Analysis of the Signaling Pathway Involved in the Regulation of Hexokinase II Gene Transcription by Insulin

Abstract

The hexokinases, by converting glucose to glucose 6-phosphate, help maintain the glucose concentration gradient that results in the movement of glucose into cells through the facilitative glucose transporters. Hexokinase II (HKII) is the major hexokinase isoform in skeletal muscle, heart, and adipose tissue. Insulin induces HKII gene transcription in L6 myotubes, and this, in turn, increases HKII mRNA and the rates of HKII protein synthesis and glucose phosphorylation in these cells. Inhibitors of distinct insulin signaling pathways were used to dissect the molecular mechanism by which HKII gene expression is induced by insulin in L6 myotubes. Treatment with wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), or with rapamycin, an inhibitor of the pathway from the insulin receptor to p70/p85 ribosomal S6 protein kinase (p70 s6k ), prevented the induction of HKII mRNA by insulin. In contrast, treatment with PD98059, an inhibitor of mitogen-activated protein kinase activation, had no effect on insulin-induced HKII mRNA. In addition, rapamycin blocked the insulin-induced expression of an HKII promoter-chloramphenicol acetyltransferase fusion gene transiently transfected into L6 myotubes, whereas PD98059 had no such effect. These results suggest that a phosphatidylinositol 3-kinase/p70 s6k -dependent pathway is required for regulation of HKII gene transcription by insulin and that the Ras-mitogen-activated protein kinase-dependent pathway is probably not involved.The four mammalian hexokinases (ATP:D-hexose-6-phosphotransferase, EC 2.7.1.1; designated HKI through IV) 1 are a family of closely related enzymes that convert glucose to glucose 6-phosphate (for review, see Ref. 1). This enzymatic step initiates glucose metabolism and ensures the glucose concentration gradient that results in the movement of glucose into the cell through the facilitative glucose transporters (designated GLUT1 through 5) (2). Although HKI and HKII are both expressed in skeletal muscle and adipose tissue, HKII is the predominant isoform in these tissues (1). HKII catalytic activity is increased by insulin, whereas that of HKI is unaffected (3, 4). L6 myotubes have been used to analyze this action of insulin. In these cells insulin selectively increases HKII mRNA and protein synthesis, and this is associated with an increased rate of glucose utilization (4, 5). Hyperinsulinemic clamp studies have also demonstrated that HKII mRNA is increased by insulin in human and rat skeletal muscle in vivo (6, 7).The binding of insulin to a specific cell-surface receptor results in the activation of an intrinsic receptor tyrosine kinase (for review, see Ref. 8). The activated receptor phosphorylates insulin receptor substrate-1 (IRS-1) and this phosphoprotein, through interaction with specific Src homology 2 (SH2) domaincontaining proteins, initiates divergent signaling cascades (for review, see Ref. 9). For example, the enzyme phosphatidylinositol 3-kinase (PI 3-kinase; EC 2.7.1.67) is activated when it binds to IRS-1 (9). This leads...

Hexokinase II integrates energy metabolism and cellular protection: Akting on mitochondria and TORCing to autophagy

“…[41][42][43][44] The correlation between Akt activity and HK-II expression suggest the transcriptional regulation of HK-II by Akt, which has been supported by many studies. Insulin treatment increases HK-II mRNA and protein in various cell types and the increase is blocked by inhibition of PI3K, an upstream kinase of Akt, as well as inhibition of mTORC1, suggesting Akt/mTORC1 contribution 44,[46][47][48][49][50][51] (Figure 2). Hyperactivity of mTORC1 is sufficient to increase HK-II expression 52 and a recent comprehensive and unbiased analysis also supports mTORC1-mediated HK-II upregulation and further demonstrated that mTORC1 signaling activates the genes encoding nearly every step of glycolysis.…”

“…This is in sharp contrast to HK-I, which is unregulated by Akt. 20,49,179,180 HK-I more tightly binds to mitochondria with less sensitivity to G-6P-dependent dissociation than HK-II, rendering it primarily a glycolytic enzyme 4,5 which provides logical support for its predominant expression in the brain, a tissue highly dependent on glucose for energy generation. MitoHK-I displays similar anti-apoptotic characteristics to mitoHK-II thus HK-I appears to be a more specialized isoform geared primarily to facilitate glycolysis and mitochondrial protection.…”

The human hexokinase II gene promoter: functional characterization and detection of variants among patients with NIDDM

“…Osawa et al [65] recently observed that insulin induced by 3.5-fold the activity of a reporter gene driven by 487 bp of the proximal rat HKII promoter transfected into rat L6 myotubes but did not define the insulin response elements within this construct. Interestingly, these authors also showed that the induction of HKII by insulin was blocked by treatment with either wortmannin or rapamycin, suggesting that a phosphatidylinositol 3-kinase (PI 3-kinase)/ p70/p85 ribosomal S6 protein kinase (p70 s6 k )-dependent pathway may transduce the action of insulin on HKII gene expression.…”

Platelet-derived Growth Factor Stimulation of Monocyte Chemoattractant Protein-1 Gene Expression Is Mediated by Transient Activation of the Phosphoinositide 3-Kinase Signal Transduction Pathway

“…Glucose-6-phosphate dehydrogenase (48) and hexokinase II (49) are examples of genes whose stimulated expression is PI 3-kinase-dependent. The stimulation of these genes by insulin is rapamycin-sensitive, indicating the involvement of pp70S6K in this stimulatory mechanism.…”