Suppressor of cytokine signaling-3
Observations pertaining to suppressor of cytokine signaling-3 (SOCS-3) point to a new mechanism for insulin signal modulation . The SOCS family of proteins (CIS and SOCS 1 – 7) were originally described as a negative feedback loop for cytokine receptor signaling through Janus kinase (JaK). JaK phosphorylates and activates signal transducer and activator of transcription (STAT), which is a transcription factor that augments SOCS gene expression. SOCS proteins then feedback to inhibit tyrosine-phosphorylated cytokine receptors, via either competitive binding through their SH2 domain preventing phosphorylation of cytokine receptor substrates or by binding and inhibiting Jak tyrosine kinases. Evidence suggests that a similar mechanism may be operative for the insulin receptor . Insulin stimulates STAT5B activation by direct tyrosine phosphorylation after STAT5B binds to insulin receptor phosphotyrosine 972 via its SH2 domain (i.e., this interaction does not involve a PTB as is the case with IRS). STAT5B induces expression of SOCS-3, which then competes with STAT5B binding to the insulin receptor inhibiting further STAT5B activation. Thus, SOCS-3 could function as a negative regulator for insulin signaling, not only with respect to STAT5B activation, but also for other signaling pathways to the extent that SOCS-3 could prevent the interaction between the PTB domain of IRS and the insulin receptor. It is intriguing that several factors that induce cellular insulin resistance also induce SOCS-3 expression, including TNFα , growth hormone , and leptin .
Plasma differentiation factor-1 (PC-1)
PC-1 is a membrane glycoprotein with pyrophosphatase activity that appears to act as an intrinsic inhibitor of the insulin receptor tyrosine kinase through an undefined mechanism [232–234]. Muscle expression of PC-1 is elevated in diabetes and in obesity, and correlates with diminished insulin receptor tyrosine phosphorylation and muscle glucose uptake .
Regulation of gene transcription
Insulin response elements
Insulin regulates the expression of many individual proteins through effects on translation, mRNA stability, and gene transcription. The stimulatory effect on translation generally results from increases in the initial phase of translation of mRNA by ribosomes, and is discussed in Chapter 16: Insulin regulation of protein metabolism. Regulation of gene transcription can be due to effects on transcription factors that are directly modified via insulin signaling mechanisms, or that are indirect as the result of stimulation of glucose metabolism. For example, genes encoding pyruvate kinase, fatty acid synthase, acetyl-CoA carboxylase, and stearoyl-CoA desaturase require increases in both insulin and glucose for induction, and PEPCK expression can be suppressed independently by insulin or glucose. The promoter elements (cis acting sequences) that mediate changes in gene transcription are referred to as insulin response sequences (IRS) or insulin response elements (IRE). There clearly exist multiple distinct classes of IREs that participate in the regulation of different gene promoters, rather than a single consensus nucleotide sequence . These IREs can mediate either positive effects (GAPDH, pyruvate kinase, fatty acid synthase, somatostatin, c-fos, glucokinase, apoA1) or negative effects (e.g. PEPCK, glucose-6 phosphatase, tyrosine aminotransferase, apoCIII, glucagon) on gene transcription. It is also important to consider the identity of the transcription factors (trans acting factors) activated by insulin that bind to the IREs and alter gene transcription rates. While the identification of transcription factors has progressed more slowly than with IREs, there has been progress in identifying transcription factors and signaling mechanisms involved in insulin regulation of specific genes.
Insulin regulation of gene expression has been most readily attributed to the mitogenic MAP kinase pathway. However, insulin inhibition of PEPCK, IGFBP-1, and glucose-6 phosphatase genes employs the PEPCK-like IRE with consensus sequence T(G/A)TTT(T/G)(G/T) in the core motif, and this occurs through a PI-3 kinase-dependent pathway . The downstream targets of PI-3 kinase are somewhat controversial with studies arguing for and against the involvement of Akt/PKB and a recent study supporting a role for GSK-3 in the inhibition of PEPCK and glucose-6-phosphatase genes . Nevertheless, PI-3 kinase pathways can also regulate gene transcription, and this calls into question notions of distinct and separate metabolic and mitogenic insulin action pathways in the web of insulin signal transduction.
TPA and serum response elements
The ability of insulin to alter gene expression via the mitogenic MAP kinase pathway has long been recognized . This classic mechanism involves phosphorylation of transcription factors in the activating protein-1 (AP-1) family such as c-Jun and c-fos which interact with the TPA response element (TRE), and ternary complex factors such as Elk-1, SAP-1, and SAP-2 which interact with the serum response element (SRE). Both elements can be found in single gene promoters, for example, in the c-fos gene which is induced by insulin through activation of both c-Jun and Elk-1.
Forkhead transcription factors
Genetic studies in the nematode, Caenorhababtidis elegans, have led to the discovery of an additional pathway for insulin-regulated gene expression . One of the developmental stages in C. elegans is the dauer stage characterized by increased longevity, reduced metabolic activity, and increased body fat. A series of specific gene mutations, termed Daf alleles, has defined a signal transduction pathway leading to a constitutive dauer phenotype. Daf genes include the homologues of the insulin/IGF-1 receptor, PI-3 kinase, PDK-1, and Akt/PKB isoform genes in mammals, as well as Daf-16 which encodes a transcription factor with homology to the mammalian forkhead family of transcription factors. Forkhead transcription factors were first identified as being involved in the forkhead mutation in Drosophila, and the subclass of mammalian forkhead proteins with greatest homology to Daf-16 was first identified in alveolar rhabdomyosarcoma; this latter subclass is referred to as forkhead in human rhabdomyosarcoma (FKHR). The FKHR family is comprised of three expressed genes, FKHR or FOXO1, FKHRL1 or FOXO3, and AFX or FOXO4 . FOXO1 is the most highly expressed FKHR isoform in insulin-responsive tissues such as liver, adipose tissue, and pancreatic β cells.
In nematodes and in mammals, the FKHR proteins represent an important signal transduction pathway by which insulin receptors modulate gene expression. Insulin stimulation results in PI-3 kinase-dependent phosphorylation of FKHR transcription factors by Akt/PKB at three different phosphorylation sites (Thr-24, Ser-256, and Ser-319 in FOXO1) . Under basal conditions, FKHR proteins reside within the nucleus, but, upon phosphorylation, dissociate from their DNA binding site, and are then excluded from the nucleus and retained in the cytosol. Replacement of Thr-24 or Ser-256 by alanine results in loss of phosphorylation by Akt/PKB, lack of FOXO1 nuclear export, and failure of insulin-mediated promoter suppression . The relocation of FKHR out of the nucleus represents an effective mechanism for insulin in the suppression of transcriptional activity. For example, FKHR proteins bind to cis elements in the promoters of the PEPCK, glucose-6-phosphatase, and IGFBP-1 genes and augment transcriptional activity. Transactivation by FKHR is prevented when cells are stimulated by insulin [241 – 243]. The effects on PEPCK and glucose-6-phosphatase genes have led some investigators to the conclusion that FKRH could help coordinate the overall suppression of gluconeogenesis and hepatic glucose production by insulin. However, FKHR proteins are not involved in the regulation of all genes that are suppressed by insulin. Furthermore, even in the case of PEPCK and glucose-6-phosphatase, FKHR cannot by itself account for all of transcriptional inhibition under physiologic conditions. FKHR can clearly mediate transactivation and insulin suppression of glucose-6-phosphatase and PEPCK when FKHR is overexpressed in cells; however, at physiologic levels, FKHR may not be essential for gene regulation, suggesting that other transcription factors predominate in the regulation of these two genes [243,244].
Another transcription factor, peroxisome proliferative activated receptor-γ co-activator 1 (PGC-1), was first identified as a factor involved in brown fat adipogenesis . PGC-1 also plays an important role in the regulated expression of gluconeogenic enzymes, even at physiologic concentrations within hepatocytes [246,247]. In addition, PGC-1 is induced in the liver by glucagon and glucocorticoids in the context of fasting, insulin deficiency, and diabetes, and then participates in the induction of the gluconeogenic program. However, in regulating hepatic gluconeogenesis, PGC-1 may physically interact with FOXO1 and function as its co-activator . In this role, FOXO1 function appears to be necessary for full induction of gluconeogenic genes by PGC-1 in hepatic cells and mouse liver. Several observations indicate that insulin may suppress gluconeogenic genes by interfering with the interaction between PGC-1 and FOXO1. For example, Akt/PKB is able to phosphorylate FOXO1, and this prevents the binding and coactivation by PGC-1. Also, insulin is able to suppress the induction of gluconeogenic genes by PGC-1 but not in the presence of a constitutively active FOXO1 mutant that is insensitive to insulin . These studies indicate that a physiologic role for FOXO1 in regulating gluconeogenic gene expression may only become evident when this is examined in the context of coactivation by PGC-1.
FKHR may also play other import roles in the biology of insulin-responsive cells. FOXO1 is involved in the stimulation of pancreatic β-cell proliferation and regulation of Pdx1 expression by insulin . FOXO1 also appears to contribute to the complex coordination of transcriptional events involved in adipocyte differentiation. Expression of a constitutively-active FOXO1 mutant prevents differentiation of preadipocytes, and
FOXO1 haploinsufficiency in mice protects from diet-induced glucose intolerance and hyperinsulinemia .
Sterol response element binding protein-1c (SREBP-1c)
Another exciting observation is the role of SREBP-1c in insulin-regulated gene expression. The sterol response element binding protein family of transcription factors is classically viewed as being involved in the regulation of genes in response to the cellular availability of cholesterol . This family includes SREBP-1a and SREBP-1c, which are encoded by a single gene and differ only in their first exon through use of alternative transcription start sites, and the homologous SREBP-2 encoded by a second gene . Evidence has accumulated that SREBP-1c is regulated primarily by insulin rather than cholesterol availability, and is also involved in adipocyte differentiation which explains the factor’s alternative designation as adipocyte determination and differentiation factor-1 (ADD1) . SREBP-1c/ADD1 is most highly expressed in liver, white adipose tissue, muscle, adrenal gland, and brain.
SREBPs contain an NH2-terminal domain consisting of a basic helix-loop-helix leucine zipper transcription factor, a central domain comprised of two transmembrane-spanning regions, and a COOH-terminal regulatory domain. In this form, SREBPs are associated with membranes in the endoplasmic reticulum. When membrane cholesterol concentrations are decreased, SREBP-1a and SREBP-2 undergo two sequential proteolytic cleavages, releasing the transcriptional domain, and allowing for its translocation into the nucleus . These transcription factors then bind sterol responsive elements (SREs) in specific gene promoters and induce genes involved in cholesterol biosynthesis (i.e., HMG-CoA reductase, HMG-CoA synthase) and the LDL receptor, resulting in the cellular replenishment of cholesterol. Primarily regulation of SREBP-1c by insulin was first manifested in livers of fasted rodents re-fed with a high carbohydrate diet [255–257]. Subsequently, insulin was demonstrated to stimulate the expression and transcription of SREBP-1c in cell lines and in adipose, liver, and muscle tissues. This insulin effect is mediated through IRS-1 and PI-3 kinase, and possibly Akt/PKB , although downstream targets and transcription factors mediating induction of SREBP-1c gene transcription have not been elucidated. Regardless, SREBP-1c appears to play an important role in the regulation of specific genes in response to insulin. In experiments involving overexpression of wild-type, constitutively active, and dominant interfering mutants, SREBP-1c has been shown to be involved in the induction of fatty acid synthase and leptin in adipose cells, and glucokinase, pyruvate kinase, fatty acid synthase, and acetyl-CoA carboxylase in the liver . Thus, SREBP-1c mediates a positive transcriptional effect on genes promoting glycolysis and lipogenesis, and seems to prepare the liver for carbohydrate availability following a meal. While containing SREs in their promoter regions, the above-mentioned genes are regulated by multiple hormones as well as metabolic substrates, and the relative contribution of SREBP-1c in overall transactivation by insulin has not been defined.
Signal transducer and activator of transcription 5B (STAT5B)
As discussed earlier in relationship to SOCS-3, the insulin receptor directly activates the STAT5B transcription factor via tyrosine phosphorylation . STAT5B binds phospho tyrosine 972 of the insulin receptor, which is the same juxtamembrane phosphotyrosine involved in IRS binding via their PTB domains. Binding of STAT5B is the result of a SH2 domain interaction as opposed to the NPXpY-consensus sequence characterizing PTB domain interactions. Direct tyrosine phosphorylation of STAT5B by the insulin receptor kinase allows for dimerization through SH2 domain interactions and translocation into the nucleus [260,261]. This is in contrast to the general mechanism of STAT factor activation by cytokine receptors, which employs JaK kinase as the activating kinase. The complement of genes regulated through insulin activation of STAT5B has not been extensively studied. However, both glucokinase and SOCS-3 genes have STAT5 binding sites in their promoter regions, and transcriptional activity is stimulated by insulin via STAT5B [41,261].
The work was supported by research grants from the National Institutes of Health [DK-38765 (WTG), DK-083562 (WTG), DK047936 (LJM), DK066483 (LJM)], the UAB Diabetes Research Center (P60-DK079626), the American Diabetes Association, and the Merit Review program of the Department of Veterans Affairs.