maintained in organ culture

Concordant glucose induction of glucokinase, glucose usage, and glucose-stimulated insulin release in pancreatic islets maintained in organ culture

Identification of a 64-kDa protein phosphorylated with glucose in human polymorphonuclear leukocytes in a cell-free system

We previously reported that a 64-kDa protein (p64) in human polymorphonuclear leukocytes (PMN) was phosphorylated with [gamma-32P]ATP under a micromolar concentration of glucose in a cell-free system. The present paper presents the results of analysis of phosphorylation reaction and the identification of phosphoprotein. The findings that p64 was also phosphorylated with glucose-6-[32P]phosphate and that phosphorylation was inhibited with mannoheptulose suggested that the reaction was mediated by hexokinase. In fact, it was found that [32P]phosphate in glucose-6-[32P]phosphate was incorporated into either p64 or rabbit muscle phosphoglucomutase and that glucose-6-phosphate formation from glucose and ATP was detected in over 100-kDa fraction of PMN cytosol. These results showed that p64 was phosphoglucomutase in PMN and that phosphate incorporation into p64 was a conversion of a phosphate group in glucose-6-phosphate produced by hexokinase. It was further demonstrated by analysis of two-dimensional electrophoresis that p64 phosphorylated with glucose induction was different from another 64-kDa protein phosphorylated by stimulation with formyl-methionyl-leucyl-phenylalanine in vivo.

Yeast SKO1 gene encodes a bZIP protein that binds to the CRE motif and acts as a repressor of transcription.

We have cloned a yeast gene, SKO1, which in high copy number suppresses lethal overexpression of cAMP-dependent protein kinase. SKO1 encodes a bZIP protein that binds to the CRE motif, TGACGTCA. We found that SKO1 also binds to a CRE-like site in SUC2, a yeast gene encoding invertase which is under positive control by cAMP. A disruption of the SKO1 gene causes a partial derepression of SUC2, indicating that SKO1 is a negative regulator of the SUC2 gene. SKO1 interacts positively with MIG1, a zinc finger protein that mediates glucose repression of SUC2. A kinetic analysis revealed a complex regulation of the SUC2 mRNA in response to glucose. First, MIG1 mediates a rapid and strong repression of SUC2, which is complete within 10 minutes. Second, a MIG1-independent process causes a further slow reduction in the mRNA. Third, in the absence of MIG1, there is also a rapid but transient glucose induction of the SUC2 mRNA. This induction is correlated with a transient loss of SKO1-dependent repression.

Effect of Okadaic Acid in Rat Adipocytes: Differential Stimulation of Glucose and Lipid Metabolism and Induction of Refractoriness to Insulin and Vanadate*

The insulin-like effects of okadaic acid (OKA) in rat adipocytes were further characterized. Okadaic acid did not alter insulin receptor function. This includes undisturbed insulin binding and receptor-mediated ligand internalization in OKA-treated cells. Also, the tyrosine kinase activity of the insulin receptor was not modified in a cell-free system. The stimulating effects of OKA were significantly increased by preincubating (40 min) the cells at 37 C. At lower temperatures (i.e. 26-30 C), OKA did not mimic insulin. Maximal stimulation of lipogenesis occurred at 0.5 microM and then declined at higher concentrations. The insulin-like effects of OKA on lipogenesis did not persist after removal of the agent by washing at 37 C. Okadaic acid maximally stimulated the incorporation of [1-14C]glucose into lipids and the oxidation of [6-14C]glucose into 14CO2, but unlike insulin, it had little if any effect of oxidizing [1-14C]glucose to 14CO2 or incorporating [6-14C]glucose into lipids. Okadaic acid was equivalent to insulin in stimulating 3-O-methyl-glucose uptake. Since the insulin-like effects of OKA did not persist after preincubation and washing, the effects of insulin in OKA-treated cells could be evaluated. The adipocytes were found to be fully refractory to the modulating actions of insulin. Thus, insulin did not stimulate glucose transport, its oxidation, or its incorporation into lipids, and failed to reverse lipolysis. Unresponsiveness was fully developed after 40-min preincubation at 37 C with 3 microM OKA and was half-maximal at 0.13 microM OKA. It persisted at least over a period of 150 min. The effect of OKA was restricted to the stimulating actions of insulin and vanadate. Basal activities were not altered, nor was the ability of the desensitized cells to respond to isoproterenol. The lack of an insulin-like effect of OKA on some metabolic pathways enabled us to demonstrate that OKA (0.25 microM) also rendered adipocytes fully unresponsive to insulin in the continuous presence of the agent. Western blotting of the 40,000 x g pellets with antibodies to phosphotyrosine revealed the appearance of a protein with an apparent mol wt of 43,000 in OKA-desensitized cells. In summary, OKA mimics some of insulin bioeffects, but concomitantly renders the cells tolerant to the modulating action of the hormone itself.

Cyclic AMP as a determinant for glucose induction of fast Ca2+ oscillations in isolated pancreatic beta-cells

The effect of glucose on the cytoplasmic Ca2+ concentration ([Ca2+]i) of pancreatic beta-cells from ob/ob-mice was examined by dual wavelength recordings of the 340/380 nm fluorescence excitation ratio of fura-2. Single beta-cells responded to 11-20 mM glucose with an initial lowering of [Ca2+]i, followed by an increase usually manifested as large amplitude oscillations (300-500 nm) with a frequency of 0.2-0.5/min (a-type). Particularly in freshly isolated beta-cells, there were also superimposed fast oscillations with frequencies of 2-8/min amplitudes in the 70-250 nM range (b-type) and sometimes pronounced [Ca2+]i transients exceeding 250 nM with durations below 10 s (c-type). After addition of 1-100 nM glucagon or 1 mM of the dibutyryl or 8-bromo derivatives of cyclic AMP, glucose generated numerous b-type oscillations superimposed on those of the a-type or on an elevated steady-state level. The duration of the b-type oscillations increased slightly when glucose was raised from 11 to 16 mM. The c-type transients probably represent a separate reaction predominantly seen when raising cyclic AMP much above its normal concentration. It is concluded that glucose can induce fast oscillations of [Ca2+]i also in isolated beta-cells, especially when measures are taken to increase their cyclic AMP content.