A fragment of metabolic prehistory.
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Animals↗
Biomedical subjects
J Tepperman
Publications and source records attributed to J Tepperman.
phyB is evolutionarily conserved and constitutively expressed in rice seedling shoots.
Southern blot analysis indicates that the rice genome contains single copies of genes encoding type A (phyA) and type B (phyB) phytochromes. We have isolated overlapping cDNA and genomic clones encoding the entire phyB polypeptide. This monocot sequence is more closely related to phyB from the dicot, Arabidopsis (73% amino acid sequence identity), than it is to the phyA gene in the rice genome (50% identity). These data support the proposal that phyA and phyB subfamilies diverged early in plant evolution and that subsequent divergence accompanied the evolution of monocots and dicots. Moreover, since rice and Arabidopsis phyB polypeptides are more closely related to one another (73% identity) than are monocot and dicot phyA sequences (63-65% identity), it appears that phyB has evolved more slowly than phyA. Sequence conservation between phyA and phyB is greatest in a central core region surrounding the chromophore attachment site, and least toward the amino-terminal and carboxy-terminal ends of the polypeptides, although hydropathy analysis suggests that the overall structure of the two phytochromes has been conserved. Gene-specific Northern blot analysis indicates that, whereas phyA is negatively regulated by phytochrome in rice seedling shoots in the manner typical of monocots, phyB is constitutively expressed irrespective of light treatment. In consequence, phyA and phyB transcripts are equally abundant in fully green tissue. Since Arabidopsis phyB mRNA levels are also unaffected by light, the present results suggest that this mode of regulation is evolutionarily conserved among phyB genes, perhaps reflecting differences in the functional roles of the different phytochrome subfamilies.
Amino Acid Sequence↗
The molecular basis of sulfonylurea herbicide resistance in tobacco.
The enzyme acetolactate synthase (ALS) is the target enzyme for the sulfonylurea and imidazolinone herbicides. We describe the isolation and characterization of the ALS genes from two herbicide-resistant mutants, C3 and S4-Hra, of Nicotiana tabacum. There are two distinct ALS genes in tobacco which are 0.7% divergent at the amino acid sequence level. The C3 mutant has a single Pro-Gln replacement at amino acid 196 in one ALS gene. This gene is termed the class I gene and is equivalent to the SuRA locus. The S4-Hra mutant has two amino acid changes in the other ALS gene. This gene is termed the class II gene or the SuRB locus. The S4-Hra mutant includes a Pro-Ala substitution at amino acid 196 and a Trp-Leu substitution at amino acid 573. Gene reintroduction experiments have confirmed that these amino acid substitutions are responsible for the herbicide resistance phenotypes. Transgenic plants carrying these genes are highly resistant to sulfonylurea herbicide applications.
Journal Article↗
Effect of a high fat diet on rat adipocyte lipolysis: responses to epinephrine, forskolin, methylisobutylxanthine, dibutyryl cyclic AMP, insulin and nicotinic acid.
An earlier report from this laboratory showed that feeding rats a high fat diet decreased epinephrine-stimulated lipolysis in their adipose tissue. Experiments were designed to explore further the effects of such diets on adipocyte response to epinephrine and to several other lipolytic and antilipolytic agents. Rats were fed diets with 67% of energy consisting of glucose or lard for 5 to 7 d. Adipocytes were prepared from epididymal fat pads and lipolysis measured by the release of glycerol into the medium during 1-h incubations. The cells from the rats fed the high fat diet showed lower lipolytic responses to stimulation by epinephrine, forskolin and dibutyryl cyclic AMP than those from rats fed the high glucose diet. The lard diet effect on the lipolytic response to isobutylmethylxanthine varied among experiments, but it also decreased it in some of them. However, the high fat diet did not induce decreased sensitivity or responsiveness to the antilipolytic effect of insulin, although previous reports have demonstrated resistance to other actions of insulin in rats fed a high fat diet. The antilipolytic effect of nicotinic acid was also similar in cells from rats fed a high fat diet to that found for cells from rats fed the high glucose diet.
1-Methyl-3-isobutylxanthine↗
Effect of trifluoperazine on the action of insulin in rat adipocytes.
Trifluoperazine (TFP), a potent inhibitor of calmodulin action, at a concentration of 12 microM decreased the stimulating effects of insulin on 1) fat cell pyruvate dehydrogenase (PDH) activation, 2) generation/action of PDH activator by adipocyte plasma membranes, and 3) insulin-induced loss of insulin receptors, without altering spermine-induced activation of fat cell PDH or preventing insulin stimulation of glucose oxidation. In addition to these effects on insulin action, TFP abolished several biological actions of the insulin-generated PDH stimulator from liver particulate fractions. These actions include fat cell PDH activation and decrease in receptors. These data indicate that TFP inhibits both membrane-associated and intracellular components of insulin action. The results suggest involvement of calcium-binding protein (calmodulin) and/or phospholipid dependent-calcium activated protein kinase C in some of the actions of insulin in fat cells. The insulin effect on glucose oxidation appears to be less dependent on these mediators.
Adipose Tissue↗
Effect of acute exercise on insulin generation of pyruvate dehydrogenase activator by rat liver and adipocyte plasma membranes.
Groups of young adult rats with body weights of 125-135 g (group A) or 300-400 g (group B) were subjected to one bout of prolonged exercise to exhaustion on a treadmill and were studied 2 h postexercise. Liver glycogen levels were markedly depleted in the exercised rats. Adipocytes from group A exercised rats showed a significantly greater increase in pyruvate dehydrogenase (PDH) activity in response to insulin than those from sedentary controls. Incubation with insulin of liver particulate fractions from exercised group A rats resulted in an increased production of a mitochondrial PDH activator compared with preparations from sedentary controls. The tissues of both exercised and sedentary group B rats were less responsive to insulin than those of the smaller rats. A significant effect of exercise on increased production of a PDH activator in response to insulin was found only in experiments in which adipocyte plasma membranes were coincubated with mitochondria and insulin. For group B rats exercise provided no significant enhancement of insulin activation of intact adipocyte PDH or stimulation of the production of a PDH activator by liver particulate preparations. Insulin binding to fat cells was not affected by exercise. Group A rats made insulin resistant by a high-fat diet did not respond to exercise by significantly increasing the insulin stimulation of PDH activator by liver membranes. The enhancing effect of a single bout of exercise on insulin response was not readily demonstrable in rats resistant to insulin either in association with age and weight or with a high-fat diet.(ABSTRACT TRUNCATED AT 250 WORDS)
Adipose Tissue↗
Studies on the effects of protease substrate analogues on some of the actions of insulin.
Added TAME (N alpha-p-tosyl-1-anginine methyl ester) or BAME (benzoyl-anginine methyl ester) inhibited insulin induced activation of glucose oxidation and fat cell PDH activation without affecting spermine action on PDH activation and glucose oxidation in fat cells. BAME inhibited insulin-induced generation of both PDH stimulator and PDH inhibitor from liver particulate fraction. In contrast, insulin-induced internalization of insulin receptors and negative cooperativity of insulin receptors were not affected by protease substrate inhibitors. These results suggest that certain actions of insulin (glucose oxidation, generation of PDH regulators) are mediated by proteolytic events, while insulin-induced down regulation and negative cooperativity of insulin receptors are not mediated by activation of endogenous proteases.
Adipose Tissue↗
Membranes and the response to insulin.
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Adipose Tissue↗
Studies on the possible involvement of prostaglandins in insulin generation of pyruvate dehydrogenase activator.
Insulin-exposed liver particulate fraction supernatants from control rats stimulated mitochondrial pyruvate dehydrogenase (PDH) activity by 26%, while the stimulation by similar preparations from indomethacin-injected rats (5 mg/kg twice daily, i.p., for 2 days) was 4%. In vitro addition of indomethacin to the particulate fraction during insulin exposure also inhibited stimulation of PDH by insulin. This inhibitory effect of indomethacin was completely overcome by the in vitro addition of prostaglandin E2 (PGE2) to the liver particulate incubation mixture. Intact adipocytes showed a similar (62%) decrease in insulin activation of PDH in the presence of indomethacin. In a cell-free adipocyte system (co-incubation of mitochondria and plasma membrane), indomethacin addition resulted in 90% decrease in insulin-stimulated PDH response. PGE2 addition completely reversed this inhibition. In contrast to its effects on PDH activation, indomethacin had no effect on insulin-stimulated glucose oxidation. In vitro incubation of fat cells with dexamethasone (1 microM) also resulted in decreased insulin activation of PDH. Inclusion of arachidonic acid during dexamethasone exposure of fat cells resulted in partial restoration of the insulin effect on PDH in fat cells and in cell-free preparations. However, addition of PGE2 during insulin exposure of plasma membranes from dexamethasone-treated preparations showed no significant restoration of the insulin effect on PDH. These studies suggest that: (1) PG metabolism is involved in insulin's generation of the second messenger, and (2) the mechanism of dexamethasone-induced inhibition of insulin effect on PDH is a complex phenomenon involving the synthesis and action of eicosanoids.
Adipose Tissue↗
Insulin-induced internalization and replacement of insulin receptors in adipocytes of rats adapted to fat feeding.
To elucidate the mechanisms of the previously observed decrease in adipocyte surface insulin binding in fat diet-induced insulin resistance (Ip et al., J. Lipid Res. 1976; 17:588-99), we performed studies on the distribution of insulin receptors between the cell surface and cell interior, and the extent of internalization and recycling of insulin receptors in adipocytes obtained from fat-fed and glucose-fed rats. Intact cell binding and binding to solubilized cells (total) was decreased by 50% in rats fed fat for 7 days when compared with glucose-fed rats. Incubation of adipocytes with insulin in Tris buffer (100 ng/ml) resulted in a 40-60% decrease in cell surface insulin binding capacity. In two separate experiments, it was found that this insulin-induced receptor loss (%) in fat-fed rat adipocyte preparations was either comparable to that of glucose-fed rats or somewhat decreased. The degradation of the receptors was not affected, as seen by the lack of difference in the chloroquine effect between the two groups. Incubation of fat cells with insulin in tissue culture medium promoted complete reinsertion of receptors into the cell membrane in glucose-fed rat adipocytes, while fat-fed rat preparations demonstrated a significant decrease (37%) in the extent of reinsertion of insulin receptors. Thus, the decrease in cell surface insulin binding and receptor number in fat-fed rat adipocytes is related to an adaptive decrease in the total receptor content coupled with an impairment in the ability to reinsert insulin receptors from the cell interior after insulin-induced internalization.
Adipose Tissue↗
Mechanisms of the fasting-induced dissociation of insulin binding from its action in isolated rat hepatocytes.
Fasting leads to an increase in insulin binding to isolated rat hepatocytes from 12 to 17%. This increase was accounted for by changes in the affinity of insulin receptors without alteration in their number. In contrast, the responsiveness of hepatocytes to insulin was markedly diminished in fasted rats. Both basal and insulin-stimulated rates of 14C-glucose incorporation into glycogen were significantly decreased in fasted animals. When insulin-induced 14C-glucose incorporation into glycogen was expressed as a percent above the basal rate, hepatocytes isolated both from control and fasted animals showed the same magnitude of maximal response (66 +/- 13% in fed and 59 +/- 12% in fasted animals, respectively). However, more insulin must be bound to hepatocytes isolated from fasted animals in order to elicit the same percent of insulin's maximal effect. Incubation of 'fed' hepatocytes in the serum obtained from fasted rats significantly diminished their responsiveness to insulin. An addition of insulin (100 ng/ml), glucose (10 mM) and antibodies to glucagon (1:100) eliminated the inhibitory effect of 'fasted' serum on 'fed' hepatocytes. A 48-hour fast increased significantly the microviscosity (decreased fluidity) of hepatocyte plasma membranes and altered membrane phospholipid composition. These changes correlated with enhanced insulin binding to isolated membranes. Moreover, in response to insulin, plasma membranes isolated from 'fasted' hepatocytes generated only one half the amount of the second messenger (PDH activator) observed in membranes of fed animals. The amount of PDH activator generated by incubation of plasma membranes with insulin correlated inversely with both insulin binding and membrane microviscosity. We conclude that 1) fasting induces both coupling defect and post-receptor changes in insulin's action; 2) both extracellular and intracellular factors contribute to fasting-induced dissociation of insulin binding from insulin action; 3) insulin/glucagon ratio may influence hepatocyte responsiveness to insulin; 4) alterations in plasma membrane fluidity and phospholipid composition may alter insulin binding and contribute to its dissociation from the subsequent action; 5) membranes isolated from 'fasted' hepatocytes generate less mediator of insulin action than do membranes isolated from 'fed' hepatocytes.
Animals↗
Photobiology of diagravitropic maize roots.
Light-induced modification of gravitropism in etiolated roots of Zea mays cv Bear x W38 is a low fluence response mediated by phytochrome. This cultivar has a threshold of 10(-6) mol m(-2) and becomes saturated with 10(-2) mol m(-2) of red light. The maximum light-mediated response of 32 degrees downward from horizontal occurs in roots 10 to 30 millimeters in length, 120 to 165 minutes after irradiation. Reciprocity is valid from 2 to at least 9,000 seconds and the response can be about 90% reversed by far red light. Photoreversibility is lost (;escape' occurs) about 20 minutes after red irradiation but appears to be regained 60 to 80 minutes later. A red light-induced (or synchronized) nutation in the apparent curvature rather than unusual escape characteristics may explain these results.
Journal Article↗
Effect of dexamethasone on adipose tissue and liver pyruvate dehydrogenase and its stimulation by insulin-generated chemical mediator.
Rats were treated with dexamethasone (50 micrograms/day, sc) for 4 days. Total pyruvate dehydrogenase (PDH) and insulin-stimulated PDHa activities were decreased in fat pads from dexamethasone-treated rats compared to control values. Coincubation experiments with adipocyte mitochondria, plasma membrane, and insulin demonstrated decreased stimulation of PDH in preparations from dexamethasone-treated rats. The responsiveness of the mitochondrial PDH system to insulin and control rat plasma membranes was not different in glucocorticoid-treated adipocyte preparations compared to controls. Liver mitochondria from dexamethasone-treated rats demonstrated decreased basal enzyme activity and a decreased percentage of stimulation of PDH when supernatants from insulin-exposed liver particulate fractions were tested. These experiments suggest that insulin resistance produced by glucocorticoid treatment, like that resulting from fat feeding, is accompanied by a decrease in the capacity of adipocyte and liver plasma membranes to generate PDH activator in response to insulin.
Adipose Tissue↗
Decreased insulin-generation of pyruvate dehydrogenase inhibitor in insulin resistant states.
Insulin resistance produced in rats by feeding a high fat diet or by dexamethasone administration (50 micrograms/day, sc for 4 days) resulted in 50-70% decrease in the generation of pyruvate dehydrogenase inhibitor by insulin exposed liver particulate fractions. The inhibition was dose dependent. Treatment of insulin mediator preparations with neuraminidase and B-D-galactosidase resulted in inactivation of the pyruvate dehydrogenase inhibitor. Presence of exogenous enzyme substrates during enzyme digestion partially protected the inhibitor from inactivation. Protease treatment did not affect the inhibitor while the stimulatory activity of the insulin mediator was abolished by trypsin treatment. These results together with the previous report suggest that insulin resistance results in a decrease in the generation of both of the mediators of insulin action. This may result from a decrease in insulin binding, shown earlier, or from a decrease in precursor availability.
Animals↗
Effects of high fat and high carbohydrate diets on liver pyruvate dehydrogenase and its activation by a chemical mediator released from insulin-treated liver particulate fraction: effect of neuraminidase treatment on the chemical mediator activity.
Rats were fed a high fat diet or a high glucose diet for 5-7 days. Basal pyruvate dehydrogenase activity (both the active form and the total enzyme activity) was decreased in liver homogenates from fat diet-adapted rats as compared to those fed the glucose diet. Supernatants from insulin-exposed liver particulate fractions from fat-fed rats showed decreased stimulation of pyruvate dehydrogenase activity as compared to those from glucose-fed rats. There was no difference in the response of the mitochondria from the two groups when they were stimulated by supernatants from insulin-treated liver particulate fractions from stock diet-fed rats. Liver particulate fractions from fat-fed rats showed decreased generation of the chemical activator in response to Concanavalin A and trypsin stimulation. This suggests that fat feeding results in a decrease in membrane protease substrate availability. Treatment of the insulin mediator with neuraminidase and beta-D-galactosidase resulted in inactivation of the mediator. Presence of exogenous enzyme substrates during enzyme digestion protected the mediator from inactivation, suggesting that carbohydrate residues are important in the action of the insulin mediator. This fat diet-induced decrease in the generation of a chemical mediator of insulin action may result from 1) a decrease in insulin binding, shown earlier; 2) a decrease in the amount of protease substrate; and 3) an alteration in its carbohydrate composition, which is important in its ability to activate pyruvate dehydrogenase.
Animals↗
The effects of streptozotocin diabetes on the activities of rat liver glycosyltransferases.
The effects of streptozotocin diabetes on the activities of rat liver glycosyltransferase enzymes have been investigated. Liver microsomal fractions were prepared from rats that had been injected with streptozotocin (65 mg/kg, i.v.) 3 wk to 2 mo earlier. Preparations from diabetic rats had decreased activities of N-acetylglucosaminyl transferase compared with those of age-matched controls (0.98 +/- 0.11 nmol transferred per mg protein in 30 min versus 3.19 +/- 0.34 for controls, P less than 0.001). Galactosyltransferase activity was also lower in diabetic rat livers (1.48 +/- 0.26 nmol transferred per mg protein in 30 min versus 3.32 +/- 0.56 for controls, P less than 0.025). Sialytransferase activities were not significantly different between diabetic and control rat livers. There were no significant differences between the diabetic and control rat liver microsomes in the activities of UDP N-acetylglucosamine pyrophosphatase, UDP galactose pyrophosphatase, or CMP sialic acid phosphatase. The glycosidases, N-acetylglucosaminidase and galactosidase, had similar activities in the livers of both groups of rats. Sialidase activity could not be detected in microsomal preparations from either diabetic or control rat livers. These results are discussed in relation to our previously reported alterations in glycosyltransferase activities, and plasma membrane glycoprotein composition in the livers of rats made insulin-resistant by a carbohydrate-free, high-fat diet and to the observation of Carter and his colleagues (FEBS Lett. 1979; 104:389-92.) that streptozotocin diabetes alters the glycoprotein composition of rat liver plasma membranes.
Animals↗
Symptomatic relief in herpes zoster.
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Bandages↗
Effect of high fat and high carbohydrate diets on adipose tissue pyruvate dehydrogenase and its activation by a plasma membrane-enriched fraction and insulin.
Rats were fed a high lard diet or a high glucose diet for 5--7 days. Basal and insulin-stimulated epididymal fat pad pyruvate dehydrogenase (PDH) activities were decreased in fat diet-adapted rats compared to those fed the glucose diet. When adipocyte plasma membranes and mitochondria were incubated together with and without insulin, it was found that the insulin stimulation of PDH activity was lower in preparations from fat-fed rats on both an absolute and percentage basis. Supernatant fractions from insulin-stimulated glucose-fed rat plasma membranes activated mitochondrial PDH to a greater extent than those from lard-fed rat preparations. There was no difference in the response of mitochondria from the two groups when they were stimulated by insulin-treated plasma membranes from stock diet-fed rat adipose tissue. These experiments suggest that fat feeding results in adaptive changes in adipocyte plasma membranes which are involved in the generation of the insulin-stimulated chemical activator of PDH. This adaptive change is in addition to those described earlier.
Adipose Tissue↗