Inhibition of insulin-dependent lipogenesis and anti-lipolysis by protein tyrosine kinase inhibitors.

Protein tyrosine kinase (PTK) blockers which competitively inhibit the kinase activity of insulin receptors were synthesized and their properties examined. The best insulin receptor kinase (IRK) inhibitors possess either one hydroxyphenyl ring and two carboxyl groups or two phenyl rings and one carboxyl group. All the inhibitors, except tBoc-tyrosine aminomalonate, effectively block the IRK-catalyzed phosphorylation of exogenous substrate, but only partially block receptor autophosphorylation. These PTK blockers inhibit the insulin induced [14C]glucose assimilation into lipids (lipogenesis), but fail to inhibit the anti-lipolytic effect of the hormone. Only tBocTyr-aminomalonate was found to inhibit all the effects of insulin measured: insulin-stimulated phosphorylation of exogenous substrate, IRK autophosphorylation, insulin-dependent lipogenesis and the insulin-dependent anti-lipolytic effect. This inhibitor is the first blocker which is reported to block insulin-dependent anti-lipolysis. The inhibitors examined are devoid of general adverse effects since they have no effect on insulin-independent lipolysis, on [U14C]fructose assimilation or on (-)isoproterenol-stimulated lipolysis. These studies suggest that insulin-dependent lipogenesis and anti-lipolysis may be mediated by two distinguishable signalling pathways. This study also suggests that PTK inhibitors may become useful tools in the investigation of the signalling pathways of PTKs.     

Receptor binding properties and insulin-like effects of human growth hormone and its 20 kDa-variant in rat adipocytes

The natural 20 kDa-variant of human growth hormone (hGH) binds with high affinity to IM-9 human lymphocyte receptors, in agreement with its potency in biological assays for growth promoting and lactogenic activities. In contrast, 20 kDa-hGH has only 3% of the potency of 22 kDa-hGH in binding to the receptors of normal and hypophysectomized rat adipocytes. In agreement with the binding potency, 20 kDa-hGH is only 3% as potent as 22 kDa-hGH in stimulating lipogenesis in normal rat adipocytes preincubated for a few hours in hGH-free medium. The 20 kDa-hGH is also much weaker than 22 kDa-hGH in stimulating lipogenesis in adipocytes from hypophysectomized rats. These data strongly support the concept that the rat adipocyte receptor, which mediates the insulin-like effects of growth hormone, is different from the receptor found on human IM-9 lymphocytes. Preincubation of rat adipocytes with hGH induces a refractoriness to subsequent activation of lipogenesis by hGH but does not abolish the response to insulin, while preincubation with insulin slightly potentiates the hGH response and does not change the insulin response. Additivity studies and a detailed comparison of the lipogenic effects of insulin and hGH suggest that hGH shares only a subset of the metabolic pathways activated by insulin.     

In vitro insulin-like actions of the growth factor from the tapeworm, Spirometra mansonoides

In vitro actions of purified plerocercoid growth factor (PGF) were compared with those of insulin and human growth hormone (hGH) in adipose tissue from normal male rats. Insulin-like effects were measured by the ability of PGF, insulin, or hGH to stimulate oxidation of [U-14C]glucose to 14CO2, to stimulate lipogenesis, and to inhibit epinephrine-induced lipolysis. PGF and insulin stimulated significant increases in glucose oxidation and lipogenesis in adipose tissue that had not been preincubated as well as in tissue that had been preincubated. hGH stimulated insulin-like effects only in tissue that had been preincubated for 3 hr. Insulin, hGH, and PGF inhibited epinephrine-induced lipolysis of preincubated (3 hr) adipose tissue. hGH produced a dramatic lipolytic response in tissue freshly removed from normal rats but no dose of PGF was lipolytic. PGF did not displace 125I-insulin from its receptors on adipocytes but did competitively inhibit 125I-hGH binding to adipocytes. These results suggest that PGF has direct insulin-like actions which are initiated by binding a GH receptor, but PGF had no anti-insulin action and the insulin-like activity of PGF was unaffected by refractoriness of adipose tissue to GH.     

Biochemical and cytochemical studies of preadipocyte differentiation in serum-free culture of porcine stromal-vascular cells: interaction of dexamethasone and growth hormone.

Stromal-vascular cells from adipose tissue of pigs 5-7 days of age were grown in serum for 2-3 days and switched to serum-free (insulin, transferrin and selenium) conditions +/- test hormones for 6-7 days. The interaction of dexamethasone (DEX) and human growth hormone (hGH) was evaluated since glucocorticoids augment and hGH antagonizes the effect of insulin. Low levels (1-10 nM) of DEX with insulin doubled (p less than 0.05) specific activity of glycerol phosphate dehydrogenase (GPDH) and doubled (p less than 0.05) the number of detectable fat cells relative to insulin alone. DEX with insulin enhanced the morphological differentiation of preadipocytes and markedly increased fat cell cluster numbers in the presence of hGH. Furthermore, 1-10 nM of DEX partially blocked (p greater than 0.05) the inhibitory effect of 10 nM hGH on GPDH activity, but 1-100 nM DEX had no effect (p greater than 0.05) on the ability of hGH to compromise lipid deposition. DEX alone (no insulin or hGH) induced the appearance of esterase-reactive but lipid-free cells. Cells with these characteristics were increased in number by DEX in the presence of hGH but were nearly absent in the presence of insulin and DEX. Therefore, transient exposure to GH in vivo may have no permanent effect on adipose tissue development in the continued presence of glucocorticoids.     

Acridine orange, an inhibitor of protein kinase C, abolishes insulin and growth hormone stimulation of lipogenesis in rat adipocytes

To determine whether protein kinase C plays a role in the actions of insulin and growth hormone in rat adipocytes, we tested the effect of acridine orange, a potent inhibitor of kinase C, on the lipogenic activity of both hormones. This compound completely inhibited the effects of insulin, growth hormone and phorbol ester 12-myristate 13-acetate, whereas 9-acridine carboxylic acid, an analog of acridine orange which does not inhibit kinase C, had no effect. Acridine orange did not act through inhibition of hormone binding. These data are consistent with the involvement of kinase C in the action of insulin and growth hormone on lipogenesis in rat fat cells.     

Dependence on pH of substrate binding to a mutant lactose carrier, lacYun, in Escherichia coli. A model for H+/lactose symport.

The potential role of guanine nucleotide regulatory proteins (G-proteins) in acute insulin regulation of glucose transport was investigated by using bacterial toxins which are known to modify these proteins. Cholera-toxin treatment of isolated rat adipocytes had no effect on either 2-deoxyglucose transport or insulin binding. Pertussis-toxin treatment resulted in an inhibition of both insulin binding and glucose transport. Insulin binding was decreased in pertussis-toxin-treated cells by up to 40%, owing to a lowering of the affinity of the receptor for hormone, with no change in hormone internalization. The dose-response curve for insulin stimulation of glucose transport was strongly shifted to the right by pertussis-toxin treatment [EC50 (half-maximally effective insulin concn.) = 0.31 +/- 0.04 ng/ml in control cells; 2.29 +/- 1.0 in treated cells), whereas cholera toxin had only a small effect (EC50 = 0.47 +/- 0.02 ng/ml). Correcting for the change in hormone binding, pertussis toxin was found to decrease the coupling efficiency of occupied receptors (50% of maximal insulin effect with 928 molecules bound/cell in control and 3418 in treated cells). Pertussis-toxin inhibition of insulin sensitivity was slow in onset, requiring 2-3 h for completion. Under conditions where pertussis-toxin inhibition of insulin sensitivity was maximal, a 41,000 Da protein similar to the alpha subunit of Gi (the inhibitory G-protein) was found to be fully ribosylated. These results are consistent with the concept that pertussis-toxin-sensitive G-protein(s) can modify the insulin-receptor/glucose-transport coupling system.     

Regulation of growth hormone and epidermal growth factor receptors by progestins in breast cancer cells

A 24 hr incubation of T-47D human breast cancer cells with R5020, a synthetic progestin, resulted in a 200-250% increase in the specific binding of human growth hormone (hGH) and epidermal growth factor (EGF) by these cells. This effect was specific for progestins in that similar responses were observed with progesterone, medroxyprogesterone acetate and ORG 2058 but no significant increases in hGH or EGF binding were observed in cells incubated with testosterone, estradiol or hydrocortisone. Increased binding was due to an increase in the concentration of receptors (hGH, control = 6,490 +/- 500, progestin treated = 13,180 +/- 3,270 sites/cell; EGF, control = 33,380 +/- 7,410, progestin treated = 67,460 +/- 20,330 sites/cell) while the affinity constants for the hormone-receptor interactions were unchanged by progestin treatment. The specific binding of insulin, calcitonin, transferrin and concanavalin A was unaffected by these treatments. It is concluded that expression of hGH and EGF receptors in this breast cancer cell line is regulated by progestins.     

Comparison of the acute metabolic effects of 22,000-dalton and 20,000-dalton growth hormone in human subjects

The acute metabolic effects of 20,000-dalton human growth hormone (hGH20K) in man have not previously been tested. We compared changes in concentrations of free fatty acids (FFA), glucose, and insulin in nine growth hormone deficient children following injection of 22,000-dalton intact human growth hormone (hGH22K) and the smaller variant, hGH20K. There was a significant decline (37%) in the mean FFA concentration from baseline to 1/2 hour post-injection and from baseline to 1 hour post-injection (36%) in the children given hGH22K, but no such decline was seen after injection of hGH20K. No significant differences in mean insulin or glucose concentrations were noted between the two treatment groups, and glucose and insulin concentrations did not acutely change after injection of either hormone. The results of this study indicate that hGH20K has a diminished activity for suppression of FFA as compared to hGH22K. This suggests that GH residues 32-46, missing in hGH20K, constitute all or part of the region of hGH22K producing this response, or that the different primary structures of the two hormones result in tertiary structural differences and altered biological activity.     

Hormonal regulation of expression of the endogenous and transfected human growth hormone gene

Expression of the endogenous human GH (hGH) gene in response to glucocorticoids, thyroid hormone, and insulin was studied in cultures of dispersed GH-secreting human pituitary adenomas. Results were compared to those obtained when the hGH gene was transfected into rat pituitary tumor cells (GC). In the human pituitary cells the glucocorticoid dexamethasone [(Dex) 10(-6) M] increased the release of GH and the levels of GH mRNA by 2 to 4-fold (P less than 0.05). T3 (10(-8) M) had no effect on GH mRNA but increased hGH release by 2- to 6-fold (P less than 0.01). Insulin (5 x 10(-9) M) alone had no significant effect on either hGH mRNA or protein, but blunted the effect of Dex. Among 11 of 18 GC cell clones transfected with the hGH gene with detectable hGH mRNA expression, Dex increased hGH mRNA levels in seven and T3 treatment reduced hGH mRNA levels in eight. Conversely, rat GH mRNA levels from the endogenous rat gene were increased by either Dex or T3 in all 18 clones. Insulin alone or in combination with T3 or Dex was found to increase hGH mRNA levels in some cell lines and to decrease hGH mRNA levels in others; these effects were correlated strongly (r = 0.88; P less than 0.001) with the influence of insulin on the endogenous rat GH gene, implying that individual cellular differences can simultaneously affect the insulin responsiveness of both genes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of hypophysectomy and the insulin-like and anti-insulin pituitary peptides on carbohydrate metabolism in yellow Avy/A (BALB/c x VY)F1 hybrid mice

The amino-terminal portion of human growth hormone, residues 1-43 (hGH1-43), has insulin-potentiating action, while a hyperglycemic pituitary peptide (HP), which co-purifies with human growth hormone (hGH), is antagonistic to the action of insulin. The effects of hGH, hGH1-43, and HP on glucose metabolism were assessed in young (4-5 weeks) and adult (6-8 months) hypophysectomized yellow Avy/A mice which lacked any interfering endogenous pituitary hormones, and compared with age-matched intact obese yellow Avy/A and lean agouti A/a mice. Treatment with hGH1-43 or HP did not promote body growth in hypophysectomized yellow mice; but after 2 weeks of treatment with hGH, there was a significant increase in body weight (P less than 0.05). Treatment with HP raised blood glucose and lowered insulin concentrations in obese yellow mice, but not in agouti or hypophysectomized yellow mice. The severely impaired glucose tolerance of the hypophysectomized yellow mice was improved by acute (60 min) and chronic (3 days) treatment with hGH1-43 as well as by 2 weeks of treatment with hGH; in contrast, HP had no effect. Glucose oxidation in adipose tissue from obese yellow mice was low and showed essentially no response to stimulation by insulin at doses lower than 1000 microunits/ml. Basal glucose oxidation rates in adipose tissue taken from agouti and hypophysectomized yellow mice were significantly higher (P less than 0.001) than those in tissue from obese yellow mice, and the rates responded significantly (P less than 0.05) to 100 microunits/ml insulin. The insulin binding affinities in liver membranes from agouti mice were higher than those from either obese or hypophysectomized yellow mice. The insulin receptor densities were similar in both agouti and obese yellow mice, but higher in hypophysectomized yellow mice (P less than 0.05). Treatment with hGH1-43 slightly increased, although not significantly, the insulin receptor density in yellow obese mice while hGH showed essentially no change. Therefore, hypophysectomy appeared to increase tissue response and decrease insulin resistance by increasing receptor numbers and lowering the circulating insulin levels. Furthermore, the insulin-like action of hGH was elicited directly in vivo by hGH1-43 in hypophysectomized yellow mice.     

A comparison of subcutaneous and intramuscular administration of human growth hormone (hGH) and increased growth rate by daily injection of hGH in GH deficient children

Plasma human growth hormone (hGH) profiles and biological activities of recombinant hGH were compared after im and sc injection in 8 normal volunteers. The time to reach maximal plasma GH and plasma hGH concentrations and the areas under the curve of hGH profiles did not differ significantly after im and sc injections. The biological effect of hGH in increasing nonesterified fatty acid and insulin-like growth factor-I (IGF-I) was the same after both im and sc injections. During 6 months of daily sc administration of recombinant hGH in 20 naive patients, their height increased between 5 and 16.5 cm with a mean of 11.0 +/- 3.0 cm/year. In 27 patients who switched from hGH injections of 2-4 times/week to daily injections, the height increased between 5.3 and 16.5 cm with a mean of 8.3 +/- 2.2 cm/year. These values were greater than those observed in a previous study in which the same amount of hGH was injected in 2-4 doses per week. Plasma IGF-I increased more with daily sc administration than with 2-4 doses per week. The rate of appearance of an antibody to hGH was low (0.5%) and there were no notable changes in blood cell count, urinalysis and/or routine chemistries during the 6 months of daily recombinant hGH treatment. These results show that sc daily administration of hGH is safe, has a greater growth promoting effect, and can be recommended for the treatment of patients with GH deficiency.     

Mitogenic and binding properties of monoclonal antibodies to the prolactin receptor in Nb2 rat lymphoma cells. Selective enhancement by anti-mouse IgG

Three monoclonal antibodies (mAbs) (T6, U5, and U6) against prolactin (PRL) receptors in rat liver were studied in the rat lymphoma lactogen-dependent (Nb2-11C) and autonomous (Nb2-SP) cell lines. The mAbs had strong affinity for lactogen receptors (Ka = 12-14 nM-1), similar to that of human growth hormone (hGH) which is a lactogenic hormone. T6 and hGH competed for the same binding site, while U5 and U6 interacted with another epitope. The 125I-hGH-receptor complex could be immunoprecipitated by either U5 or U6, but not by T6. Affinity labeling and immunoblotting revealed that hGH and U6 bind to a protein of 63-65 kDa. T6, U5, and U6 were mitogenic in Nb2-11C cells but their respective potencies were 185-, 70-, and 4700-fold lower than that of hGH. Anti-mouse IgG enhanced the mitogenic effect of all three mAbs and almost completely abolished the differences between them, although their mitogenic activity was still 60-120-fold lower than hGH. Des-13-hGH, a competitive antagonist of hGH which hardly effected the binding of 125I-U5, inhibited the U5-stimulated proliferation of Nb2-11C cells in a noncompetitive manner, indicating that simultaneous binding of both ligands fixed the receptor in a nonactive conformation. A Fab fragment of T6 was not mitogenic, and inhibited the hGH-induced mitogenesis in a competitive manner, but its mitogenicity could be restored by anti-mouse IgG. We suggest that the dimerization or oligomerization of the lactogen receptor in Nb2-11C cells is an obligatory step in the transduction of the mitogenic signal. It may be induced by binding of the mAb to a site, which can be either identical or may even be distinct from that which binds the lactogenic hormone.     

Degradation, receptor binding affinity, and potency of insulin from the Atlantic hagfish (Myxine glutinosa) determined in isolated rat fat cells

Insulin from the Atlantic hagfish, Myxine glutinosa, a primitive vertebrate, was studied with respect to degradation, receptor binding, and stimulation of glucose transport and metabolism in isolated rat adipocytes. The degradation was studied in a concentrated suspension with about 100mul of cells/ml of suspension. 125I-labeled hagfish insulin and 125I-labeled pig insulin were degraded at the same rate when present in concentrations of 0.3nM. Native hagfish insulin inhibited the rate of degradation of 125I-labeled pig insulin half-maximally at a concentration of 12+/-2 nM (S.D., n=6) as compared to 130+/-32 nM (S.D.,n=6) for pig insulin. Native hagfish insulin in a concentration of 130 nM was biologically inactivated at a rate several times slower than pig insulin in the same concentration. The results indicate that the maximal velocity (Vmax) of degradation of hagfish insulin as well as the concentration causing half-maximal velocity (Km) are about 10 times lower for hagfish insulin than for pig insulin. The receptor binding and the biological effects of hagfish insulin were studied in dilute cell suspensions where the degradation of hormone in the medium was negligible. The receptor binding affinity of hagfish insulin was 23+/-7 per cent (S.D., n=10) of that of pig insulin. Hagfish insulin was able to elicit the same maximal stimulation of both 3-o-methylglucose exchange and lipogenesis from glucose as pig insulin. However, the potency of hagfish insulin with respect to activation of lipogenesis was only 4.6+/-0.6 per cent (S.D., n=15) of that of pig insulin. Hagfish insulin thus constitutes the first described insulin which exhibits a discrepancy between relative binding affinity and relative potency. This discrepancy was not due to the methionine residue (B31) at the COOH-terminal end of the B chain of hagfish insulin, since removal of this residue caused no marked change in the binding affinity or the potency. The results indicate that the receptor occupancy must be 5 times higher with hagfish insulin than with pig insulin to cause a particular degree of activation of lipogenesis. Hagfish insulin might therefore be characterized as a "partial antagonist" on the receptors. However, it was not possible to demonstrate antagonistic properties of hagfish insulin on the cells. The effect of hagfish insulin plus pig insulin in submaximally stimulating concentrations was additive. Furthermore, the decay of activation of adipocytes after incubation with hagfish insulin followed the same time course as the decay of activation after incubation with pig insulin in a concentration of equal potency. These phenomena are in agreement with the concept that adipocytes possess a large excess of receptors which can mediate the effect of insulin on lipogenesis from glucose.     

Effects of insulin secretagogues on protein kinase C-catalyzed phosphorylation of an endogenous substrate in isolated pancreatic islets

The influence of the insulin secretagogues, carbachol and glucose, on protein kinase C activation in isolated pancreatic islets has been examined by determination of the phosphorylation state of an endogenous 80-kDa protein substrate of protein kinase C. The islet 80-kDa protein was identified as the myristoylated alanine-rich C kinase substrate previously described (Stumpo D. J., Graff, J. M., Albert, K. A., Greengard, P., and Blackshear, P. J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4012-4016) by immunoprecipitation studies. The muscarinic agonist, carbachol (500 microM), induced insulin secretion and a time-dependent increase in the phosphorylation state of this protein in islets. This phosphorylation was maximal (220 +/- 24% of control) at 5 min and was suppressed by the protein kinase C inhibitor, staurosporine. Concentrations of glucose (28 mM) which induce maximal insulin secretion did not induce a statistically significant increase in 80-kDa phosphorylation. The combination of carbachol and a submaximally stimulatory concentration of glucose (10 mM), when added simultaneously, exerted a marked synergistic effect on insulin secretion and a synergistic effect on the phosphorylation of the 80-kDa protein kinase C substrate. These data suggest that the activation of protein kinase C may play an important role in carbachol-induced insulin secretion and in the potentiation by carbachol of insulin secretion induced by glucose. However, the activation of protein kinase C does not appear to be a primary determinant of insulin secretion induced by glucose alone.     

Effects of cortisol on lipolysis and regional interstitial glycerol levels in humans

Cortisol's effects on lipid metabolism are controversial and may involve stimulation of both lipolysis and lipogenesis. This study was undertaken to define the role of physiological hypercortisolemia on systemic and regional lipolysis in humans. We investigated seven healthy young male volunteers after an overnight fast on two occasions by means of microdialysis and palmitate turnover in a placebo-controlled manner with a pancreatic pituitary clamp involving inhibition with somatostatin and substitution of growth hormone, glucagon, and insulin at basal levels. Hydrocortisone infusion increased circulating concentrations of cortisol (888 +/- 12 vs. 245 +/- 7 nmol/l). Interstitial glycerol concentrations rose in parallel in abdominal (327 +/- 35 vs. 156 +/- 30 micromol/l; P = 0.05) and femoral (178 +/- 28 vs. 91 +/- 22 micromol/l; P = 0.02) adipose tissue. Systemic [(3)H]palmitate turnover increased (165 +/- 17 vs. 92 +/- 24 micromol/min; P = 0.01). Levels of insulin, glucagon, and growth hormone were comparable. In conclusion, the present study unmistakably shows that cortisol in physiological concentrations is a potent stimulus of lipolysis and that this effect prevails equally in both femoral and abdominal adipose tissue.     

Metabolic studies of a synthetic lipolytic domain (AOD9604) of human growth hormone

A synthetic analogue (AOD9604) of the lipolytic domain of human growth hormone (hGH) has been studied for its metabolic actions in obese Zucker rats. Daily treatment with an oral dose of AOD9604 of 500 microg/kg body weight for 19 days reduced over 50% (15.8 +/- 0.6 vs. 35.6 +/- 0.8 g) body weight gain of the animals in comparison with the control. The adipose tissues of the AOD9604--treated animals were found to have an increase in lipolytic activity. In contrast to chronic treatment with intact hGH, chronic treatment with AOD9604 showed no adverse effect on insulin sensitivity of the animals, as demonstrated with euglycemic clamp techniques. The results in the present study suggest that the analogue of the hGH lipolytic domain may have the potential to be developed into an orally usable and safe therapeutic agent for obesity.     

Insulin and phorbol ester stimulate phosphorylation of a 15,000 dalton membrane protein in rat diaphragm in a similar manner

The effects of insulin on the phosphorylation of a 15 kilodalton (kDa) membrane protein in rat diaphragm in situ have been investigated. Incubation of the diaphragm with insulin or tumor-promoting phorbol ester increased the 32P-labelling of the 15 kDa protein at serine residues by 50 +/- 8% and 64 +/- 11%, (mean +/- S.E.), respectively. Thermolytic peptide mapping of the 15 kDa protein after insulin treatment of the diaphragm yielded two major phosphopeptides, one of which was absent from digests from control diaphragms. The same two phosphopeptides were identified after incubation of the diaphragm with phorbol ester and after phosphorylation of sarcolemma in vitro with [gamma-32P]ATP and protein kinase C. Additional experiments indicated that pretreatment of diaphragms with insulin or phorbol ester both increased the state of phosphorylation of the 15 kDa sarcolemma protein on phosphorylation sites regulated by protein kinase C. The stimulatory effect of insulin was decreased by staurosporine or by preincubation of the diaphragms with phorbol esters. These results indicate that the insulin-induced increases in protein kinase C activity previously found in rat diaphragm (Walaas et al. (1987) FEBS Lett. 220, 311-318) may be involved in insulin-mediated regulation of phosphorylation of the 15 kDa protein in situ.     

Role of glucose utilization in the restoration of hypophysectomy-induced hepatic cytochrome P450 2E1 by growth hormone in rats

Growth hormone and insulin are the primary determinants for cytochrome P450 2E1 (CYP2E1) expression. The role of glucose on the induction of CYP2E1 by hypophysectomy and on the restorative effect by growth hormone was investigated in the rat liver. Western and Northern blot analyses revealed that hypophysectomy induced CYP2E1 by 5-fold at 1-4 weeks, relative to control, with a concomitant increase in CYP2E1 mRNA. Hypophysectomized rats (HXR) showed a 20% reduction in the plasma glucose level. Hypophysectomy-induced increase in the CYP2E1 mRNA was completely abolished by glucose feeding in drinking water (10%) for 7 days. Treatment of HXR with hGH (2 I.U./kg, twice a day, for 7 days) inhibited the increases in CYP2E1 protein and mRNA levels with restoration of the plasma glucose level. In contrast to the effect of human growth hormone (hGH) on CYP2E1 in HXR with free access to foods, CYP2E1 expression failed to be restored by hGH in starving HXR. However, glucose feeding of starving HXR abolished the induction of CYP2E1. Effects of hypophysectomy and hGH treatment were studied in streptozotocin-induced diabetic rats. Insulin, but not hGH, prevented an increase in CYP2E1 mRNA in diabetic rats. The hepatic CYP2E1 induction in hypophysectomized diabetic rats was inhibited by hGH treatment, indicating that the hGH effect on CYP2E1 expression did not involve insulin production. These results provide evidence that the induction of hepatic CYP2E1 by hypophysectomy may result from reduced glucose utilization, and that the effect of hGH on CYP2E1 expression may be mediated with enhanced glucose utilization, but not with insulin production.     

Effects of phorbol esters on insulin receptor function and insulin action in hepatocytes: evidence for heterogeneity

We investigated the effect of phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator on insulin receptors and insulin action in freshly isolated and primary cultures of rat hepatocytes. PMA (1 x 10^–7 M) did not alter insulin receptor numbers or affinity either acutely or chronically but within 60 minute inactivated insulin stimulated tyrosine kinase of the insulin receptor. PKC activation inhibitied insulin (1 x 10^–7M) stimulation of glycogen and lipid synthesis with a decrease or no change in basal glycogenesis and lipogenesis respectively. However, PKC activation did not alter insulin stimulated or basal amino acid transport even though PCK activation inhibited insulin stimulation of the insulin. receptor tyrosine kinase. Thus, within one tissue, PKC activation has differential effect on insulin action depending on which pathway is examined. Furthermore, insulin stimulation of the insulin receptor tyrosine kinase may not be a necessary step for all insulin signaling pathways.     

Effect of aging on the kinetic characteristics of the insulin receptor autophosphorylation in rat adipocytes.

The effect of aging on the insulin binding parameters and on the kinetic characteristics of the insulin receptor autophosphorylation in rat adipose tissue has been investigated. Using solubilized receptors from adipocyte plasma membranes, no significant differences were identified in either affinity or receptor number in adult vs old rats. Time courses for in vitro receptor phosphorylation revealed that both the initial rate of autophosphorylation and the maximal 32P incorporation were decreased by 40% in old (24-month) animals as compared to adult (3-month) control rats. The tyrosine phosphatase activity associated with the adipocyte plasma membranes does not account for the decreased kinase activity found in old rats. Insulin sensitivity (measured as the dose of insulin required for 50% maximal stimulation of kinase activity) was similar in both groups of rats. However, the kinase activity showed a decreased responsiveness to the hormone in the old rats. Double reciprocal plot analysis of receptor phosphorylation revealed that the Km for ATP was not modified. In contrast, the insulin-stimulated Vmax value was decreased by two-fold in 24-month-old rats. The decrease in Vmax does not appear to be related to an increased basal phosphorylation level on Ser/Thr residues of the C terminus of the receptor beta-subunit. Thus, we conclude that the reduced insulin receptor kinase activity in adipose tissue from old rats is due, at least in part, to a defect of the intrinsic kinase activity of the insulin receptor.