Successful pregnancy outcomes in a patient with type A insulin resistance syndrome.

BACKGROUND: The management of severe insulin resistance during pregnancy is challenging because of the increased risk of perinatal complications for both mother and fetus. We describe two consecutive pregnancies in a patient with severe insulin resistance caused by a mutation in the ß subunit of the insulin receptor. CASE REPORT: A non-obese Japanese woman was diagnosed as having diabetes mellitus during her first pregnancy at age 31 years. She presented at 6 weeks' gestation with a fasting plasma glucose concentration of 15.1 mmol/l and an HbA(1c) level of 95 mmol/mol (10.8%). Fasting insulin concentration was high at 68.8 µU/ml, suggesting severe insulin resistance. Anti-insulin and insulin-receptor antibodies were both negative. Genetic analysis revealed an in-frame heterozygous deletion mutation (∆Leu(999)) in the insulin receptor gene. Despite large daily doses (up to 480 units per day) of insulin aspart and isophane, the patient's postprandial plasma glucose level exceeded 11.1 mmol/l. In the patient's second pregnancy, the addition of metformin at a dose of 2250 mg per day achieved tighter glycaemic control, with lower doses of insulin lispro and isophane (up to 174 units/day). Both newborns, who were found to carry the same mutation, were small for gestational age and developed transient hypoglycaemia after birth. CONCLUSION: Adding metformin to the conventional insulin regimen effectively achieved tight glycaemic control with a lower dose of insulin. The mutation of the insulin receptor gene might underlie the intrauterine growth retardation of the newborns. To our knowledge, this is the first report of successful management of diabetes mellitus in a pregnant woman with type A insulin resistance syndrome.

Restored insulin-sensitivity in IRS-1-deficient mice treated by adenovirus-mediated gene therapy.

Insulin resistance is commonly observed both in overt diabetes and in individuals prone to, but not yet manifesting, diabetes. Hence the maintenance or restoration of insulin sensitivity may prevent the onset of this disease. We previously showed that homozygous disruption of insulin receptor substrate-1 (IRS-1) in mice resulted in insulin resistance but not diabetes. Here, we have explored the mechanism of systemic insulin resistance in these mice and used adenovirus-mediated gene therapy to restore their insulin sensitivity. Mice expressing the IRS-1transgene showed almost normal insulin sensitivity. Expression of an IRS-1 mutant (IRS-1Deltap85) lacking the binding site for the p85 subunit of phosphatidylinositol 3-kinase (PI3K) also restored insulin sensitivity, although PI3K is known to play a crucial role in insulin's metabolic responses. Protein kinase B (PKB) activity in liver was decreased in null mice compared with the wild-type and the null mice expressing IRS-1 or IRS-1Deltap85. In primary hepatocytes isolated from null mice, expression of IRS-1 enhanced both PI3K and PKB activities, but expression of IRS-1Deltap85 enhanced only PKB. These data suggest that PKB in liver plays a pivotal role in systemic glucose homeostasis and that PKB activation might be sufficient for reducing insulin resistance even without full activation of PI3K.

Troglitazone increases the number of small adipocytes without the change of white adipose tissue mass in obese Zucker rats.

Troglitazone (CS-045) is one of the thiazolidinediones that activate the peroxisome proliferator-activated receptor gamma (PPARgamma), which is expressed primarily in adipose tissues. To elucidate the mechanism by which troglitazone relieves insulin resistance in vivo, we studied its effects on the white adipose tissues of an obese animal model (obese Zucker rat). Administration of troglitazone for 15 d normalized mild hyperglycemia and marked hyperinsulinemia in these rats. Plasma triglyceride level was decreased by troglitazone in both obese and lean rats. Troglitazone did not change the total weight of white adipose tissues but increased the number of small adipocytes (< 2,500 micron2) approximately fourfold in both retroperitoneal and subcutaneous adipose tissues of obese rats. It also decreased the number of large adipocytes (> 5,000 micron2) by approximately 50%. In fact, the percentage of apoptotic nuclei was approximately 2.5-fold higher in the troglitazone-treated retroperitoneal white adipose tissue than control. Concomitantly, troglitazone normalized the expression levels of TNF-alpha which were elevated by 2- and 1.4-fold in the retroperitoneal and mesenteric white adipose tissues of the obese rats, respectively. Troglitazone also caused a dramatic decrease in the expression levels of leptin, which were increased by 4-10-fold in the white adipose tissues of obese rats. These results suggest that the primary action of troglitazone may be to increase the number of small adipocytes in white adipose tissues, presumably via PPARgamma. The increased number of small adipocytes and the decreased number of large adipocytes in white adipose tissues of troglitazone-treated obese rats appear to be an important mechanism by which increased expression levels of TNF-alpha and higher levels of plasma lipids are normalized, leading to alleviation of insulin resistance.

Development of non-insulin-dependent diabetes mellitus in the double knockout mice with disruption of insulin receptor substrate-1 and beta cell glucokinase genes. Genetic reconstitution of diabetes as a polygenic disease.

Non-insulin-dependent diabetes mellitus (NIDDM) is considered a polygenic disorder in which insulin resistance and insulin secretory defect are the major etiologic factors. Homozygous mice with insulin receptor substrate-1 (IRS-1) gene knockout showed normal glucose tolerance associated with insulin resistance and compensatory hyperinsulinemia. Heterozygous mice with beta cell glucokinase (GK) gene knockout showed impaired glucose tolerance due to decreased insulin secretion to glucose. To elucidate the interplay between insulin resistance and insulin secretory defect for the development of NIDDM, we generated double knockout mice with disruption of IRS-1 and beta cell GK genes by crossing the mice with each of the single gene knockout. The double knockout mice developed overt diabetes. Blood glucose levels 120 min after intraperitoneal glucose load (1.5 mg/g body wt) were 108 +/- 24 (wild type), 95 +/- 26 (IRS-1 knockout), 159 +/- 68 (GK knockout), and 210 +/- 38 (double knockout) mg/dl (mean +/- SD) (double versus wild type, IRS-1, or GK; P < 0.01). The double knockout mice showed fasting hyperinsulinemia and selective hyperplasia of the beta cells as the IRS-1 knockout mice (fasting insulin levels: 0.38 +/- 0.30 [double knockout], 0.35 +/- 0.27 [IRS-1 knockout] versus 0.25 +/- 0.12 [wild type] ng/ml) (proportion of areas of insulin-positive cells to the pancreas: 1.18 +/- 0.68%; P < 0.01 [double knockout], 1.20 +/- 0.93%; P < 0.05 [IRS-1 knockout] versus 0.54 +/- 0.26% [wild type]), but impaired insulin secretion to glucose (the ratio of increment of insulin to that of glucose during the first 30 min after load: 31 [double knockout] versus 163 [wild type] or 183 [IRS-1 knockout] ng insulin/mg glucose x 10(3)). In conclusion, the genetic abnormalities, each of which is nondiabetogenic by itself, cause overt diabetes if they coexist. This report provides the first genetic reconstitution of NIDDM as a polygenic disorder in mice.

Hypertension, hypertriglyceridemia, and impaired endothelium-dependent vascular relaxation in mice lacking insulin receptor substrate-1.

Insulin resistance is often associated with atherosclerotic diseases in subjects with obesity and impaired glucose tolerance. This study examined the effects of insulin resistance on coronary risk factors in IRS-1 deficient mice, a nonobese animal model of insulin resistance. Blood pressure and plasma triglyceride levels were significantly higher in IRS-1 deficient mice than in normal mice. Impaired endothelium-dependent vascular relaxation was also observed in IRS-1 deficient mice. Furthermore, lipoprotein lipase activity was lower than in normal mice, suggesting impaired lipolysis to be involved in the increase in plasma triglyceride levels under insulin-resistant conditions. Thus, insulin resistance plays an important role in the clustering of coronary risk factors which may accelerate the progression of atherosclerosis in subjects with insulin resistance.

Insulin signalling and insulin actions in the muscles and livers of insulin-resistant, insulin receptor substrate 1-deficient mice.

We and others recently generated mice with a targeted disruption of the insulin receptor substrate 1 (IRS-1) gene and demonstrated that they exhibited growth retardation and had resistance to the glucose-lowering effect of insulin. Insulin initiates its biological effects by activating at least two major signalling pathways, one involving phosphatidylinositol 3-kinase (PI3-kinase) and the other involving a ras/mitogen-activated protein kinase (MAP kinase) cascade. In this study, we investigated the roles of IRS-1 and IRS-2 in the biological action in the physiological target organs of insulin by comparing the effects of insulin in wild-type and IRS-1-deficient mice. In muscles from IRS-1-deficient mice, the responses to insulin-induced PI3-kinase activation, glucose transport, p70 S6 kinase and MAP kinase activation, mRNA translation, and protein synthesis were significantly impaired compared with those in wild-type mice. Insulin-induced protein synthesis was both wortmannin sensitive and insensitive in wild-type and IRS-1 deficient mice. However, in another target organ, the liver, the responses to insulin-induced PI3-kinase and MAP kinase activation were not significantly reduced. The amount of tyrosine-phosphorylated IRS-2 (in IRS-1-deficient mice) was roughly equal to that of IRS-1 (in wild-type mice) in the liver, whereas it only 20 to 30% of that of IRS-1 in the muscles. In conclusion, (i) IRS-1 plays central roles in two major biological actions of insulin in muscles, glucose transport and protein synthesis; (ii) the insulin resistance of IRS-1-deficient mice is mainly due to resistance in the muscles; and (iii) the degree of compensation for IRS-1 deficiency appears to be correlated with the amount of tyrosine-phosphorylated IRS-2 (in IRS-1-deficient mice) relative to that of IRS-1 (in wild-type mice).

Csk enhances insulin-stimulated dephosphorylation of focal adhesion proteins.

Insulin has pleiotropic effects on the regulation of cell physiology through binding to its receptor. The wide variety of tyrosine phosphorylation motifs of insulin receptor substrate 1 (IRS-1), a substrate for the activated insulin receptor tyrosine kinase, may account for the multiple functions of insulin. Recent studies have shown that activation of the insulin receptor leads to the regulation of focal adhesion proteins, such as a dephosphorylation of focal adhesion kinase (pp125FAK). We show here that C-terminal Src kinase (Csk), which phosphorylates C-terminal tyrosine residues of Src family protein tyrosine kinases and suppresses their kinase activities, is involved in this insulin-stimulated dephosphorylation of focal adhesion proteins. We demonstrated that the overexpression of Csk enhanced and prolonged the insulin-induced dephosphorylation of pp125FAK. Another focal adhesion protein, paxillin, was also dephosphorylated upon insulin stimulation, and a kinase-negative mutant of Csk was able to inhibit the insulin-induced dephosphorylation of pp125FAK and paxillin. Although we have shown that the Csk Src homology 2 domain can bind to several tyrosine-phosphorylated proteins, including pp125FAK and paxillin, a majority of protein which bound to Csk was IRS-1 when cells were stimulated by insulin. Our data also indicated that tyrosine phosphorylation levels of IRS-1 appear to be paralleled by the dephosphorylation of the focal adhesion proteins. We therefore propose that the kinase activity of Csk, through the insulin-induced complex formation of Csk with IRS-1, is involved in insulin's regulation of the phosphorylation levels of the focal adhesion proteins, possibly through inactivation of the kinase activity of c-Src family kinases.

Insulin effect during embryogenesis determines fetal growth: a possible molecular link between birth weight and susceptibility to type 2 diabetes.

Low birth weight has been reported to be associated with impaired insulin secretion and insulin resistance. It has been proposed that this association results from fetal programming in response to the intrauterine environment (the thrifty phenotype hypothesis). To elucidate the relationship between birth weight and genetically determined defects in insulin secretion, we measured the birth weights of neonates derived from crosses of male pancreatic beta-cell type glucokinase knockout (Gck+/-) mice and female wild-type (WT) or Gck+/- mice. In 135 offspring, birth weights were lower in the presence of a fetal heterozygous mutation and higher in the presence of a maternal heterozygous mutation. Moreover, Gck-/- neonates had significantly smaller birth weights than WT or Gck+/- neonates (means +/- SE 1.49+/-0.03 [n = 30] vs. 1.63+/-0.03 [n = 30] or 1.63+/-0.02 [n = 50] g, respectively; P<0.01). Thus, Gck mutations in beta-cells may impair insulin response to glucose and alter intrauterine growth as well as glucose metabolism after birth. This study has confirmed the results of a previous report that human subjects carrying mutations in Gck had reduced birth weights and has provided direct evidence for a link between insulin and fetal growth. Moreover, birth weights were reduced in insulin receptor substrate-1 knockout mice despite normal insulin levels. Taken together, these results suggest that a genetically programmed insulin effect during embryogenesis determines fetal growth and provides a possible molecular link between birth weight and susceptibility to type 2 diabetes.

Subcellular localization of insulin receptor substrate family proteins associated with phosphatidylinositol 3-kinase activity and alterations in lipolysis in primary mouse adipocytes from IRS-1 null mice.

To clarify the roles of insulin receptor substrate (IRS) family proteins in phosphatidylinositol (PI) 3-kinase activation and insulin actions in adipocytes, we investigated the intracellular localization of IRS family proteins and PI 3-kinase activation in response to insulin by fractionation of mouse adipocytes from wild-type and IRS-1 null mice. In adipocytes from wild-type mice, tyrosine-phosphorylated IRS-1 and IRS-2, which were found to associate with PI 3-kinase in response to insulin, were detected in the plasma membrane (PM) and low-density microsome (LDM) fractions. By contrast, tyrosine-phosphorylated IRS-3 (pp60), which was found to associate with PI 3-kinase, was predominantly localized in the PM fraction. In adipocytes from IRS-1-null mice, insulin-stimulated PI 3-kinase activity in anti-phosphotyrosine (alphaPY) immunoprecipitates in the LDM fraction was almost exclusively mediated via IRS-2 and was reduced to 25%; however, insulin-stimulated PI 3-kinase activity in the PM fraction was primarily mediated via IRS-3 and was reduced to 60%. To determine the potential functional impact of the distinct subcellular localization of IRSs and associating PI 3-kinase activity on adipocyte-specific metabolic actions, we examined lipolysis in IRS-1 null mice. The level of isoproterenol-induced lipolysis was increased 5.1-fold in adipocytes from IRS-1 null mice as compared with wild-type mice. Moreover, hormone-sensitive lipase (HSL) protein was increased 4.3-fold in adipocytes from IRS-1-null mice compared with wild-type mice, and HSL mRNA expression was also increased. The antilipolytic effect of insulin in IRS-1 null adipocytes, however, was comparable to that in wild-type mice. Thus, discordance between these two insulin actions as well as the transcriptional and translational effect (HSL mRNA and protein regulation) and the PM effect (antilipolysis) of insulin may be explained by distinct roles of both PI 3-kinase activity associated with IRS-1/IRS-2 and PI 3-kinase activity associated with IRS-3 in insulin actions related to their subcellular localization.

Insulin receptor-related receptor is expressed in pancreatic beta-cells and stimulates tyrosine phosphorylation of insulin receptor substrate-1 and -2.

The receptor-type protein tyrosine kinases in murine pancreatic islets were screened to identify possible growth/differentiation factors in pancreatic beta-cells. The analysis revealed that insulin receptor-related receptor (IRR) is highly expressed in the islets as well as in several highly differentiated beta-cell lines derived from transgenic mice. Islets predominantly contain IRR as uncleaved proreceptors compared with IRR as processed forms in the beta-cell lines, suggesting that the activity of IRR is regulated on the level of processing proteases in vivo. To examine the IRR signaling pathway, a chimeric receptor consisting of the extracellular domain of insulin receptor and the intracellular domain of IRR was expressed in Chinese hamster ovary cells. The hybrid receptor is functional because insulin is capable of tyrosine-phosphorylating the catalytic domain in these cells. It also stimulates the tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2, indicating that both proteins serve as substrates of IRR-protein tyrosine kinase in intact cells. The phenotype of the IRS-2 knockout mouse recently reported suggests that an IRS-2-mediated signaling pathway controls the compensatory increase in pancreatic beta-cell mass in insulin-resistant states. From our findings of the specific expression of IRR and its ability of signaling to IRS-2, we speculate that this receptor might play a role in the regulation of beta-cell mass.

Nicorandil improves diabetes and rat islet beta-cell damage induced by streptozotocin in vivo and in vitro.

OBJECTIVE: N-(2-hydroxyethyl)-nicotinamide nitrate (nicorandil) is a unique anti-anginal agent, reported to act as both an ATP-sensitive K(+) channel opener (PCO) and a nitric oxide donor. It also has an anti-oxidant action. We examined the effects of nicorandil on streptozotocin (STZ)-induced islet beta-cell damage both in vivo and in vitro. DESIGN AND METHODS: STZ-induced diabetic Brown Norway rats (STZ-DM) were fed with nicorandil-containing chow from day 2 (STZ-DM-N48), 3 (STZ-DM-N72), and 4 (STZ-DM-N96) to day 30. Body weight, blood glucose, and plasma insulin were measured every week. For the in vitro assay, neonatal rat islet-rich cultures were performed and cells were treated with nicorandil from 1 h before to 2 h after exposure to STZ for 30 min. Insulin secretion from islet cells was assayed after an additional 24 h of culture. We also observed the effect of nicorandil on the generation of reactive oxygen species (ROS) from rat inslinoma cells (RINm5F). RESULTS: Body weight loss and blood glucose levels of STZ-DM-N48 rats were significantly lower than those of STZ-DM rats. Immunohistochemical staining of insulin showed preservation of insulin-secreting islet beta-cells in STZ-DM-N48 rats. Nicorandil also dose-dependently recovered the insulin release from neonatal rat islet cells treated with STZ in in vitro experiments. Nicorandil did not act as a PCO on neonatal rat islet beta-cells or RINm5F cells, and did not show an inhibitory effect on poly(ADP-ribose) polymerase-1. However, the drug inhibited the production of ROS stimulated by high glucose (22.0 mmol/l) in RINm5F cells. CONCLUSIONS: These results suggested that nicorandil improves diabetes and rat islet beta-cell damage induced by STZ in vivo and in vitro. It protects islet beta-cells, at least partly, via a radical scavenging effect.

Anti-IRS-1 monoclonal antibody, 6G5, cross-reacts to IRS-2.

We previously reported the production of anti-IRS-1 monoclonal antibodies against human IRS-1 C-terminal portion. One of anti IRS-1 monoclonal antibodies, 6G5, was found to immunologically cross-react with IRS-2, which has been reported recently. The data presented here provide information arising from the use of the anti-IRS-1 MAb, 6G5; this MAb was found to be a useful reagent in studying the functional role of IRSs in the insulin-signaling system.

[The role of phosphorylation cascade in insulin action].

Insulin induces a wide variety of growth and metabolic response in many cell types. Insulin initiates its biological effects by activation of tyrosine kinase in the beta-subunit and phosphorylates several proteins, such as insulin receptor substrate-1 (IRS-1), Shc. thereby activating phosphatidyl inositol 3-kinase activity, and ras activity. MAP kinase cascade activated by ras, 70kDaS6 kinase lying downstream of PI3-kinase, and the regulation of glycogen synthase have been discussed.

Vasopressin-dependent upregulation of aquaporin-2 gene expression in aged rats with glucocorticoid deficiency.

AIM: The study was undertaken to determine whether ageing affects kidney expression of the aquaporin-2 (AQP2) water channel in glucocorticoid-deficient rats. METHODS: After adrenalectomy, 6- and 52-week-old Sprague-Dawley rats received aldosterone via osmotic minipumps (glucocorticoid-deficient rats). Aldosterone and dexamethasone were administered to control rats of the same age. RESULTS: An acute water load test verified impairment of water excretion in both young and aged rats with glucocorticoid deficiency, with a more serious impairment in the older rats. Despite the presence of hypoosmolality, non-suppressible release of arginine vasopressin (AVP) was particularly evident in the aged rats with glucocorticoid deficiency in comparison with the young rats. The expression levels of AQP2 mRNA and protein were lower in the aged rats, with a particularly large reduction in AQP2 protein expression. AQP2 expression levels were significantly augmented in the glucocorticoid-deficient rats compared with the controls under both basal and water-loaded conditions. Acute water loading did not suppress expression of AQP2 mRNA and protein, and the percentage increases in AQP2 mRNA and protein expression vs. the respective controls were more pronounced in the 52-week-old glucocorticoid-deficient rats compared with the 6-week-old rats. CONCLUSION: The findings indicate that upregulation of AQP2 expression is maintained dependent upon non-suppressible release of AVP in rats with glucocorticoid deficiency, and that AQP2 plays a crucial role in persistent impairment of water excretion in aged rats with glucocorticoid deficiency.

Identification of a 190-kDa protein as a novel substrate for the insulin receptor kinase functionally similar to insulin receptor substrate-1.

Recently, we generated mice with a targeted disruption of the insulin receptor substrate-1 (IRS-1) gene and demonstrated that they exhibited growth retardation and mild insulin resistance, suggesting the presence of IRS-1-independent pathway that partially substitutes for IRS-1 in IRS-1-deficient mice (Tamemoto, H., Kadowaki, T., Tobe, K., Yagi, T., Sakura, H., Hayakawa, T., Terauchi, Y., Ueki, K., Kaburagi, Y., Satoh, S., Sekihara, H., Yoshioka, S., Horikoshi, H., Furuta, Y., Ikawa, Y., Kasuga, M., Yazaki, Y., and Aizawa, S. (1994) Nature 372, 182-186). We have examined the insulin-stimulated tyrosine-phosphorylated proteins in livers of wild type and IRS-1-deficient mice. Tyrosine phosphorylation of an 190-kDa protein (pp190) by insulin was significantly stimulated in livers of IRS-1-deficient mice, which was weakly observed in wild type mice in addition to IRS-1. We also demonstrated that pp190 was immunologically distinct from IRS-1 and was associated with both the 85-kDa subunit of phosphatidylinositol 3-kinase and the Grb2/Ash molecule as IRS-1. We identified pp190 as a novel substrate for insulin receptor kinase (IRS-2), which can bind both PI3-kinase and Ash/Grb2, and whose tyrosine phosphorylation is specifically induced in IRS-1-deficient mice. These data suggested that pp190 may play some physiological roles in insulin's signal transduction; furthermore, induction of tyrosine phosphorylation of pp190 may be one of the compensatory mechanisms that substitute for IRS-1 in IRS-1-deficient mice.

Insulin and platelet-derived growth factor stimulate phosphorylation of the c-raf product at serine and threonine residues in intact cells.

We have examined the phosphorylation of the serine threonine kinase, the product of c-raf proto-oncogene in response to insulin or platelet-derived growth factor in intact cells. Both insulin and platelet-derived growth factor stimulated phosphorylation of the c-raf protein about 2- to 3-fold. The phosphorylation occurred exclusively on serine and threonine residues; phosphotyrosine was not detected. In immune-complex kinase assays, treatment with insulin, and platelet-derived growth factor increased autophosphorylation of the c-raf kinase, suggesting activation of its kinase activity. To investigate whether the phosphorylation of the c-raf protein in intact cells results from an autophosphorylation event or from the phosphorylation by other cellular kinase(s), we replaced lysine 375 in the putative ATP-binding domain of the c-raf protein with alanine using oligonucleotide site-directed mutagenesis and expressed the mutated protein in NIH3T3 cells. The substitution resulted in the inactivation of the serine/threonine-specific autophosphorylation in immune-complex kinase assays. In intact cells, however, although phosphorylation of the mutant protein in response to insulin and platelet-derived growth factor occurred to a lesser extent than that of the wild-type protein, the phosphopeptide maps were indistinguishable. These results suggest that serine threonine phosphorylation might be responsible for the activation of c-raf kinase upon treatment of cells with insulin and platelet-derived growth factor, and most of the phosphate associated with the c-raf protein results from its phosphorylation by as yet uncharacterized cellular serine/threonine kinase(s).

A novel negative selection for homologous recombinants using diphtheria toxin A fragment gene.

In producing mutant mice by gene targeting in embryonic stem (ES) cells, the efficient isolation of the homologous recombinants is still a critical step. We previously reported on a negative selection using the diphtheria toxin A (DT-A) fragment gene for homologous recombinants (1). It was efficient but limited to gene loci expressed in ES cells. For wider applicability of this negative selection to many gene loci not expressed or expressed at low levels in ES cells, we exploited a novel targeting vector composed of a polyA-less neo gene, a mRNA destabilizing signal, a pausing signal for RNA polymerase II from the minute virus of mice, and the DT-A gene. There was about a 30-fold decrease in frequency of G418-resistant colonies with this strategy against that using only the neo gene in the vector, and homologous recombinants were obtained at frequencies of more than 1/50 among G418 resistant cells at fyn, csk, c-mos, and insulin receptor substrate-1 gene loci.

Regulatable production of insulin from primary-cultured hepatocytes: insulin production is up-regulated by glucagon and cAMP and down-regulated by insulin.

To utilize hepatocytes for insulin-producing surrogate cells, we devised a regulatory secretion system by placing proinsulin DNA under the regulatable promoter for phosphoenolpyruvate carboxykinase (PEPCK). The expression of PEPCK is down-regulated by insulin, and up-regulated by cAMP and glucagon. To express insulin in hepatocytes, we constructed an adenoviral insulin expression system. After infection, the hepatocytes secreted immunoreactive insulin (IRI) at an increasing rate. IRI secretion increased over four-fold upon stimulation with 300 microM cAMP and 500 microM of the cAMP-dependent phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). This increase was also observed with glucagon and IBMX. Production was augmented two-fold by the addition of wortmannin, phosphatidylinositol (PI)-3-kinase inhibitor, suggesting that inhibitory insulin signaling to the PEPCK promoter may be mediated through PI-3-kinase. Addition of exogenous insulin to the culture decreased insulin mRNA expression remarkably on Northern blot. Thus, by using a PEPCK promoter for insulin expression, we were able to up-regulate insulin production from hepatocytes with cAMP and glucagon, and down-regulate with insulin itself.