A Multicomponent Insulin Response Sequence Mediates a Strong Repression of Mouse Glucose-6-phosphatase Gene Transcription by Insulin

Abstract

Glucose-6-phosphatase (G6Pase) catalyzes the final step in the gluconeogenic and glycogenolytic pathways. The transcription of the gene encoding the catalytic subunit of G6Pase is stimulated by glucocorticoids, whereas insulin strongly inhibits both basal G6Pase gene transcription and the stimulatory effect of glucocorticoids. To identify the insulin response sequence (IRS) in the G6Pase promoter through which insulin mediates its action, we have analyzed the effect of insulin on the basal expression of mouse G6Pase-chloramphenicol acetyltransferase (CAT) fusion genes transiently expressed in hepatoma cells. Deletion of the G6Pase promoter sequence between ؊271 and ؊199 partially reduces the inhibitory effect of insulin, whereas deletion of additional sequence between ؊198 and ؊159 completely abolishes the insulin response. The presence of this multicomponent IRS may explain why insulin potently inhibits basal G6Pase-CAT expression. The G6Pase promoter region between ؊198 and ؊159 contains an IRS, since it can confer an inhibitory effect of insulin on the expression of a heterologous fusion gene. This region contains three copies of the T(G/A)TTTTG sequence, which is the core motif of the phosphoenolpyruvate carboxykinase (PEPCK) gene IRS. This suggests that a coordinate increase in both G6Pase and PEPCK gene transcription is likely to contribute to the increased hepatic glucose production characteristic of patients with non-insulin-dependent diabetes mellitus.While insulin has long been known to modulate intracellular metabolism by altering the activity or intracellular location of various enzymes, it is only more recently that the regulation of gene transcription by insulin has been recognized as a major action of this hormone (1). cis-Acting elements that mediate the action of insulin on gene transcription, referred to as an insulin response sequences or elements (IRSs/IREs), 1 have been identified in a number of genes but, unlike cAMP (2, 3), which regulates gene transcription predominantly through one cisacting element, it is already apparent that a single consensus IRS does not exist (1). Instead, most of the sequences identified to date appear unique, a situation that resembles that for phorbol esters that regulate gene transcription through at least eight distinct consensus sequences (4).Non-insulin-dependent diabetes mellitus (NIDDM) is characterized by defects in insulin secretion, peripheral glucose utilization, and hepatic glucose production (HGP) (5). The ability of insulin to stimulate peripheral glucose utilization and repress HGP in patients with NIDDM is reduced, a phenomenon known as insulin resistance (5). Various investigators have speculated that an alteration in the insulin-regulated expression of specific genes, a consequence of insulin resistance, may contribute to the pathophysiology of this disease (1, 5, 6). The gene encoding the glucose-6-phosphatase (G6Pase) catalytic subunit (7) is one such candidate, since it catalyzes the final step in the gluconeogenic pathway, the conversion of ...

Identification of a cAMP response element within the glucose- 6-phosphatase hydrolytic subunit gene promoter which is involved in the transcriptional regulation by cAMP and glucocorticoids in H4IIE hepatoma cells

“…This region contains the 10 bp perfect palindromic sequence 5h-TTTACG-TAAA-3h. The sequence and position of this motif relative to the previously described insulin response element [10] are conserved between the human, rat and mouse G6Pase genes ( Figure 2). …”

“…Due to the similarity of the sequence k161\k152 to the consensus sequence of a CRE, the mutations were introduced in such a way as to prevent potential CREB binding [14], while not interfering with the overlapping motif of the insulin response element located between nt k164 and k158 [10] (Figure 2). The mutagenesis of nt k157, k154 and k153 within construct k180\j4 decreased the reporter-gene expression by a similar degree to that resulting from deletion of the sequence k161 to k150, as can be seen by comparing CRE2mut with the construct k150\j4 (Figure 3).…”

“…Glucagon (acting via cAMP) and glucocorticoids increase G6Pase activity by inducing G6Pase gene expression in the liver during starvation [4][5][6][7]. The recent cloning of the promoter regions of the human [8], rat [9] and mouse [10] G6Pase genes has made it possible to study the molecular basis of control of G6Pase gene expression. An insulin response element has recently been described in the G6Pase promoter and shown to be highly similar to one in the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter [10].…”

“…The recent cloning of the promoter regions of the human [8], rat [9] and mouse [10] G6Pase genes has made it possible to study the molecular basis of control of G6Pase gene expression. An insulin response element has recently been described in the G6Pase promoter and shown to be highly similar to one in the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter [10]. In addition, the binding of the hepatic nuclear factors HNF 1α and HNF 3γ to three activation elements between nt k234 and k66 has been shown to be essential for basal G6Pase gene-promoter expression [11].…”

Sequence variation between the mouse and human glucose-6-phosphatase catalytic subunit gene promoters results in differential activation by peroxisome proliferator activated receptor gamma coactivator-1α

“…Cell culture and transient transfections Rat H4IIE hepatoma cells were cultured and transiently transfected in suspension using the calcium phosphate-DNA co-precipitation method as previously described [27].…”