help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2005-1507
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
147/6/3060    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Qiao, L.
Right arrow Articles by Shao, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Qiao, L.
Right arrow Articles by Shao, J.
Endocrinology Vol. 147, No. 6 3060-3069
Copyright © 2006 by The Endocrine Society

Knocking Down Liver CCAAT/Enhancer-Binding Protein {alpha} by Adenovirus-Transduced Silent Interfering Ribonucleic Acid Improves Hepatic Gluconeogenesis and Lipid Homeostasis in db/db Mice

Liping Qiao, Paul S. MacLean, Hanning You, Jerome Schaack and Jianhua Shao

Graduate Center for Nutritional Sciences (L.Q., H.Y., J.Sh.), University of Kentucky, Lexington, Kentucky 40536; and Departments of Medicine (P.S.M.) and Microbiology (J.Sc.), University of Colorado at Denver and Health Sciences Center, Aurora, Colorado 80010

Address all correspondence and requests for reprints to: Jianhua Shao, M.D., Ph.D., Graduate Center for Nutritional Sciences, University of Kentucky, 900 South Limestone, Lexington, Kentucky 40536-0200. E-mail: jianhuashao{at}uky.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CCAAT/enhancer-binding protein-{alpha} (C/EBP{alpha}) is a member of the basic leucine zipper transcription factor family and regulates expression of several enzymes in the liver that control glucose and lipid metabolism. Using adenovirus-transduced silent interfering (si)RNA against C/EBP{alpha}, endogenous liver C/EBP{alpha} protein was knocked down by 70–80% in 8-wk-old wild-type (WT) and db/db mice. In WT mice, fasting blood glucose concentrations were reduced approximately 24% without changes in plasma free fatty acid and triglycerides, when compared with LacZ adenovirus-treated control mice. Ad-C/EBP{alpha} siRNA treatment nearly normalized fasting glucose and significantly reduced plasma insulin and free fatty acid content, even though there was no elevation of C/EBP{alpha} protein in the livers of db/db mice. In parallel with the changes in glucose levels, hepatic glucose production was significantly reduced in C/EBP{alpha} siRNA-treated WT and db/db mice. mRNA levels of phyosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and liver glycogen synthase were decreased in the C/EBP{alpha} siRNA-treated WT and db/db mice. Interestingly, the magnitude of reduction in these enzymes was more profound in db/db mice. C/EBP{alpha} siRNA also decreased mRNA levels of proliferator activator protein-{gamma} coactivator-1{alpha} in both the WT and db/db mice but reduced cAMP response element-binding protein only in WT and did not alter hepatic nuclear factor-4{alpha} and CBP/p300 expression. Expression of genes involved in lipogenesis, such as fatty acid synthase, acetyl-CoA carboxylase, and sterol regulatory element-binding protein-1c was robustly suppressed in the C/EBP{alpha} siRNA-treated db/db mice. Taken together, these results indicate that C/EBP{alpha} plays an important role in maintaining glucose and lipid homeostasis in the liver.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CCAAT/ENHANCER-BINDING PROTEIN (C/EBP)-{alpha} is the original member of the C/EBP family, which includes transcription factors with structural as well as functional homologies (1, 2). Six different members of this family have been isolated and characterized, including C/EBP{alpha}, -ß, -{gamma}, -{delta}, -{epsilon}, and -{zeta} (2). C/EBP members are among the basic leucine zipper transcription factors, and they bind to similar DNA sequences as homo- and heterodimers. C/EBP{alpha} is preferentially expressed in the liver, adipose tissue, and certain cells of the lung and the mammary gland (2). Two isoforms of C/EBP{alpha}, 42 and 30 kDa, are generated by ribosomal scanning of the mRNA (2). Both isoforms have intact DNA binding domains, although the 30-kDa isoform possesses reduced transactivation potential relative to the full-length protein. A variety of C/EBP{alpha} target genes has been identified to date. C/EBP{alpha} is involved in different biological processes, including gluconeogenesis, adipogenesis, cell proliferation, and tissue development (1, 2, 3, 4).

The essential role of C/EBP{alpha} in development and energy homeostasis has been demonstrated in studies of C/EBP{alpha}-deficient mice. Systemic gene deletion of C/EBP{alpha} in mice results in a profound alteration of liver structure with an impairment in hepatic glycogen storage, and mice die soon after birth because of the hypoglycemia associated with the low expression of gluconeogenic enzymes, including phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) (5, 6). In addition, disruption of C/EBP{alpha} also impairs lipid accumulation in both hepatocytes and adipocytes (5, 6). These studies suggest that C/EBP{alpha} is involved in both glucose and lipid metabolism during development. Because of the lethal embryonic phenotype of C/EBP{alpha} gene deletion, several other C/EBP{alpha} gene ablation mouse models have been created for evaluating C/EBP{alpha} function in neonatal and adult mice. Hepatic-specific disruption of C/EBP{alpha} using a floxed C/EBP{alpha} allele and albumin promoter-directed Cre expression results in impaired glucose tolerance without changes in PEPCK, G6Pase, and liver glycogen synthase (7). In contrast, liver-specific deletion of C/EBP{alpha} by recombinant adenovirus encoding the Cre gene leads to reduction of PEPCK and glycogen synthase expression in the liver (8). One recent study (9) demonstrated that systemic deletion of C/EBP{alpha} of mice at d 1 after birth or 3 months later does not alter energy homeostasis immediately. However, 15 d later the mice exhibit hypophagia, fatty liver, and hypoglycemia with significant reductions of PEPCK, G6Pase, and glycogen synthase expression in the liver (9). Despite the inconsistency of these mouse models, these studies demonstrate that C/EBP{alpha} is involved in controlling glucose metabolism, particularly in the liver.

The liver is an important organ in maintaining glucose and energy homeostasis. During postabsorptive condition, hepatocytes synthesize glucose via gluconeogenesis and glycogenolysis to maintain blood glucose concentrations. In both types 1 and 2 diabetes, excessive hepatic gluconeogenesis is a major contributor to fasting hyperglycemia (10, 11, 12). C/EBP{alpha} not only helps to maintain basal rates of PEPCK and G6Pase transcription but also mediates the glucagon (via cAMP) stimulated expression of these enzymes in hepatocytes (3). However, it is still unclear whether C/EBP{alpha} is involved in the excessive hepatic gluconeogenesis in diabetes or whether it may be considered a potential therapeutic target for hyperglycemia. In the present study, adenoviral gene delivery of silent interfering (si)RNA was used to knock down C/EBP{alpha} in the liver of db/db diabetic mice to examine its role in perturbed gluconeogenesis and lipid metabolism. The results from this study indicate that knocking down C/EBP{alpha} in the liver reduced hepatic glucose production and improved glucose and lipid homeostasis in db/db mice. Furthermore, acute knocking down of C/EBP{alpha} decreased lipid accumulation in the liver by reducing lipogenic gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of the siRNA expression vector
The creation of the C/EBP{alpha} siRNA expression cassette and adenovirus vector has been described in detail previously (13). The DNA sequences (only one sequence indicated) for the C/EBP{alpha} siRNA cassette are 5'-CGACGAGTTCCTGGCCGAC-3'. Recombinant adenovirus encoding siRNA against C/EBP{alpha} was generated using the AdEasy system (14). The adenovirus vector was amplified in HEK293 cells and purified by cesium chloride density centrifugation (15).

Experimental animals and adenovirus administration
Male C57BL/6J+/+Leprdb/db(db/db) mice weighing 39–41 g and age-matched (8 wk old) wild-type (WT) littermates were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were housed in a pathogen-free animal facility under standard 12-h light, 12-h dark cycle with free access to food and water. The experiments using mouse models were carried out under the Association for Assessment and Accreditation of Laboratory Animal Care guidelines with approval of the University of Kentucky Animal Care and Use Committee. The purified adenoviruses were diluted in PBS immediately before infusion. Adenovirus vectors were injected via the tail vein at a dosage of 1 x 109 pfu/mouse in 100 µl. The effects were examined 3 or 6 d after injection. Adenovirus encoding ß-galactosidase (LacZ) was used for control. For studies in the fasting state, mice were fasted overnight. For postprandial studies, mice were fasted overnight and then fed for 2 h.

RNA extraction and quantitative real-time PCR assay
Total RNA was isolated with Trizol reagent (Invitrogen, Carlsbad, CA). cDNA was synthesized using SuperScript III reverse transcriptase and oligo(dT)12–18 primers (Invitrogen). mRNA levels of genes of interest were measured by quantitative real-time PCR using cDNA as template. The primers (Table 1Go) were designed using the Roche Applied Science web site-based Universal ProbeLibrary Assay Design Center (http://www.roche-applied-science.com). Quantitative real-time PCR was performed using the MX3000p real-time PCR system (Stratagene, La Jolla, CA) using SYBR Green dye (Molecular Probes, Eugene, OR). Relative levels of PCR products were calculated from standard curves established from each primer pair. Expression data were normalized against to the amount of 18s PCR product.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Primer sequences for real-time PCR

 
Nuclear extraction and Western blot
Total or nuclear protein was extracted as described previously (13, 16). Proteins were separated by SDS-PAGE gel and probed by specific antibody raised against C/EBP{alpha}, C/EBPß, or hepatic nuclear factor-4{alpha} (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Band densities were determined using Image Station 2000R and 1D software (Kodak, New Haven, CT).

Hepatic glucose production measurement
Hepatic glucose production rate was studied as previously described (17). Briefly, 100 µl containing 5 µCi of D-[3-3H]glucose in 0.9% NaCl were injected via the tail vein. Blood samples were collected every 15 min for 1 h. Blood glucose concentration and radioactivity were measured. Hepatic glucose production was calculated using steady-state equations.

Blood metabolite analysis
Blood samples were collected from the tail vein. Glucose concentrations were determined using a colorimetric glucose oxidase assay (Sigma, St. Louis, MO). Plasma insulin concentrations were measured with a mouse insulin ELISA kit (ALPCO, Windham, NH). Concentrations of free fatty acids and triglycerides were measured using commercial kits (Wako, Osaka, Japan; and Sigma, respectively).

Liver glycogen and triglyceride content assays
Liver glycogen content was measured as previously described (7). In short, pieces of liver tissue (~50 mg) were dissolved in 250 µl of 1 N KOH at 95 C for 30 min and neutralized with 1 N HCl. Glycogen was degraded to glucose using amyloglucosidase. Glucose concentrations were determined. Liver triglyceride content was determined as described previously (7).

Data analysis
Data are expressed as mean ± SD. Statistical analyses were performed using the Student’s t test or ANOVA analyses followed by contrast test with Tukey or Dunnett error protection. Differences were considered significant when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Liver-specific knocking down of C/EBP{alpha}
LacZ histochemistry staining was used to evaluate tissue transduction specificity and efficiency by an adenovirus vector. Three days after tail vein injection of 1 x 109 pfu, more than 85% of hepatocytes were positive for LacZ (data not shown). LacZ expression was not detected in the heart, lung, kidney, pancreas, and epididymal fat tissues and less than 0.01% of spleen cells were positive (data not shown). Thus, adenovirus vectors delivered via tail vein injection were avidly taken up by liver cells, consistent with previous findings (18).

During the 3 d of the experimental period, the mice showed no signs of illness other than those related to the diabetic phenotype for db/db mice. C/EBP{alpha} regulates bilirubin uridine 5'-diphosphate-glucuronosyltransferase expression in the liver (8). Deletion of the C/EBP{alpha} gene induces jaundice due to low bilirubin UDP-glucuronosyltransferase expression and increase in unconjugated serum bilirubin (8). No sign of jaundice was found in the mice 3 d after any adenovirus vector infusion. However, 6 d after C/EBP{alpha} siRNA vector infusion, jaundice was observed in the mice. Furthermore, no significant difference of food intake was observed between C/EBP{alpha} siRNA and control vector-treated WT or db/db mice (data not shown).

To assess the knockdown efficiency, nuclear protein levels of C/EBP{alpha} in the livers were measured by Western blot. As shown in Fig. 1Go, C/EBP{alpha} was significantly knocked down (70–80%) by adenovirus-mediated siRNA expression in both WT and db/db mice (Fig. 1AGo). Because the siRNA sequence was designed against the 3' region of C/EBP{alpha} mRNA, the two C/EBP{alpha} isoforms were equally reduced in siRNA vector transduced mice. Interestingly, the C/EBP{alpha} protein levels were comparable between WT and db/db mice (Fig. 1AGo). C/EBPß protein was significantly elevated in C/EBP{alpha} siRNA vector-injected WT and db/db mice (P < 0.001, Fig. 1AGo). Elevated C/EBPß mRNA was also observed (data not shown). In contrast, in cultured hepatoma H4IIe cells, overexpression or knocking down of C/EBP{alpha} increased or decreased C/EBPß, respectively (Fig. 1BGo), in agreement with previous studies demonstrating that C/EBP{alpha} and C/EBPß directly regulate each other’s gene expression (2). These data suggest that increased C/EBPß gene expression in C/EBP{alpha} siRNA-treated mice is mediated by a mechanism other than activation by C/EBP{alpha}. Because tail vein-infused adenovirus vector-dependent LacZ expression was nearly exclusive to hepatocytes, we refer to this C/EBP{alpha} knocking down as hepatic specific.


Figure 1
View larger version (40K):
[in this window]
[in a new window]
  		FIG. 1. Adenovirus-mediated siRNA knocks down C/EBP{alpha} in livers from WT and db/db mice. Three (A) or 6 d (C) after adenovirus vector injection, liver tissues were collected after overnight fasting. Nuclear protein was extracted and analyzed by Western blot. The autoradiograph is representative of six independent assays. B, H4IIe rat hepatoma cells were transduced with adenovirus vectors encoding green fluorescent protein (GFP) control, C/EBP{alpha}, or C/EBP{alpha} siRNA. Twenty-four hours later, nuclear proteins were extracted and C/EBP{alpha} and C/EBPß protein levels were measured by Western blot.

 
Knocking down C/EBP{alpha} decreased hepatic glucose production and improved glucose homeostasis in db/db mice
To investigate the role of hepatic C/EBP{alpha} in energy homeostasis, the biomarkers of glucose and lipid metabolism were measured 3 d after vector administration. For WT mice, fasting blood glucose concentrations were approximately 24% lower in C/EBP{alpha} siRNA vector-treated mice, compared with control vector-infused mice (P < 0.05, Table 2Go). For db/db diabetic mice, fasting blood glucose was nearly normalized by C/EBP{alpha} siRNA (P < 0.001, Table 2Go). Although fasting glucose concentrations were significantly reduced in siRNA vector-treated WT mice, there were no significant changes in insulin concentrations (P > 0.05). However, significantly reduced fasting insulin concentrations were observed in siRNA vector-treated db/db mice (P < 0.01). No significant alterations of serum triglyceride were found in C/EBP{alpha} siRNA-treated WT and db/db mice, but free fatty acid was significantly reduced in db/db mice (Table 1Go). Blood glucose concentrations after overnight fasting followed by 2 h of feeding were also studies in these mice. C/EBP{alpha} siRNA treatment significantly reduced blood glucose levels, compared with vector control-treated db/db mice. However, no differences in postprandial blood glucose concentrations were observed in C/EBP{alpha} siRNA vector- or control vector-treated WT mice. To test whether the changes in blood glucose concentrations in db/db mice coincide with C/EBP{alpha} expression levels, mice were tested 6 d after injection of adenovirus vectors in two groups of mice. As shown in Fig. 1CGo, C/EBP{alpha} protein levels were still significantly lower (~40%, P < 0.05) in siRNA vector-treated mice. However, the inhibitory effect was less than that in the livers after 3 d of treatment (comparative data not shown). Fasting glucose concentrations were also higher, compared with mice treated for 3 d with siRNA (9.8 ± 1.3 vs. 8.6 ± 2.1 mM).


View this table:
[in this window]
[in a new window]
  		TABLE 2. Plasma metabolic parameters in Ad-C/EBP{alpha} siRNA transducted WT and db/db mice

 
Hepatic glucose output is the main contributor to the maintenance of fasting blood glucose concentration, particularly after prolonged fasting. C/EBP{alpha} plays an important role in controlling gluconeogenesis. Therefore, we measured hepatic glucose production in mice 3 d after adenovirus infusion. As expected, the fasting hepatic glucose production rate was significantly elevated in db/db mice (Fig. 2Go). In both WT and db/db mice, knocking down C/EBP{alpha} significantly reduced hepatic glucose production (P < 0.05, Fig. 2Go). Furthermore, the reductions in hepatic glucose production were almost identical with the changes of fasting blood glucose concentrations. These observations are consistent with previous studies demonstrating that hepatic glucose production is one of the determining factors for fasting blood glucose concentration. Of interest, our results showed that C/EBP{alpha} protein levels were comparable between WT and db/db diabetic mice. However, knocking down C/EBP{alpha} almost normalized the hepatic glucose output in db/db mice. These findings imply that knocking down C/EBP{alpha} could be used as a strategy for reducing hepatic gluconeogenesis in these diabetic mice.


Figure 2
View larger version (11K):
[in this window]
[in a new window]
  		FIG. 2. Knocking down C/EBP{alpha} suppresses hepatic glucose production (HGP). After overnight fasting, 5 µCi D-[3-3H]glucose in saline were injected via the tail vein. Blood samples were collected at the indicated times. Glucose concentration and 3H radioactivity were measured. Hepatic glucose production rates were calculated as described previously (17 ). Data are means ± SD, n = 6. *, P < 0.05, compared with control mice treated with LacZ adenovirus.

 
Decreased PEPCK and G6Pase gene expression with C/EBP{alpha} siRNA administration
PEPCK and G6Pase are rate-limiting enzymes for gluconeogenesis. Physiologically, expression of these two key enzymes is rapidly regulated at the transcription level. Both in vivo and in vitro studies have indicated that C/EBP{alpha} can directly regulate transcription of these genes in hepatocytes. Therefore, we measured mRNA levels of PEPCK and G6Pase to determine whether C/EBP{alpha} siRNA-suppressed hepatic glucose production is regulated through gluconeogenesis. As expected, significant elevations of PEPCK and G6Pase mRNA levels were observed in the livers of db/db mice (Fig. 3Go).


Figure 3
View larger version (18K):
[in this window]
[in a new window]
  		FIG. 3. C/EBP{alpha} siRNA treatment reduces the expression of the rate-limiting enzymes of glucose metabolism. Mice were treated with purified adenoviruses encoding C/EBP{alpha} siRNA or LacZ. Liver tissues were collected 3 d after vector administration. cDNA was synthesized from total RNA and used as template for real-time PCR. mRNA levels of PEPCK, G6Pase, GS and LGP were measured by real-time PCR using a standard curve created for each target gene. Relative mRNA levels are presented as mean ± SD for six mice performed in triplicate. *, P < 0.05 vs. LacZ adenovirus-treated WT or db/db mice, respectively.

 
Comparing the mRNA levels of PEPCK or G6Pase between control vector- and C/EBP{alpha} siRNA vector-treated mice after 3 d of treatment, C/EBP{alpha} siRNA vector infusion led to lower PEPCK and G6Pase mRNA levels in both WT and db/db mice (P < 0.05, Fig. 3Go). The results also reveal that the PEPCK mRNA levels are almost at the same levels for C/EBP{alpha} siRNA vector-treated WT and db/db (Fig. 3Go). However, the magnitude of C/EBP{alpha} siRNA-suppressed G6Pase transcription was significantly higher in db/db mice, compared with WT mice (P < 0.05). Decreased PEPCK and G6Pase mRNA levels were also observed in the livers from the both WT and db/db mice 6 d after receiving the C/EBP{alpha} siRNA vector (data not shown).

Another source of glucose for the maintenance of blood glucose concentration is hepatic glycogenolysis. Consistent with results from a previous study (19), there was no significant difference of glycogen content in the livers of WT and db/db mice (data not shown). However, fasting glycogen synthase (GS) mRNA levels in the livers of db/db mice were significant lower than in WT mice (Fig. 3Go). In both WT and db/db C/EBP{alpha} siRNA adenovirus-treated mice, GS mRNA levels were significantly lower than in the livers of control vector-injected mice (Fig. 3Go). Despite this robust reduction of GS mRNA, the accompanying decrease in hepatic glycogen contents in C/EBP{alpha} siRNA vector-treated mice was modest (data not shown). The balance of synthesis and glycogenolysis determines glycogen content in the liver. Glycogen phosphorylase is a key enzyme in the control of glycogenolysis. Our study also found that the mRNA levels of liver glycogen phosphorylase were significantly reduced in C/EBP{alpha} siRNA vector-treated mice (Fig. 3Go). Therefore, we speculate that decreased glycogen turnover may partially contribute to low hepatic glucose production in C/EBP{alpha} siRNA-treated mice. The detailed mechanism is under investigation.

C/EBP{alpha} siRNA inhibits lipid accumulation in the livers of db/db mice
Elevated lipid accumulation in the liver (fatty liver) has been a consistent characteristic of the diabetic phenotype in db/db or other obese diabetic murine models. To investigate the role of C/EBP{alpha} in altered hepatic lipid metabolism in db/db mice, hepatic lipid content and triglyceride levels were measured. Consistent to previous reports, an elevation in hepatic lipid accumulation is evident in db/db mice with both histological staining (Fig. 4AGo) and biochemical analysis (P < 0.001, Fig. 4BGo). Three days after C/EBP{alpha} siRNA vector injection, little change was observed in the hepatic lipid accumulation in WT mice, but a profound decrease was observed in db/db mice (P < 0.001, Fig. 4Go). These results strongly indicate that C/EBP{alpha} plays an important role in hepatic lipid metabolism.


Figure 4
View larger version (94K):
[in this window]
[in a new window]
  		FIG. 4. C/EBP{alpha} siRNA suppresses lipid accumulation and triglyceride (TG) levels in the livers of db/db mice. A, Liver lipid content was examined using Oil Red O staining (x200). B, Hepatic triglyceride content was measured as described in Materials and Methods. Data are mean ± SD. *, P < 0.05, compared with LacZ vector-treated db/db mice (n = 6).

 
Knocking down C/EBP{alpha} leads to lower expression of lipogenic genes
The liver, which is intimately involved in lipid metabolism, takes up, stores, synthesizes, and secretes lipid into circulation. To determine whether knocking down C/EBP{alpha} alters hepatic lipid synthesis, the expression of several key lipogenic genes was measured at the transcription level. As shown in Fig. 5Go, mRNA levels of fatty acid synthase (FASn), acetyl-coenzyme A carboxylase (ACC)1, and serum conjugate diluent, stearoyl-CoA-desaturase (SCD)1 were dramatically elevated in the livers of db/db mice, compared with control vector-treated WT mice. These data demonstrate that lipogenesis is up-regulated in the liver of db/db mouse. The results also showed that after 3 d of C/EBP{alpha} siRNA treatment, the RNA levels of FASn, ACC, and SCD1 were significantly decreased in both WT and db/db mice (Fig. 5Go). C/EBP{alpha} siRNA treatment not only decreased FASn mRNA of db/db mice to the same levels as identically treated WT mice but also suppressed it to a lower level than that of WT mice treated with control vector. Surprisingly, SCD1 mRNA levels in the livers of C/EBP{alpha} siRNA-treated db/db mice were significantly lower than those in WT mice receiving the same treatment (Fig. 5Go). We also measured the FASn, ACC, and SCD1 RNA levels in the livers from db/db mice 6 d after vector injection. Similar to the above from mice treated for 3 d, the RNA levels of FASn, ACC, and SCD1 were significantly lower, compared with those of mice that received the control vector (data not shown).


Figure 5
View larger version (15K):
[in this window]
[in a new window]
  		FIG. 5. Reduction of hepatic lipogenic enzymes expression in the livers of C/EBP{alpha} siRNA-treated WT and db/db mice. The mRNA levels of FASn, ACC1, and SCD1 were measured by real-time PCR, using reverse-transcribed cDNA as template. PCR was performed in triplicate for each sample. Values are mean ± SD from six mice in each group. *, P < 0.05, compared with LacZ vector-treated WT or db/db mice, respectively.

 
Potential mediators of C/EBP{alpha} siRNA-altered hepatic glucose and lipid metabolism
In addition to C/EBP{alpha}, several other transcription factors and coactivators have been shown to regulate the expression of enzymes in gluconeogenic and lipogenic pathways, including peroxisomal proliferator-activated receptor (PPAR){alpha}, proliferator activator protein-{gamma} coactivator (PGC)-1{alpha}, PGC-1ß, liver X receptor (LXR){alpha}, cAMP response element-binding protein (CREB), sterol regulatory element-binding protein-1 (SREBP1)c, and CREB binding protein (CBP). Generally, these proteins interact and coordinately control transcriptional activation at the regulatory region in the promoter of target genes. To investigate whether the effects of knocking down C/EBP{alpha} in the liver may be mediated by changes in these critical regulatory factors, we examined the expression of several of these factors.

PPAR{alpha} plays an important role in the regulation of genes involved in fatty acid oxidation and peroxisomal proliferation in the liver (20). PPAR{alpha} also enhances hepatic gluconeogenesis by increasing PEPCK and G6Pase expression (21, 22). Consistent with results from a previous study (23), PPAR{alpha} mRNA levels in the livers of db/db mice were more than 2-fold higher than those in WT mice treated with the control vector (Fig. 6AGo). C/EBP{alpha} siRNA treatment led to decreased PPAR{alpha} mRNA in both WT and db/db mice (P < 0.05, Fig. 6AGo). Thus, elevated PPAR{alpha} expression in the liver of db/db mouse and C/EBP{alpha} siRNA-induced decrease of PPAR{alpha} may directly contribute to perturbed hepatic glucose production in these diabetic mouse models, and a portion of the normalization that comes with siRNA treatment could be mediated via PPAR{alpha}. These data are consistent with previous reports indicating that C/EBP{alpha} up-regulates PPAR{alpha} gene expression and deletion of C/EBP{alpha} gene dramatically reduces PPAR{alpha} mRNA (9). However, these observations would appear to be in conflict with PPAR{alpha}’s link with increased lipid oxidation. Long-term C/EBP{alpha} gene disruption reduces PPAR{alpha} and increases hepatic lipids (7, 9). What this paradox suggests is that if PPAR{alpha} is involved, it may exert a more profound effect on carbohydrate metabolism in db/db mice, and it may not be able to counteract the control that other regulatory factors exert on lipid metabolism (i.e. SREBP1c). Whereas the selective sensitivity of carbohydrate metabolism, but not lipid metabolism, to PPAR{alpha}-mediated regulation remains unproven, it may be important for explaining the coordinated changes observed in db/db mice in C/EBP{alpha} siRNA-treated mice.


Figure 6
View larger version (32K):
[in this window]
[in a new window]
  		FIG. 6. Effects of knocking down C/EBP{alpha} on the expression of transcription factors in the livers of WT and db/db mice. Treatment and sample preparation are described in detail in Materials and Methods and preceding figure legends. cDNA was synthesized and used as template. The mRNA levels of PPAR{alpha}, CREB, CBP, PGC-1{alpha}, PGC-1ß, SREBP1c, SREBP1a, and LXR{alpha} were measured by real-time PCR. Relative mRNA quantities were determined using standard curves established for each target gene for each PCR. Values are mean ± SD for six mice. *, P < 0.05, compared with LacZ vector-treated WT or db/db control mice, respectively.

 
Similar to a previous report (24), PGC-1{alpha} mRNA were significantly elevated in the livers of db/db mice (Fig. 6AGo). However, both CREB and CBP expression were markedly lower in the livers of db/db control mice (P < 0.05, Fig. 6AGo). C/EBP{alpha} siRNA vector administration decreased PGC-1{alpha} mRNA in the livers of both WT and db/db mice (P < 0.05, Fig. 6AGo). Interestingly, knocking down C/EBP{alpha} decreased only CREB in WT mice (P < 0.05, Fig. 6AGo). There was a slight increase of CBP in the livers of C/EBP{alpha} siRNA vector-treated WT and db/db mice (Fig. 6AGo).

SREBP1c, LXR{alpha}, and PGC-1ß regulate hepatic lipogenesis by regulating transcription of genes that encode enzymes in lipogenic cascades (25, 26). SREBP1c mRNA was significantly increased in the livers of db/db mice, without difference in SREBP1a, LXR{alpha}, and PGC-1ß (Fig. 6BGo). Compared with control vector-treated mice, C/EBP{alpha} siRNA adenovirus infusion produced a marked decrease in SREBP1c in the livers of db/db mice (P < 0.05, Fig. 6BGo). C/EBP{alpha} siRNA vector-treated WT and db/db mice exhibited a significant decrease of LXR{alpha} in the liver tissues (Fig. 6BGo). C/EBP{alpha} siRNA treatment decreased only PGC-1ß in db/db mice (Fig. 6BGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The novel observations of this study are that whereas C/EBP{alpha} was not elevated in the livers of db/db mice, transduction with C/EBP{alpha} siRNA reduced hepatic glucose production and lipid accumulation that are characteristic of this diabetic phenotype. These adjustments with siRNA treatment were accompanied by alterations in the expression of key transcription factors known to control glucose and lipid metabolism in the liver and key enzymes in the pathways of gluconeogenesis and lipogenesis. These observations indicate that whereas C/EBP{alpha} may not solely explain the perturbations in glucose and lipid metabolism that are found in this model of diabetes, altering its expression had a dramatic effect in normalizing these perturbations. From a practical perspective, C/EBP{alpha} may provide a viable target for the treatment of diabetes and its association impairments in glucose and lipid metabolism.

Previous studies have indicated that C/EBP{alpha} services as a central regulator of both carbohydrate and lipid metabolism in both liver and adipose tissue (5, 6, 9, 27). However, the role or function of C/EBP{alpha} in the regulation of energy metabolism in adult liver remains uncertain. Acute disruption of hepatic C/EBP{alpha} gene expression in adult mice produces a phenotype similar to conventional C/EBP{alpha}–/– mice (8). In contrast, the same group recently reported that using albumin-Cre deletion of hepatic C/EBP{alpha} gene does not alter hepatic PEPCK and G6Pase gene expression in mice (28). The reasons for these observational differences are unclear, but they suggest that different approaches to eliminating C/EBP{alpha} function might produce slightly different phenotypes. In this study, we used adenovirus-mediated siRNA acute knockdown of C/EBP{alpha} in hepatocytes in db/db diabetic and WT mice. Our study revealed that knocking down hepatic C/EBP{alpha} suppresses hepatic gluconeogenesis and lipogenesis in both WT and db/db mice. Our observations join the growing body of evidence that indicates that C/EBP{alpha} plays a critical role in maintaining hepatic energy metabolism in adult mice.

C/EBP{alpha}, C/EBPß, and C/EBP{delta} are expressed at relatively high levels in liver. Both C/EBP{alpha} and C/EBPß are involved in maintaining energy homeostasis (3). Deletion of the C/EBPß gene in mice creates two phenotypes (29). Similar to conventional C/EBP{alpha} gene knockout mice, some C/EBPß–/– mice die immediately after birth from hypoglycemia. The rest of the mice survive with decreased hepatic glucose production and hypoglycemia. By expressing C/EBPß from the C/EBP{alpha} locus, one study has showed that C/EBPß can functional replace C/EBP{alpha} (30). Because all C/EBP transcription factors share a DNA binding consensus sequence, they can bind at the same target gene. Therefore, it has been hypothesized that the members of this family (except C/EBP{zeta}) can functionally compensate for each other, particularly with respect to C/EBP{alpha} and C/EBPß in the liver. Our study showed that knocking down C/EBP{alpha} led to a robust increase in C/EBPß expression in the liver but led to a distinctly different phenotype with respect to glucose and lipid metabolism. This type of phenomenon has been observed in other C/EBP{alpha} knockout models (8, 9) and suggests that, although there may be some functional overlap of C/EBP{alpha} and C/EBPß in the liver, C/EBPß does not replace C/EBP{alpha} in controlling glucose and lipid metabolism.

C/EBP{alpha} regulates hepatic glucose production and gluconeogenesis mainly through controlling the expression of key enzymes. Several C/EBP{alpha}-responding elements have been identified in both PEPCK and G6Pase promoters (29, 31). C/EBP{alpha} not only regulates basal transcription of these genes but also mediates the regulatory effects of hormones (32). For example, cAMP is the secondary signal of glucagon or other hormones that stimulate PEPCK and G6Pase gene expression in the liver. Multiple regulatory elements are required to mediate the stimulatory effect of cAMP (33, 34). However, for both genes a CRE is the main regulatory motif that mediates the regulatory effect of cAMP. Interestingly, in response to cAMP stimulation, not only CREB but also C/EBP{alpha} and C/EBPß bind to the CRE site (29, 31, 35). Our observations indicate that hepatic CREB mRNA level was already reduced in db/db mice, and knocking down C/EBP{alpha} reduced only CREB in WT mice. Thus, reduced CREB with C/EBP{alpha} siRNA treatment may contribute to the decrease in PEPCK and G6Pase expression and hepatic glucose production of WT, but it cannot explain the differences observed in db/db mice.

The list of transcription factors and coactivators that are involved in gluconeogenesis has grown dramatically over recent years (4). In responding to regulatory stimulation, these transcription factors or coactivators assemble at the regulatory region of the promoter and form a transcription complex. The complex coordinately modulates PEPCK or G6Pase gene transcription. PGC-1{alpha} is a transcriptional coactivator and is critical in maintaining PEPCK and G6Pase gene expression in the liver (24, 36). PGC-1{alpha} accesses the PEPCK promoter via interaction with Foxo1 or hepatic nuclear factor-4{alpha} (24, 37). Our study reveals that knocking down C/EBP{alpha} leads to a significant decrease of PGC-1{alpha} in the livers from both WT and db/db mice, implying that C/EBP{alpha} may regulate PGC-1{alpha} expression in liver cells. In other studies in our laboratory, a putative C/EBP{alpha} regulatory region has been found within the PGC-1{alpha} promoter, and we observed that ectopic expression of C/EBP{alpha} increases PGC-1{alpha} gene transcription (Qiao, L., and J. Shao, unpublished data). Taken together, a portion of the effect of C/EBP{alpha} siRNA on PEPCK and G6Pase gene transcription and hepatic gluconeogenesis may be mediated through PGC-1{alpha}.

Elevated hepatic lipid accumulation has been reported in both diabetic animal models and human subjects. Matsusue et al. (28) recently reported that C/EBP{alpha} plays a role in accelerated lipogenesis in ob/ob mice. Observations from the present study provide further evidence of this link between C/EBP{alpha} and hepatic lipid metabolism and provide some indication as to the underlying mechanisms of this relationship. Whereas C/EBP{alpha} on its own cannot explain the elevated mRNA levels of FASn, ACC1, and SCD1 in db/db mice, knocking down C/EBP{alpha} in the liver led to marked reduction of FASn and SCD1 mRNA in WT mice and normalized FASn and ACC1 transcript levels in db/db mice. Therefore, our study strongly suggests that C/EBP{alpha} has the potential, either directly or indirectly, to enhance increasing lipogenic genes expression.

Hepatic lipids come from circulation and de novo synthesis. To study the contribution of de novo lipid synthesis to the fatty liver, we measured lipogenic gene expression in the livers of db/db and WT mice. As expected, the mRNA levels of FASn and ACC1 were strikingly increased in db/db mice, indicating that elevated hepatic lipid synthesis plays an important role in the development of fatty liver in this diabetic mouse model. Furthermore, knocking down C/EBP{alpha} in the liver led to marked decrease of FASn and SCD1 mRNA in WT mice and normalized FASn and ACC1 transcript levels in db/db mice. SCD1 mRNA in C/EBP{alpha} siRNA vector-treated db/db mice was significantly lower than that in WT mice. Therefore, our study suggests that C/EBP{alpha} regulates hepatic lipid metabolism by increasing lipogenic gene expression.

SREBP1c and LXR{alpha} are the main regulators of lipogenic gene expression in the liver in response to insulin and glucose stimulation (25). Observations from the present study showed that only SREBP1c mRNA was significantly increased in db/db mice, an effect that has also been observed in the livers of ob/ob diabetic mice (38). Knocking down C/EBP{alpha} significantly reduced both SREBP1c and LXR{alpha} in db/db mice, but our studies in cultured hepatocytes yield little evidence to support a direct effect of C/EBP{alpha} on the transcription of these genes (Qiao, L., and J. Shao, unpublished observations). Instead, these effects are more likely to mediate indirectly via changes in circulating glucose and insulin levels. Insulin and elevated glucose enhance SREBP1c and LXR{alpha} gene expression and increase the expression of a number of genes in the lipogenic pathway (25). Both insulin and fasting glucose concentrations were reduced by C/EBP{alpha} siRNA treatment, particularly in db/db mice (Table 2Go). Whereas other factors may be involved, it is likely that the reduction of serum insulin and glucose with C/EBP{alpha} siRNA treatment plays a critical role in the accompanying reduction of SREBP1c and LXR{alpha}.

PGC-1ß is a coactivator of SREBP in controlling hepatic lipogenic gene transcription (26). PGC-1ß binds to FASn promoter while docking with SREBP1c and up-regulates the promoter activity (26). In a similar fashion, it has also been shown that PGC-1ß coactivates LXR{alpha} in the activation of lipoprotein export (26). Taken together, the reduction of PGC-1ß in C/EBP{alpha} siRNA vector-treated db/db mice may contribute to the lower hepatic lipogenic potential in these mice and suppress very low-density lipoprotein secretion. Whereas these observations were accompanied by reduced hepatic lipid accumulation, a more persistent deficiency of C/EBP{alpha} induced low expression of PGC-1ß and LXR{alpha} and increased hepatic lipid accumulation lipid (7, 9). Whereas these different observations may appear paradoxical, this could be a difference between acute and chronic alterations in C/EBP{alpha} expression. Acutely knocking down C/EBP{alpha} may significantly alter lipid turnover and result in decreased lipid content, whereas prolonged deficiency of C/EBP{alpha} and the low expression of PGC-1ß, LXR{alpha}, and PPAR{alpha} may result in the gradual lipid accumulation and eventually fatty liver.

In summary, by using adenovirus-transduced siRNA in vivo, our study demonstrates that C/EBP{alpha} serves as a key regulator of glucose and lipid metabolism in the liver. Our results also indicate that C/EBP{alpha} regulates hepatic gluconeogenesis and lipogenesis by controlling rate-limiting enzyme expression through both direct and indirect transcription regulatory mechanisms.


   Footnotes
 
This work was supported by Grants 1-04-JF-44 from the American Diabetes Association (to J.S.) and Grant DK67403 from the National Institutes of Health (to P.S.M.).

Disclosure summary: L.Q., P.S.M., H.Y., J.Sc., and J.S. have nothing to declare.

First Published Online March 16, 2006

Abbreviations: ACC, Acetyl-coenzyme A carboxylase; CBP, CREB-binding protein; C/EBP, CCAAT/enhancer-binding protein; CRE, cAMP response element; CREB, cAMP response element-binding protein; FASn, fatty acid synthase; G6Pase, glucose-6-phosphatase; GS, glycogen synthase; LXR, liver X receptor; PEPCK, phosphoenolpyruvate carboxykinase; PGC, proliferator activator protein-{gamma} coactivator; PPAR, peroxisomal proliferator-activated receptor; SCD, stearoyl- coenzyme A-desaturase; si, silent interfering; SREBP1, sterol regulatory element-binding protein-1; WT, wild type.

Received November 29, 2005.

Accepted for publication March 7, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Johnson PF, Landschulz WH, Graves BJ, McKnight SL 1987 Identification of a rat liver nuclear protein that binds to the enhancer core element of three animal viruses. Genes Dev 1:133–146[Abstract/Free Full Text]
  2. McKnight SL 2001 McBindall—a better name for CCAAT/enhancer binding proteins? Cell 107:259–261[CrossRef][Medline]
  3. Croniger C, Leahy P, Reshef L, Hanson RW 1998 C/EBP and the control of phosphoenolpyruvate carboxykinase gene transcription in the liver. J Biol Chem 273:31629–31632[Free Full Text]
  4. Hanson RW 2005 Metabolism in the era of molecular biology. J Biol Chem 280:1705–1715[Free Full Text]
  5. Flodby P, Barlow C, Kylefjord H, Ahrlund-Richter L, Xanthopoulos KG 1996 Increased hepatic cell proliferation and lung abnormalities in mice deficient in CCAAT/enhancer binding protein {alpha}. J Biol Chem 271:24753–24760[Abstract/Free Full Text]
  6. Wang ND, Finegold MJ, Bradley A, Ou CN, Abdelsayed SV, Wilde MD, Taylor LR, Wilson DR, Darlington GJ 1995 Impaired energy homeostasis in C/EBP{alpha} knockout mice. Science 269:1108–1112[Abstract/Free Full Text]
  7. Inoue Y, Inoue J, Lambert G, Yim SH, Gonzalez FJ 2004 Disruption of hepatic C/EBP{{alpha}} results in impaired glucose tolerance and age-dependent hepatosteatosis. J Biol Chem 279:44740–44748[Abstract/Free Full Text]
  8. Lee YH, Sauer B, Johnson PF, Gonzalez FJ 1997 Disruption of the c/ebp {alpha} gene in adult mouse liver. Mol Cell Biol 17:6014–6022[Abstract]
  9. Yang J, Croniger CM, Lekstrom-Himes J, Zhang P, Fenyus M, Tenen DG, Darlington GJ, Hanson RW 2005 Metabolic response of mice to a postnatal ablation of CCAAT/enhancer-binding protein {alpha} (C/EBP{alpha}). J Biol Chem 280:38689–38699[Abstract/Free Full Text]
  10. DeFronzo RA, Simonson D, Ferrannini E 1982 Hepatic and peripheral insulin resistance: a common feature of type 2 (non-insulin-dependent) and type 1 (insulin-dependent) diabetes mellitus. Diabetologia 23:313–319[Medline]
  11. Bogardus C, Lillioja S, Howard BV, Reaven G, Mott D 1984 Relationships between insulin secretion, insulin action, and fasting plasma glucose concentration in nondiabetic and noninsulin-dependent diabetic subjects. J Clin Invest 74:1238–1246[Medline]
  12. DeFronzo RA 1988 Lilly lecture 1987. The triumvirate: ß-cell, muscle, liver. A collusion responsible for NIDDM. Diabetes 37:667–687[Medline]
  13. Qiao L, Maclean PS, Schaack J, Orlicky DJ, Darimont C, Pagliassotti M, Friedman JE, Shao J 2005 C/EBP{alpha} regulates human adiponectin gene transcription through an intronic enhancer. Diabetes 54:1744–1754[Abstract/Free Full Text]
  14. He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B 1998 A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA 95:2509–2514[Abstract/Free Full Text]
  15. Orlicky DJ, Schaack J 2001 Adenovirus transduction of 3T3-L1 cells. J Lipid Res 42:460–466[Abstract/Free Full Text]
  16. Shao J, Qiao L, Janssen RC, Pagliassotti M, Friedman JE 2005 Chronic hyperglycemia enhances PEPCK gene expression and hepatocellular glucose production via elevated liver activating protein/liver inhibitory protein ratio. Diabetes 54:976–984[Abstract/Free Full Text]
  17. Croniger CM, Millward C, Yang J, Kawai Y, Arinze IJ, Liu S, Harada-Shiba M, Chakravarty K, Friedman JE, Poli V, Hanson RW 2001 Mice with a deletion in the gene for CCAAT/enhancer-binding protein ß have an attenuated response to cAMP and impaired carbohydrate metabolism. J Biol Chem 276:629–638[Abstract/Free Full Text]
  18. Smith TA, Mehaffey MG, Kayda DB, Saunders JM, Yei S, Trapnell BC, McClelland A, Kaleko M 1993 Adenovirus mediated expression of therapeutic plasma levels of human factor IX in mice. Nat Genet 5:397–402[CrossRef][Medline]
  19. Roesler WJ, Khandelwal RL 1985 Age-related changes in hepatic glycogen metabolism in the genetically diabetic (db/db) mouse. Diabetes 34:395–402[Abstract]
  20. Lee CH, Olson P, Evans RM 2003 Minireview: lipid metabolism, metabolic diseases, and peroxisome proliferator-activated receptors. Endocrinology 144:2201–2207[Abstract/Free Full Text]
  21. Chakravarthy MV, Pan Z, Zhu Y, Tordjman K, Schneider JG, Coleman T, Turk J, Semenkovich CF 2005 "New" hepatic fat activates PPAR{alpha} to maintain glucose, lipid, and cholesterol homeostasis. Cell Metab 1:309–322[CrossRef][Medline]
  22. Koo SH, Satoh H, Herzig S, Lee CH, Hedrick S, Kulkarni R, Evans RM, Olefsky J, Montminy M 2004 PGC-1 promotes insulin resistance in liver through PPAR{alpha}-dependent induction of TRB-3. Nat Med 10:530–534[CrossRef][Medline]
  23. Memon RA, Tecott LH, Nonogaki K, Beigneux A, Moser AH, Grunfeld C, Feingold KR 2000 Up-regulation of peroxisome proliferator-activated receptors (PPAR{alpha}) and PPAR{gamma} messenger ribonucleic acid expression in the liver in murine obesity: troglitazone induces expression of PPAR{gamma}-responsive adipose tissue-specific genes in the liver of obese diabetic mice. Endocrinology 141:4021–4031[Abstract/Free Full Text]
  24. Yoon JC, Puigserver P, Chen G, Donovan J, Wu Z, Rhee J, Adelmant G, Stafford J, Kahn CR, Granner DK, Newgard CB, Spiegelman BM 2001 Control of hepatic gluconeogenesis through the transcriptional coactivator PGC-1. Nature 413:131–138[CrossRef][Medline]
  25. Horton JD, Goldstein JL, Brown MS 2002 SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest 109:1125–1131[CrossRef][Medline]
  26. Lin J, Yang R, Tarr PT, Wu PH, Handschin C, Li S, Yang W, Pei L, Uldry M, Tontonoz P, Newgard CB, Spiegelman BM 2005 Hyperlipidemic effects of dietary saturated fats mediated through PGC-1ß coactivation of SREBP. Cell 120:261–273[CrossRef][Medline]
  27. Darlington GJ, Ross SE, MacDougald OA 1998 The role of C/EBP genes in adipocyte differentiation. J Biol Chem 273:30057–30060[Free Full Text]
  28. Matsusue K, Gavrilova O, Lambert G, Brewer Jr HB, Ward JM, Inoue Y, LeRoith D, Gonzalez FJ 2004 Hepatic CCAAT/enhancer binding protein-{alpha} mediates induction of lipogenesis and regulation of glucose homeostasis in leptin-deficient mice. Mol Endocrinol 18:2751–2764[Abstract/Free Full Text]
  29. Croniger C, Trus M, Lysek-Stupp K, Cohen H, Liu Y, Darlington GJ, Poli V, Hanson RW, Reshef L 1997 Role of the isoforms of CCAAT/enhancer-binding protein in the initiation of phosphoenolpyruvate carboxykinase (GTP) gene transcription at birth. J Biol Chem 272:26306–26312[Abstract/Free Full Text]
  30. Chen SS, Chen JF, Johnson PF, Muppala V, Lee YH 2000 C/EBPß, when expressed from the C/ebp{alpha} gene locus, can functionally replace C/EBP{alpha} in liver but not in adipose tissue. Mol Cell Biol 20:7292–7299[Abstract/Free Full Text]
  31. Hornbuckle LA, Everett CA, Martin CC, Gustavson SS, Svitek CA, Oeser JK, Neal DW, Cherrington AD, O’Brien RM 2004 Selective stimulation of G-6-Pase catalytic subunit but not G-6-Pase transporter gene expression by glucagon in vivo and cAMP in situ. Am J Physiol Endocrinol Metab 286:E795–E808
  32. Chakravarty K, Cassuto H, Reshef L, Hanson RW 2005 Factors that control the tissue-specific transcription of the gene for phosphoenolpyruvate carboxykinase-C. Crit Rev Biochem Mol Biol 40:129–154[CrossRef][Medline]
  33. Roesler WJ 2000 What is a cAMP response unit? Mol Cell Endocrinol 162:1–7[CrossRef][Medline]
  34. Koo SH, Flechner L, Qi L, Zhang X, Screaton RA, Jeffries S, Hedrick S, Xu W, Boussouar F, Brindle P, Takemori H, Montminy M 2005 The CREB coactivator TORC2 is a key regulator of fasting glucose metabolism. Nature 437:1109–1111[CrossRef][Medline]
  35. O’Brien RM, Lucas PC, Yamasaki T, Noisin EL, Granner DK 1994 Potential convergence of insulin and cAMP signal transduction systems at the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter through CCAAT/enhancer binding protein (C/EBP). J Biol Chem 269:30419–30428[Abstract/Free Full Text]
  36. Herzig S, Long F, Jhala US, Hedrick S, Quinn R, Bauer A, Rudolph D, Schutz G, Yoon C, Puigserver P, Spiegelman B, Montminy M 2001 CREB regulates hepatic gluconeogenesis through the coactivator PGC-1. Nature 413:179–183[CrossRef][Medline]
  37. Puigserver P, Rhee J, Donovan J, Walkey CJ, Yoon JC, Oriente F, Kitamura Y, Altomonte J, Dong H, Accili D, Spiegelman BM 2003 Insulin-regulated hepatic gluconeogenesis through FOXO1-PGC-1{alpha} interaction. Nature 423:550–555[CrossRef][Medline]
  38. Shimomura I, Bashmakov Y, Horton JD 1999 Increased levels of nuclear SREBP-1c associated with fatty livers in two mouse models of diabetes mellitus. J Biol Chem 274:30028–30032[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 DiabetesHome page
L. Qiao, C. Zou, D. R. van der Westhuyzen, and J. Shao
Adiponectin Reduces Plasma Triglyceride by Increasing VLDL Triglyceride Catabolism
Diabetes, July 1, 2008; 57(7): 1824 - 1833.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. Qiao, C. Zou, P. Shao, J. Schaack, P. F. Johnson, and J. Shao
Transcriptional Regulation of Fatty Acid Translocase/CD36 Expression by CCAAT/Enhancer-binding Protein {alpha}
J. Biol. Chem., April 4, 2008; 283(14): 8788 - 8795.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. M. Schroeder-Gloeckler, S. M. Rahman, R. C. Janssen, L. Qiao, J. Shao, M. Roper, S. J. Fischer, E. Lowe, D. J. Orlicky, J. L. McManaman, et al.
CCAAT/Enhancer-binding Protein beta Deletion Reduces Adiposity, Hepatic Steatosis, and Diabetes in Leprdb/db Mice
J. Biol. Chem., May 25, 2007; 282(21): 15717 - 15729.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. Qiao, O. A. MacDougald, and J. Shao
CCAAT/Enhancer-binding Protein {alpha} Mediates Induction of Hepatic Phosphoenolpyruvate Carboxykinase by p38 Mitogen-activated Protein Kinase
J. Biol. Chem., August 25, 2006; 281(34): 24390 - 24397.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
147/6/3060    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed