PGC-1ß and ChREBP partner to cooperatively regulate hepatic lipogenesis in a glucose concentration-dependent manner.

Peroxisome proliferator-activated receptorγ coactivators (PGC-1α and PGC-1ß) play important roles in the transcriptional regulation of intermediary metabolism. To evaluate the effects of overexpressing PGC-1α or PGC-1ß at physiologic levels in liver, we generated transgenic mice with inducible overexpression of PGC-1α or PGC-1ß. Gene expression array profiling revealed that whereas both PGC-1 family proteins induced mitochondrial oxidative enzymes, the expression of several genes involved in converting glucose to fatty acid was induced by PGC-1ß, but not PGC-1α. The increased expression of enzymes involved in carbohydrate utilization and de novo lipogenesis by PGC-1ß required carbohydrate response element binding protein (ChREBP). The interaction between PGC-1ß and ChREBP, as well as PGC-1ß occupancy of the liver-type pyruvate kinase promoter, was influenced by glucose concentration and liver-specific PGC-1ß(-/-) hepatocytes were refractory to the lipogenic response to high glucose conditions. These data suggest that PGC-1ß-mediated coactivation of ChREBP is involved in the lipogenic response to hyperglycemia.

Glucose induces protein targeting to glycogen in hepatocytes by fructose 2,6-bisphosphate-mediated recruitment of MondoA to the promoter.

In the liver, a high glucose concentration activates transcription of genes encoding glucose 6-phosphatase and enzymes for glycolysis and lipogenesis by elevation in phosphorylated intermediates and recruitment of the transcription factor ChREBP (carbohydrate response element binding protein) and its partner, Mlx, to gene promoters. A proposed function for this mechanism is intracellular phosphate homeostasis. In extrahepatic tissues, MondoA, the paralog of ChREBP, partners with Mlx in transcriptional induction by glucose. We tested for glucose induction of regulatory proteins of the glycogenic pathway in hepatocytes and identified the glycogen-targeting proteins, G(L) and PTG (protein targeting to glycogen), as being encoded by Mlx-dependent glucose-inducible genes. PTG induction by glucose was MondoA dependent but ChREBP independent and was enhanced by forced elevation of fructose 2,6-bisphosphate and by additional xylitol-derived metabolites. It was counteracted by selective depletion of fructose 2,6-bisphosphate with a bisphosphatase-active kinase-deficient variant of phosphofructokinase 2/fructosebisphosphatase 2, which prevented translocation of MondoA to the nucleus and recruitment to the PTG promoter. We identify a novel role for MondoA in the liver and demonstrate that elevated fructose 2,6-bisphosphate is essential for recruitment of MondoA to the PTG promoter. Phosphometabolite activation of MondoA and ChREBP and their recruitment to target genes is consistent with a mechanism for gene regulation to maintain intracellular phosphate homeostasis.

Cyclin D1 inhibits hepatic lipogenesis via repression of carbohydrate response element binding protein and hepatocyte nuclear factor 4α.

Following acute hepatic injury, the metabolic capacity of the liver is altered during the process of compensatory hepatocyte proliferation by undefined mechanisms. In this study, we examined the regulation of de novo lipogenesis by cyclin D1, a key mediator of hepatocyte cell cycle progression. In primary hepatocytes, cyclin D1 significantly impaired lipogenesis in response to glucose stimulation. Cyclin D1 inhibited the glucose-mediated induction of key lipogenic genes, and similar effects were seen using a mutant (D1-KE) that does not activate cdk4 or induce cell cycle progression. Cyclin D1 (but not D1-KE) inhibited the activity of the carbohydrate response element-binding protein (ChREBP) by regulating the glucose-sensing motif of this transcription factor. Because changes in ChREBP activity could not fully explain the effect of cyclin D1, we examined hepatocyte nuclear factor 4α (HNF4α), which regulates numerous differentiated functions in the liver including lipid metabolism. We found that both cyclins D1 and D1-KE bound to HNF4α and significantly inhibited its recruitment to the promoter region of lipogenic genes in hepatocytes. Conversely, knockdown of cyclin D1 in the AML12 hepatocyte cell line promoted HNF4α activity and lipogenesis. In mouse liver, HNF4α bound to a central domain of cyclin D1 involved in transcriptional repression. Cyclin D1 inhibited lipogenic gene expression in the liver following carbohydrate feeding. Similar findings were observed in the setting of physiologic cyclin D1 expression in the regenerating liver. In conclusion, these studies demonstrate that cyclin D1 represses ChREBP and HNF4α function in hepatocytes via Cdk4-dependent and -independent mechanisms. These findings provide a direct link between the cell cycle machinery and the transcriptional control of metabolic function of the liver.

Fructose 2,6-bisphosphate is essential for glucose-regulated gene transcription of glucose-6-phosphatase and other ChREBP target genes in hepatocytes.

Glucose metabolism in the liver activates the transcription of various genes encoding enzymes of glycolysis and lipogenesis and also G6pc (glucose-6-phosphatase). Allosteric mechanisms involving glucose 6-phosphate or xylulose 5-phosphate and covalent modification of ChREBP (carbohydrate-response element-binding protein) have been implicated in this mechanism. However, evidence supporting an essential role for a specific metabolite or pathway in hepatocytes remains equivocal. By using diverse substrates and inhibitors and a kinase-deficient bisphosphatase-active variant of the bifunctional enzyme PFK2/FBP2 (6-phosphofructo-2-kinase-fructose-2,6-bisphosphatase), we demonstrate an essential role for fructose 2,6-bisphosphate in the induction of G6pc and other ChREBP target genes by glucose. Selective depletion of fructose 2,6-bisphosphate inhibits glucose-induced recruitment of ChREBP to the G6pc promoter and also induction of G6pc by xylitol and gluconeogenic precursors. The requirement for fructose 2,6-bisphosphate for ChREBP recruitment to the promoter does not exclude the involvement of additional metabolites acting either co-ordinately or at downstream sites. Glucose raises fructose 2,6-bisphosphate levels in hepatocytes by reversing the phosphorylation of PFK2/FBP2 at Ser32, but also independently of Ser32 dephosphorylation. This supports a role for the bifunctional enzyme as the phosphometabolite sensor and for its product, fructose 2,6-bisphosphate, as the metabolic signal for substrate-regulated ChREBP-mediated expression of G6pc and other ChREBP target genes.

Elevated glucose represses liver glucokinase and induces its regulatory protein to safeguard hepatic phosphate homeostasis.

OBJECTIVE: The induction of hepatic glucose 6-phosphatase (G6pc) by glucose presents a paradox of glucose-induced glucose intolerance. We tested whether glucose regulation of liver gene expression is geared toward intracellular homeostasis. RESEARCH DESIGN AND METHODS: The effect of glucose-induced accumulation of phosphorylated intermediates on expression of glucokinase (Gck) and its regulator Gckr was determined in hepatocytes. Cell ATP and uric acid production were measured as indices of cell phosphate homeostasis. RESULTS: Accumulation of phosphorylated intermediates in hepatocytes incubated at elevated glucose induced rapid and inverse changes in Gck (repression) and Gckr (induction) mRNA concomitantly with induction of G6pc, but had slower effects on the Gckr-to-Gck protein ratio. Dynamic metabolic labeling in mice and liver proteome analysis confirmed that Gckr and Gck are low-turnover proteins. Involvement of Max-like protein X in glucose-mediated Gck-repression was confirmed by chromatin immunoprecipitation analysis. Elevation of the Gck-to-Gckr ratio in hepatocytes was associated with glucose-dependent ATP depletion and elevated urate production confirming compromised phosphate homeostasis. CONCLUSIONS: The lowering by glucose of the Gck-to-Gckr ratio provides a potential explanation for the impaired hepatic glucose uptake in diabetes. Elevated uric acid production at an elevated Gck-to-Gckr ratio supports a role for glucose regulation of gene expression in hepatic phosphate homeostasis.

Regulator of G protein signaling (RGS16) inhibits hepatic fatty acid oxidation in a carbohydrate response element-binding protein (ChREBP)-dependent manner.

G protein-coupled receptor (GPCR) pathways control glucose and fatty acid metabolism and the onset of obesity and diabetes. Regulators of G protein signaling (RGS) are GTPase-activating proteins (GAPs) for G(i) and G(q) α-subunits that control the intensity and duration of GPCR signaling. Herein we determined the role of Rgs16 in GPCR regulation of liver metabolism. Rgs16 is expressed during the last few hours of the daily fast in periportal hepatocytes, the oxygen-rich zone of the liver where lipolysis and gluconeogenesis predominate. Rgs16 knock-out mice had elevated expression of fatty acid oxidation genes in liver, higher rates of fatty acid oxidation in liver extracts, and higher plasma ß-ketone levels compared with wild type mice. By contrast, transgenic mice that overexpressed RGS16 protein specifically in liver exhibited reciprocal phenotypes as well as low blood glucose levels compared with wild type littermates and fatty liver after overnight fasting. The transcription factor carbohydrate response element-binding protein (ChREBP), which induces fatty acid synthesis genes in response to high carbohydrate feeding, was unexpectedly required during fasting for maximal Rgs16 transcription in liver and in cultured primary hepatocytes during gluconeogenesis. Thus, RGS16 provides a signaling mechanism for glucose production to inhibit GPCR-stimulated fatty acid oxidation in hepatocytes.

Activation and repression of glucose-stimulated ChREBP requires the concerted action of multiple domains within the MondoA conserved region.

Carbohydrate response element-binding protein (ChREBP) is a glucose-dependent transcription factor that stimulates the expression of glycolytic and lipogenic genes in mammals. Glucose regulation of ChREBP has been mapped to its conserved NH(2)-terminal region of 300 amino acids, designated the MondoA conserved region (MCR). Within the MCR, five domains (MCR1-5) have a particularly high level of conservation and are likely to be important for glucose regulation. We carried out a large-scale deletion and substitution mutational analysis of the MCR domain of ChREBP. This analysis revealed that MCRs 1-4 function in a concerted fashion to repress ChREBP activity in basal (nonstimulatory) conditions. Deletion of the entire MCR1-4 segment or the combination of four specific point mutations located across this region leads to a highly active, glucose-independent form of ChREBP. However, deletion of any individual MCR domain and the majority of point mutations throughout MCR1-4 rendered ChREBP inactive. These observations suggest that the MCR1-4 region interacts with an additional coregulatory factor required for activation. This possibility is supported by the observation that the MCR1-4 region can compete for activity with wild-type ChREBP in stimulatory conditions. In contrast, mutations in the MCR5 domain result in increased activity, suggesting that this domain may be the target of intramolecular repression in basal conditions. Thus, the MCR domains act in a complex and coordinated manner to regulate ChREBP activity in response to glucose.

Overlapping roles of the glucose-responsive genes, S14 and S14R, in hepatic lipogenesis.

The Spot 14 (S14; Thrsp) gene has been implicated in supporting regulated lipogenesis in mammals. S14 gene expression in liver is controlled by a wide variety of hormones and dietary factors in parallel with the major lipogenic enzyme genes. In addition, mice deleted for the S14 gene display reduced de novo lipogenesis in the lactating mammary gland. However, no decrease in hepatic lipogenesis was observed in the S14 null mouse. It was postulated that this difference could be due to the expression of a paralogous gene called S14R (S14 related; Mig12) in the liver but not mammary tissue. To test this hypothesis, we used small interfering RNA to simultaneously reduce levels of S14 and S14R in cultured primary hepatocytes. We found that rates of lipogenesis were decreased by approximately 65% in cells treated with insulin and high glucose. This reduction was associated with a decrease in total liver triacylglycerols and an altered morphology of lipid droplets. Expression of either S14 or S14R gene products was sufficient to fully restore normal lipogenesis. No change in the hepatic expression of other major lipogenic enzyme genes occurred during manipulation of S14 and/or S14R levels. These data support the hypothesis that both S14 and S14R are directly involved in supporting hepatic lipogenesis and that the two proteins play overlapping roles in this process.

Glucose activates ChREBP by increasing its rate of nuclear entry and relieving repression of its transcriptional activity.

Carbohydrate response element-binding protein (ChREBP) is a glucose-responsive transcription factor that activates genes involved in de novo lipogenesis in mammals. The current model for glucose activation of ChREBP proposes that increased glucose metabolism triggers a cytoplasmic to nuclear translocation of ChREBP that is critical for activation. However, we find that ChREBP actively shuttles between the cytoplasm and nucleus in both low and high glucose in the glucose-sensitive beta cell-derived line, 832/13. Glucose stimulates a 3-fold increase in the rate of ChREBP nuclear entry, but trapping ChREBP in the nucleus by mutagenesis or with a nuclear export inhibitor does not lead to constitutive activation. In fact, mutational studies targeting the nuclear export signal of ChREBP also identified a distinct function essential for glucose-dependent transcriptional activation. From this, we conclude that an additional event independent of nuclear translocation is required for activation. The N-terminal segment of ChREBP (amino acids 1-298) has previously been shown to repress activity under basal conditions. This segment has five highly conserved regions, Mondo conserved regions 1-5 (MCR1 to -5). Based on activating mutations in MCR2 and MCR5, we propose that these two regions act coordinately to repress ChREBP in low glucose. In addition, other mutations in MCR2 and mutations in MCR3 were found to prevent glucose activation. Hence, we conclude that both relief of repression and adoption of an activating form are required for ChREBP activation.

Hepatic expression of the SPOT 14 (S14) paralog S14-related (Mid1 interacting protein) is regulated by dietary carbohydrate.

The Spot 14 (S14) gene is rapidly up-regulated by signals that induce lipogenesis such as enhanced glucose metabolism and thyroid hormone administration. Previous studies in S14 null mice show that S14 is required for normal lipogenesis in the lactating mammary gland, but not the liver. We speculated that the lack of a hepatic phenotype was due to the expression of a compensatory gene. We recently reported that this gene is likely an S14 paralog that we named S14-Related (S14-R). S14-R is expressed in the liver, but not in the mammary gland. If S14-R compensates for the absence of S14 in the liver, we hypothesized that, like S14, S14-R expression should be glucose responsive. Here, we report that hepatic S14-R mRNA levels increase with high-carbohydrate feeding in mice or within 2 h of treating cultured hepatocytes with elevated glucose. A potential carbohydrate response element (ChoRE) was identified at position -458 of the S14-R promoter. Deletion of or point mutations within the putative S14-R ChoRE led to 50-95% inhibition of the glucose response. Gel-shift analysis revealed that the glucose-activated transcription complex carbohydrate responsive element-binding protein/Max-like protein X (Mlx) binds to the S14-R ChoRE. Finally, S14-R glucose induction is completely blocked when a dominant-negative form of Mlx is overexpressed in primary hepatocytes. In conclusion, our results indicate that the S14-R gene is a glucose-responsive target of carbohydrate responsive element-binding protein/Mlx and suggest that the S14-R protein is a compensatory factor, at least partially responsible for the normal liver lipogenesis observed in the S14 null mouse.

Identification and function of phosphorylation in the glucose-regulated transcription factor ChREBP.

In the liver, induction of genes encoding enzymes involved in de novo lipogenesis occurs in response to increased glucose metabolism. ChREBP (carbohydrate-response-element-binding protein) is a basic helix-loop-helix/leucine zipper transcription factor that regulates expression of these genes. To evaluate the potential role of ChREBP phosphorylation in its regulation, we used MS to identify modified residues. In the present paper, we report the detection of multiple phosphorylation sites of ChREBP expressed in hepatocytes, several of which are only observed under high-glucose conditions. Mutation of each of these serine/threonine residues of ChREBP did not alter its ability to respond to glucose. However, mutation of five N-terminal phosphoacceptor sites resulted in a major decrease in activity under high-glucose conditions. These phosphorylated residues are located within a region of ChREBP (amino acids 1-197) that is critical for glucose regulation. Mutation of Ser(56) within this region to an aspartate residue resulted in increased nuclear accumulation and activity under high-glucose conditions. Together, these data suggest that ChREBP activity is regulated by complex multisite phosphorylation patterns involving its N-terminal regulatory region.

The metabolic sensor GCN2 branches out.

GCN2 is a sensor of amino acid deprivation that triggers a repression of global protein synthesis while simultaneously inducing translation of specific proteins. In this issue of Cell Metabolism, Guo and Cavener (2007) present a much broader role for GCN2 in controlling lipid homeostasis in response to amino acid deprivation.

A critical role for the loop region of the basic helix-loop-helix/leucine zipper protein Mlx in DNA binding and glucose-regulated transcription.

The carbohydrate response element (ChoRE) is a cis-acting sequence found in the promoters of genes induced transcriptionally by glucose. The ChoRE is composed of two E box-like motifs that are separated by 5 bp and is recognized by two basic helix-loop-helix/leucine zipper (bHLH/LZ) proteins, ChREBP and Mlx, which heterodimerize to bind DNA. In this study, we demonstrate that two ChREBP/Mlx heterodimers interact to stabilize binding to the tandem E box-like motifs in the ChoRE. Based on a model structure that we generated of ChREBP/Mlx bound to the ChoRE, we hypothesized that intermolecular interactions between residues within the Mlx loop regions of adjacent heterodimers are responsible for stabilizing the complex. We tested this hypothesis by preparing Mlx variants in which the loop region was replaced with that of another family member or mutated at several key residues. These Mlx variants retained their ability to bind to a single perfect E-box motif as a heterodimer with ChREBP, but no longer bound to the ChoRE nor supported glucose responsive activity. In summary, our results support a model in which the loop regions of Mlx play an important functional role in mediating the coordinate binding of ChREBP/Mlx heterodimers to the ChoRE.

ChREBP*Mlx is the principal mediator of glucose-induced gene expression in the liver.

In mammals, glucose-regulated gene expression has been best characterized in the liver, where increased glucose metabolism induces transcription of genes encoding enzymes involved in de novo lipogenesis. ChREBP and Mlx dimerize and function together as a glucose-responsive transcription factor to regulate target genes, such as liver-type pyruvate kinase, acetyl-CoA carboxylase 1, and fatty acid synthase. To identify additional glucose-responsive genes in the liver, we used microarray analysis to compare gene expression patterns in low and high glucose conditions in hepatocytes. Target genes of ChREBP.Mlx were simultaneously identified by gene profiling in the presence or absence of a dominant negative Mlx. Of 224 genes that are induced by glucose, 139 genes (62%) were also inhibited by the dominant negative Mlx. Lipogenic enzyme genes involved in the entire pathway of de novo lipogenesis were found to be glucose-responsive target genes of ChREBP.Mlx. Genes encoding enzymes in other metabolic pathways and numerous regulators of metabolism were also identified. To determine if any of these genes are direct targets of ChREBP.Mlx, we searched for ChoRE-like sequences in the 5'-flanking regions of several genes that responded rapidly to glucose. ChoRE sequences that bound to ChREBP.Mlx and supported a glucose response were identified in two additional genes. Combining all of the known ChoRE sequences, we generated a modified ChoRE consensus sequence, CAYGNGN(5)CNCRTG. In summary, ChREBP.Mlx is the principal transcription factor regulating glucose-responsive genes in the liver and coordinately regulates a family of genes required for glucose utilization and energy storage.

Glucose activation of ChREBP in hepatocytes occurs via a two-step mechanism.

Carbohydrate response element binding protein (ChREBP) is a transcription factor that mediates glucose-responsive changes in gene expression in hepatocytes. In the current model for glucose regulation, inhibition of ChREBP in low glucose occurs in response to cAMP-dependent protein kinase (PKA)-mediated phosphorylation of residues S196, S626, and/or T666. Activation of ChREBP in conditions of increased glucose results simply from reversal of these inhibitory phosphorylations. To test this model, we analyzed mutant forms of ChREBP that lack one or more of the proposed PKA sites and found that these forms of ChREBP still require glucose for activation. Additionally, cAMP levels in cultured hepatocytes were negligible in low glucose conditions, indicating PKA should not be active. Finally, overall ChREBP phosphorylation did not change in response to altered glucose levels. We conclude that in addition to its repression by PKA, glucose activation of ChREBP involves a second mechanism that is independent of PKA phosphorylation.

Glucose as a regulator of eukaryotic gene transcription.

Glucose has essential metabolic roles as both a fuel for energy and a substrate for the biosynthesis of cell components. Because of its central importance, many cells have evolved mechanisms to sense glucose levels in their environment and to adapt the expression of their genetic information to glucose availability. This glucose signaling is vital in mammalian cells where derangements in glucose utilization might contribute to conditions such as obesity and type 2 diabetes. Two crucial issues stand out in understanding pathways of glucose-regulated gene transcription. First, how do cells sense changing glucose levels? Second, how is this signal transduced to the transcriptional apparatus of the cell? In mammalian cells, glucose sensing involves the detection of changes in glucose metabolism rather than glucose itself. A transcription factor that is involved in mediating responses to glucose, ChREBP, has been identified recently and studies have begun to elucidate the molecular basis of coupling between glucose metabolism and transcription factor activity.

Direct role of ChREBP.Mlx in regulating hepatic glucose-responsive genes.

Enzymes required for de novo lipogenesis are induced in mammalian liver after a meal high in carbohydrates. In addition to insulin, increased glucose metabolism initiates an intracellular signaling pathway that transcriptionally regulates genes encoding lipogenic enzymes. A cis-acting sequence, the carbohydrate response element (ChoRE), has been found in the promoter region of several of these genes. ChREBP (carbohydrate response element-binding protein) was recently identified as a candidate transcription factor in the glucose-signaling pathway. We reported that ChREBP requires the heterodimeric partner Max-like factor X (Mlx) to bind to ChoRE sequences. In this study we provide further evidence to support a direct role of Mlx in glucose signaling in the liver. We constructed two different dominant negative forms of Mlx that could dimerize with ChREBP but block its binding to DNA. When introduced into hepatocytes, both dominant negative forms of Mlx inhibited the glucose response of a transfected ChoRE-containing promoter. The glucose response was rescued by adding exogenous wild type Mlx or ChREBP, but not MondoA, a paralog of ChREBP that can also form a heterodimer with Mlx. Furthermore, dominant negative Mlx blocked the induction of glucose-responsive genes from their natural chromosomal context under high glucose conditions. In contrast, genes induced by the insulin and thyroid hormone-signaling pathways were unaffected by dominant negative Mlx. Mlx was present in the glucose-responsive complex of liver nuclear extract from which ChREBP was purified. In conclusion, Mlx is an obligatory partner of ChREBP in regulating lipogenic enzyme genes in liver.

Mlx is the functional heteromeric partner of the carbohydrate response element-binding protein in glucose regulation of lipogenic enzyme genes.

The expression of genes encoding enzymes involved in de novo triglyceride synthesis (lipogenesis) is transcriptionally induced in the liver in response to increased glucose metabolism. The carbohydrate response element-binding protein (ChREBP) is a newly identified basic helix-loop-helix/leucine zipper transcription factor proposed to regulate the expression of the glucose-responsive gene pyruvate kinase. This gene contains a carbohydrate response element (ChoRE) consisting of two E box motifs separated by 5 bp that is necessary and sufficient for glucose regulation. We demonstrate that overexpression of ChREBP in primary rat hepatocytes activates other ChoRE-containing promoters in a manner consistent with their ability to respond to glucose. In vitro binding of ChREBP to ChoRE sequences was not detected. Because E box-binding proteins function as obligate dimers, we performed a yeast two-hybrid screen of a mouse liver cDNA library to identify potential heteromeric partners. Mlx (Max-like protein X) was selected as the only basic helix-loop-helix/leucine zipper interaction partner in this screen. When a plasmid expressing either Mlx or ChREBP was cotransfected with a ChoRE-containing reporter plasmid into human embryonic kidney 293 cells, no increase in promoter activity was observed. However, the expression of both proteins dramatically enhanced promoter activity. This activation was observed with reporters containing ChoREs from several different lipogenic enzyme genes. In contrast, reporters containing non-glucose-responsive E box elements were not activated by ChREBP-Mlx expression. In vitro binding of ChREBP to ChoRE-containing oligonucleotides was observed only in the presence of Mlx. ChREBP-Mlx binding discriminated between E box sites that are glucose-responsive and those that are not. We conclude that Mlx is a functional heteromeric partner of ChREBP in regulating the expression of glucose-responsive genes.

A potential role for fructose-2,6-bisphosphate in the stimulation of hepatic glucokinase gene expression.

The effects of fructose-2,6-bisphosphate (F-2,6-P(2)) on hepatic glucokinase (GK) and glucose-6-phosphatase (G-6-Pase) gene expression were investigated in streptozotocin-treated mice, which exhibited undetectable levels of insulin. Hepatic F-2,6-P(2) levels were manipulated by adenovirus-mediated overexpression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Streptozotocin treatment alone or with infusion of control adenovirus leads to a dramatic decrease in hepatic F-2,6-P(2) content compared with normal nondiabetic mice. This is accompanied by a 14-fold decrease in GK and a 3-fold increase in G-6-Pase protein levels, consistent with a diabetic phenotype. Streptozotocin-treated mice that were infused with adenovirus-overexpressing an engineered 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase with high kinase activity and little bisphosphatase activity showed high levels of hepatic F-2,6-P(2). Surprisingly, these mice had a 13-fold increase in GK protein and a 2-fold decrease in G-6-Pase protein compared with diabetic controls. The restoration of GK is associated with increases in the phosphorylation of Akt upon increasing hepatic F-2,6-P(2) content. Moreover, the changes in levels of F-2,6-P(2) and Akt phosphorylation revealed a pattern similar to that of streptozotocin mice treated with insulin, indicating that increasing hepatic content of F-2,6-P(2) mimics the action of insulin. Because G-6-Pase gene expression was down-regulated only after the restoration of euglycemia, the effect of F-2,6-P(2) was indirect. Also, the lowering of blood glucose by high F-2,6-P(2) was associated with an increase in hepatic nuclear factor 1-alpha protein, a transcription factor involved in G-6-Pase gene expression. In conclusion, F-2,6-P(2) can stimulate hepatic GK gene expression in an insulin-independent manner and can secondarily affect G-6-Pase gene expression by lowering the level of plasma glucose.

The role of SREBP-1c in nutritional regulation of lipogenic enzyme gene expression.

A high carbohydrate diet up-regulates the transcription of enzymes of triglyceride biosynthesis (lipogenesis) in the mammalian liver. This treatment stimulates hepatic insulin signaling, leading to transcription of sterol regulatory element-binding protein-1c (SREBP-1c). SREBP-1c has been implicated as a major factor that up-regulates lipogenic genes in response to carbohydrate feeding. However, we presented evidence for another factor, carbohydrate response factor, which is also involved in this response, and we proposed a model wherein SREBP-1c and carbohydrate response factor are independent transcription factors that act in response to insulin and glucose, respectively. In this study, we examined the contribution of SREBP-1c to the expression of lipogenic genes in glucose- and insulin-treated primary rat hepatocytes using an inducible adenovirus system. We found that SREBP-1c overexpression leads to a modest induction of fatty acid synthase, S(14), and acetyl-CoA carboxylase mRNAs to 20% (fatty acid synthase), 10% (S(14)), and 5% (acetyl-CoA carboxylase) of the induction seen by high glucose and insulin treatment. Restoring insulin to cells overexpressing SREBP-1c did not further increase these mRNA levels. In contrast, adenovirus-expressed SREBP-1c did not induce pyruvate kinase mRNA, suggesting that induction of this gene is SREBP-1c-independent. SREBP-1c does indeed play a role in the induction of lipogenic enzyme genes in response to insulin treatment, but it is not sufficient for the induction seen when hepatocytes are treated with insulin and high glucose.