PPARα (Peroxisome Proliferator-activated Receptor α) Activation Reduces Hepatic CEACAM1 Protein Expression to Regulate Fatty Acid Oxidation during Fasting-refeeding Transition*

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is expressed at high levels in the hepatocyte, consistent with its role in promoting insulin clearance in liver. CEACAM1 also mediates a negative acute effect of insulin on fatty acid synthase activity. Western blot analysis reveals lower hepatic CEACAM1 expression during fasting. Treating of rat hepatoma FAO cells with Wy14,643, an agonist of peroxisome proliferator-activated receptor α (PPARα), rapidly reduces Ceacam1 mRNA and CEACAM1 protein levels within 1 and 2 h, respectively. Luciferase reporter assay shows a decrease in the promoter activity of both rat and mouse genes by Pparα activation, and 5′-deletion and block substitution analyses reveal that the Pparα response element between nucleotides −557 and −543 is required for regulation of the mouse promoter activity. Chromatin immunoprecipitation analysis demonstrates binding of liganded Pparα to Ceacam1 promoter in liver lysates of Pparα+/+, but not Pparα−/− mice fed a Wy14,643-supplemented chow diet. Consequently, Wy14,643 feeding reduces hepatic Ceacam1 mRNA and CEACAM1 protein levels, thus decreasing insulin clearance to compensate for compromised insulin secretion and maintain glucose homeostasis and insulin sensitivity in wild-type mice. Together, the data show that the low hepatic CEACAM1 expression at fasting is mediated by Pparα-dependent mechanisms. Changes in CEACAM1 expression contribute to the coordination of fatty acid oxidation and insulin action in the fasting-refeeding transition.

Plasma insulin levels are determined by several factors, including insulin secretion from pancreatic ␤-cells and insulin clearance (1,2). Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), 4 a substrate of the insulin receptor tyrosine kinase (3), promotes insulin clearance by up-regulating receptor-mediated insulin endocytosis and degradation in a phosphorylation-dependent manner (4). Its specific inactivation in liver impairs insulin clearance to cause hyperinsulinemia and ensuing insulin resistance with increased hepatic steatosis (5).
CEACAM1 is ubiquitously produced with a predominant expression in liver by comparison to kidney and a limited expression in white adipose tissue and skeletal muscle (6). Physiologically, this is consistent with the major role of the liver in insulin clearance by comparison to kidney and the insignificant involvement of the other insulin target tissues in insulin extraction. The mechanistic underpinning of this hierarchal expression profile relates to the up-regulation of Ceacam1 promoter activity by insulin (7), the level of which is 2-3-fold higher in the portal relative to the systemic circulation (8).
In addition to CEACAM1, the hepatocyte is home to the highest level of fatty acid synthase, a lipogenic enzyme that catalyzes the conversion of malonyl-CoA to palmitic acid. Similar to CEACAM1, this high level of expression of fatty acid synthase is mediated by transcriptional up-regulation by insulin (9). However, despite the abundance of this enzyme, fatty acid synthase activity in liver is restricted even under stimulating feeding conditions, during which carbohydrates undergo glycolysis and their products are converted to fatty acids to contribute to energy sources at fasting. We have shown (10) that this counter-regulation of fatty acid synthase activity in liver is mediated by increased CEACAM1 phosphorylation and binding to fatty acid synthase in response to transient pulses of insulin (11).
At fasting, when insulin secretion is low, metabolism shifts from glycolysis to lipolysis, during which free fatty acids are released from white adipose tissue into the liver to undergo ␤-oxidation and supply energy to the heart and brain. This mechanism is largely supported by the activation of peroxisome proliferator-activated receptor ␣ (PPAR␣), a nuclear transcription factor that induces the expression of genes involved in fatty acid transport to mitochondria to undergo ␤-oxidation (12)(13)(14)(15)(16). Acutely after refeeding (ϳ8 h), PPAR␣ plays a significant role in maintaining fatty acid ␤-oxidation during the stepwise reversal of the fasting metabolic state by insulin surges until glycogen stores are replenished (17), at which point the levels of malonyl-CoA are restored, ␤-oxidation stops, and glycolysis resumes (18).
Because CEACAM1 plays an important regulatory role in insulin and fatty acid homeostasis, we herein examined whether it is itself metabolically regulated during the fastingrefeeding transition and identified the underlying mechanisms.

Experimental Procedures
Animal Husbandry-Male mice were kept in a 12-h dark/ light cycle. All procedures were approved by the Institutional Animal Care and Utilization Committee. Wild-type Ppar␣ ϩ/ϩ and Ppar␣ Ϫ/Ϫ null mice propagated on the C57BL/6 background were from Taconic Biosciences (Cambridge City, IN). Male mice (2-4 months of age) were fed ad libitum a standard chow (Harlan Teklad 2016; Harlan, Haslett, MI). In some experiments mice were fed for 3-7 days a chow diet powdered and mixed in a geometric proportion with 0.1% w/w of Wy14,643 (Enzo Life Sciences, Farmingdale, NY), a PPAR␣ agonist.
Plasma Biochemistry-After an overnight fast, mice were anesthetized at 1100 h. Whole venous blood was drawn to measure the levels of glucose, plasma insulin, and C-peptide levels (19). Glycogen content was measured as described previously (20).
Insulin Secretion and Glucose and Insulin Tolerance Tests-Awake overnight-fasted mice were injected intraperitoneally with 1.5 g/kg body weight (BW) dextrose solution before measuring glucose in tail blood. For insulin tolerance, mice were fasted for 6 h and intraperitoneally injected with human regular insulin (Novo Nordisk, 0.75 units/kg BW), and their glucose was measured as previously described (21). For glucose-stimulated insulin secretion, mice were fed a Wy-supplemented diet for 4 days, fasted overnight, and injected intraperitoneally with 3 g/kg BW of dextrose solution. Retro-orbital blood was removed at 0 -30-min post-injection to measure plasma insulin levels using RIA kit (Millipore, Temecula, CA).
Fatty Acid Synthase Activity-This was measured by the incorporation of radiolabeled malonyl-CoA into palmitate, as described previously (10). Briefly, 60 -100 mg of liver tissue was homogenized in DTT-containing Tris-buffer, pH 7.5, and centrifuged at 4°C, and the supernatant was incubated for 20 min at 37°C with 0.1 Ci of [ 14 C]malonyl-CoA and 25 nM malonyl-CoA in the absence or presence of 500 M NADPH. The reaction was stopped with 1:1 chloroform/methanol solution, mixed, and centrifuged, and the supernatant vacuum-dried was resuspended in 200 l of water-saturated butanol to be extracted. The butanol layer was counted, and values were expressed as relative cpm of 14 C-incorporated/g of protein.
Cloning of Mouse Ceacam1 Promoter-Functional mapping of the mouse Ceacam1 promoter revealed three potential peroxisome proliferator response elements (PPRE)/RXR at nucleotides (nts) Ϫ1056/Ϫ1037, Ϫ557/Ϫ543, Ϫ260/Ϫ248. In a PCR reaction, double-stranded genomic DNA spanning nt ϩ30 to Ϫ1100 was synthesized and amplified in a polymerase chain reaction (PCR) using 100 ng of mouse genomic DNA as template and 1 M concentrations of sense forward primer (nt Ϫ1100/Ϫ1086) containing an Xho1 flanking sequence (small letters in italics, 5Ј-ataccctcgagCCTAAGAAGCTTTAC-3Ј) and antisense primer (nt ϩ30/ϩ11) with BglII flanking sequence (small letters in italics, 5Ј-gaagatctTTTGTGGAGA-TGTGCTGAGG-3Ј). After the initial DNA denaturation at 94°C for 5 min, 30 cycles of PCR were carried out as described previously (7). The 5Ј deletion mutant was synthesized using the same PCR conditions but with a forward primer spanning nt Ϫ467 to Ϫ453 (5Ј-ataccctgctcgagTCAGTGACGATGGAT-3Ј). Amplified genomic DNA was subsequently purified and subcloned at the KpnI and BglII sites of pGL4.10 [luc2] BASIC promoterless firefly luciferase reporter plasmid (Promega Corp., Madison, WI).
Scanning Mutants of individual PPREs (nts Ϫ1056/Ϫ1037 (⌬1), nts Ϫ557/Ϫ543 (⌬2), and nts Ϫ260/Ϫ248 (⌬3)) were obtained in two sequential PCR reactions using the Quik-Change II XL Site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA). The first PCR reaction utilized 100 ng of the PGL4.10 plasmid containing the genomic DNA sequence between nt Ϫ1100 and ϩ30 as the template, with the reverse antisense nt ϩ30/ϩ11 primer and forward primers containing a sequence replacing each of the native PPRE site. The resulting PCR product was then used as the template to insert a mutation replacing the native RXR site downstream of each PPRE to mutate the PPRE/RXR site. The ⌬1 mutations were: 5Ј-TAAT-CGA-3Ј (for PPRE alone) and 5Ј-TAATCGAGCTAGT-3Ј (for PPRE/RXR). The ⌬2 mutations were 5Ј-CAATTCT-3Ј (for PPRE alone) and 5Ј-CAATTCTATGAAATC-3Ј (for PPRE/ RXR). The ⌬3 mutations were 5Ј-CTTTTCT-3Ј (for PPRE alone) and 5Ј-CTTTTCTGTTATG-3Ј (for PPRE/RXR). The resulting individual mutant products were used as templates to create any combinational mutations of the three PPRE/RXRs following a similar PCR-based strategy.
For luciferase assays cells were seeded at 4.4 ϫ 10 5 into 6-well plates and at ϳ60 -70% confluence, and transfection was performed with 500 ng of promoter constructs and 10 ng of Renilla luciferase (pRL-TK, Promega) using FuGENE 6 (Promega) per the manufacturer's instructions. Empty pGL4.10 vector was used as the negative control, and PPREx3-TK-luc (plasmid 1015; Addgene) (22) was used as a positive control. Twentyfour hours post-transfection, cells were serum-starved and treated with ethanol or 30 M Wy14,643 for 24 h as above. Luciferase activity was assessed using the Dual-Luciferase Reporter Assay System (Promega).
To assay for luciferase activity of the rat promoter, FAO rat hepatoma and HEK293 cells were co-transfected with 300 ng of pGL3 containing the rat Ceacam1 promoter (nts Ϫ1609/Ϫ21) (7) and 30 ng of Renilla luciferase (pRL-TK) using Lipofectamine 2000 (Invitrogen) before they were treated for 24 h, and luciferase activity was assayed.
Northern Blot Analysis-mRNA was purified using TRIzol (Invitrogen) followed by the MicroPoly (A) Pure kit (Ambion, ThermoFisher Scientific) and analysis by probing with cDNAs for Ppar␣ and Ceacam1 using the Random Primed DNA Labeling kit (Roche Applied Science) before reprobing and normalizing to ␤-actin.
Semi-quantitative Real-time RT-PCR-Total RNA was isolated with the PerfectPure RNA tissue kit (5 Prime) following the manufacturer's protocol. cDNA was synthesized with ImProm-II TM reverse transcriptase (Promega) using 1 g of total RNA and oligo(dT) primers (21). cDNA was evaluated with quantitative real-time-PCR (Step One Plus, Applied Biosystems, Waltham, MA). mRNA was normalized to Gapdh or 18S. Results are expressed in -fold change as the mean Ϯ S.E.
Statistical Analysis-Data were analyzed with SPSS software by two-way analysis of variance or two-tailed Student's t test with GraphPad Prism 4 software. p Ͻ 0.05 was statistically significant.

Results
CEACAM1 Is Regulated by Fasting/Refeeding-After an overnight fast, mice were refed a regular chow diet for up to 24 h. As previously shown (10), insulin surged at 1, 4, and 7 h of refeeding (Fig. 1A). Consistent with its ability to increase Ceacam1 promoter activity (7), hepatic CEACAM1 protein content was induced in parallel to transient insulin surges (Fig.  1B). Insulin surge during refeeding also induced CEACAM1 phosphorylation, as Western blot analysis using an antibody against tyrosyl phosphorylated CEACAM1 (␣-pCC1) shows (Fig. 1B). Moreover, the activity of Fasn is diminished in parallel to CEACAM1 phosphorylation (Fig. 1C) as previously reported (10). Of interest, the CEACAM1:Fasn protein ratio begins to drop at ϳ8 h of refeeding (Fig. 1B), pointing to the possibility that the higher CEACAM1:Fasn ratio plays a role in maintaining low fatty acid synthase activity in the early hours of refeeding (24,25). This could contribute to the regulation of Fasn substrate, malonyl-CoA, and hence fatty acid transport to mitochondria for ␤-oxidation, an essential step in glycogen repletion. As has been reported (26), replenishment of glycogen content in liver takes ϳ8 h of refeeding (Fig. 1D), at which point CEACAM1 phosphorylation dropped (Fig. 1A) and Fasn enzymatic activity reciprocally increased (Fig. 1C).
Ppar␣ Activation Decreases Ceacam1 Expression-Because PPAR␣ is activated at fasting to increase transcription of genes that are involved in fatty acid ␤-oxidation in liver (15), we then investigated whether it is implicated in the metabolically driven changes in hepatic CEACAM1 expression. Northern analysis shows a higher hepatic Ppar␣ mRNA levels at fasting than the first few hours of refeeding (Fig. 1E).
To test the hypothesis that CEACAM1 is reduced by Ppar␣ activation, we assessed the effect of PPAR␣ agonist, Wy14,643 (Wy), on rat and mouse Ceacam1 promoter activity as well as on its mRNA and protein levels. Using a luciferase reporter assay (7), we show that Wy14,643 treatment reduced rat Ceacam1 promoter activity by ϳ4-fold in rat hepatoma FAO cells ( Fig. 2A, Wy-treated versus vehicle (Veh)-treated Ϫ1609pLuc). Similarly, Wy14,643 treatment decreased the promoter activity of the rat Ceacam1 promoter (Ϫ1609pLuc) in human embryonic kidney (HEK293) cells (not shown), with an expected lower potency than in rat cells (27). Furthermore, Wy14,643 decreased Ceacam1 mRNA and CEACAM1 protein content in rat hepatoma FAO cells beginning at 1 and 2 h, respectively (Fig. 2, B and C).
Like rat promoter, Wy14,643 treatment reduced mouse Ceacam1 promoter activity by ϳ2-fold in human HepG2 hepatoma cells (Fig. 3B, Wy-treated versus Veh-treated Ϫ1100pLuc). 5Ј-Deletion analysis indicated that removing the genomic DNA region containing the two potential distal PPREs between nts Ϫ1100 and Ϫ467 abolished the repressive effect of Ppar␣ activation on mouse Ceacam1 promoter activity (Fig. 3B, Wy-treated versus Veh-treated Ϫ467pLuc). To further identify the active PPRE in the mouse Ceacam1 promoter, we then carried out mutational mapping of the three potential PPRE/ RXR sites in the promoter. As Fig. 3C indicates, mutating the sequence between nts Ϫ557 and Ϫ543 either individually (⌬2 construct) or together with the other two (⌬1,2; ⌬2,3 and ⌬1, 2,3 constructs) completely abolished the repressive effect of Wy14,643 treatment on Ceacam1 promoter activity, as opposed to mutating the other two PPRE/RXR motifs alone (⌬1;⌬3 and ⌬1,3 constructs). This points to the functional rel-FIGURE 1. Changes in hepatic CEACAM1 protein content at fasting/refeeding and physiologic implications. A, mice were fasted (F) overnight and refed ad libitum for 1-24 h (Rfd) before assessing plasma insulin levels. B, liver lysates were subjected to Western analysis by immunoblotting (Ib) with antibodies against ␣-CEACAM1 (␣-CC1) to assess changes in hepatic CEACAM1 protein levels, phospho-CEACAM1 (␣-pCC1) to detect phosphorylated CEACAM1, and Fasn to detect protein content of fatty acid synthase. Gels were reimmunoblotted (reIb) with an antibody against actin to normalize for protein loading. Liver tissues were assayed for fatty acid synthase activity relative to microgram of proteins (C) and for glycogen content relative to wet tissue weight (D). Assays were carried out in triplicate and on more than three mice per each time point. Data are presented as the mean Ϯ S.E. E, liver tissues were subjected to Northern analysis to assess hepatic Ppar␣ mRNA levels followed by ␤-actin for normalization. Both Northern and Western gels represent more than three experiments done on more than three mice per each time point.

FIGURE 2. Regulation of rat Ceacam1 expression by PPAR␣ activation.
A, a rat Ceacam1 promoter fragment spanning a genomic DNA sequence from nt Ϫ1609 to Ϫ21 was subcloned into a promoterless luciferase reporter plasmid in both sense and reverse orientations. The constructs were transiently cotransfected with Renilla luciferase (pRL) into rat FAO hepatoma cells and treated with 30 M Wy,14,463 (Wy) before luciferase activity was assayed, determined relative to that in Renilla, and expressed as the mean Ϯ S.D. of triplicate transfections in relative light units. The graph represents four separate experiments. B, rat FAO cells were treated with ethanol (Ϫ) or Wy (ϩ) for 0 -1 h and analyzed by Northern blot using a Gapdh cDNA probe for normalization. rCC1, rat CC1. C, Rat FAO cells were treated with ethanol (Veh) or Wy for 0 -12 h and subjected to Western blot (Ib) to analyze CEACAM1 protein content followed by tubulin for normalization. Each gel represents more than three different experiments. evance of the PPRE/RXR sequence located between nts Ϫ557 and Ϫ543 in the regulation of mouse Ceacam1 promoter activity.
A ChIP assay using a proximal primer (PP) set spanning the mouse Ceacam1 promoter between nts Ϫ280 and Ϫ562 showed that liganded Ppar␣ binds to Ceacam1 gene in liver lysates derived from Wy14,643-fed to a higher extent than Vehfed Ppar␣ ϩ/ϩ mice or Wy14,643-fed Ppar␣ Ϫ/Ϫ mice (Fig. 4A). A similar observation is made for the positive control, malic enzyme, a known target of Ppar␣. The distal primer set amplifying a region between nts ϩ6499 and ϩ6764 in Ceacam1 gene did not detect any binding. Together, the data revealed that activated Ppar␣ binds to Ceacam1 gene to down-regulate its transcription.
Metabolic Consequence of Ppar␣-dependent Down-regulation of CEACAM1-Feeding 2-month-old male mice a chow supplemented with Wy14,643 activated hepatic Ppar␣, as assessed by increased mRNA (Fig. 4B) and protein (Fig. 4C) levels of its target gene, CD36/FABP (16), in Ppar␣ ϩ/ϩ , but not Ppar␣ Ϫ/Ϫ mice. In parallel, this reduced Ceacam1 mRNA (Fig.  4B) and CEACAM1 protein levels (Fig. 4C) in Ppar␣ ϩ/ϩ but not Ppar␣ Ϫ/Ϫ mice. Consistently, Ppar␣ activation by Wy14,643 reduced insulin clearance in Ppar␣ ϩ/ϩ wild-type mice, as assessed by the ϳ3-fold decrease in C-peptide/insulin molar ratio relative to RD-fed mice (Table 1). This is further supported by a ϳ2-fold decrease in CEACAM1 protein content (Fig. 5A) and in [ 125 I]insulin internalization (Fig. 5B) in Wy14,643-treated primary hepatocytes derived from Ppar␣ ϩ/ϩ but not Ppar␣ Ϫ/Ϫ mice (dashed versus solid lines). Consistent with decreased insulin secretion as a result of enhanced fatty acid oxidation in ␤-cells upon Ppar␣ activation (28,29), C-peptide levels were ϳ4 -5-fold lower in Ppar␣ ϩ/ϩ mice fed with a Wy14,643-supplemented diet compared with chow-fed (Table 1). In contrast, supplementing chow with Wy14,643 did not affect C-peptide levels in Ppar␣ Ϫ/Ϫ mutants (Table 1). Furthermore, insulin release in response to glucose is also markedly reduced in Wy14,643-fed by comparison to chow-fed wild-type mice but FIGURE 3. The effect of PPAR␣ activation on mouse Ceacam1 promoter activity. A, sequence analysis of the mouse Ceacam1 promoter revealed three potential PPRE/RXR elements for potential Ppar␣ binding: nts Ϫ1056/ Ϫ1037, Ϫ557/Ϫ543, and Ϫ260/Ϫ248. B, as above, 5Ј-deletion constructs from the mouse promoter were subcloned into pGL4.10 promoterless luciferase reporter plasmid and transfected in HepG2 human hepatoma cells. Luciferase activity was determined in transfected cells treated with (black bars) or without (white bars) 30 M Wy14,643 (Wy). As the negative control, cells were transfected with the empty pGL4.10. As a positive control, cells were transfected with PPREx3-TK plasmid. C, a series of constructs from nt Ϫ1100 to ϩ30 mouse promoter bearing block mutations on individual or combinational PPRE/RXR sites were generated and subcloned into the pGL4.10 promoterless plasmid before their luciferase activity in response to ethanol (Veh) or Wy was determined as above. Luciferase light units were expressed as the mean Ϯ S.D. in relative light units. The graph represents typical results from four separate experiments.

FIGURE 4. Regulation of mouse Ceacam1 expression by PPAR␣.
A, Ppar␣ ϩ/ ϩand Ppar␣ Ϫ/Ϫ mice were fed a Wy-supplemented diet for 7 days before liver extraction and ChIP analysis as described under "Experimental Procedures." The relative location of the fragment amplified using the proximal (PP; Ϫ548/ Ϫ282) and distal (DP; ϩ6499/ϩ6764) primers in the Ceacam1 (Cc1) gene are also shown. Malic enzyme, a positive target of PPAR␣, was used as the control. The gel represents experiments on more than three mice per treatment category per mouse group. Wy, Wy14,643. reIb, reimmunoblot. B, livers were removed from mice treated with Wy for 7 days to analyze mRNA levels of Ceacam1 and CD36, a transcriptional target gene of PPAR␣, by quantitative real-time-PCR analysis. Analysis of each mouse was done in triplicate, and values are represented as the mean Ϯ S.E. C, as in B, livers were removed to determine protein content by Western blot (Ib) analysis. Analyses were done on more than five mice per treatment per mouse group. Two mice from each feeding category per mouse group are shown. remained unaffected in the mutants (Fig. 6A; dashed versus solid lines). Given the observed lowering effect of Ppar␣ activation on insulin secretion in response to glucose, it is likely that CEACAM1-dependent insulin clearance is reduced by Ppar␣ activation to compensate for the diminished insulin secretion and limit insulin deficiency, as assessed by mildly reduced plasma insulin levels in Wy14,643-fed wild-type mice (Table 1). This maintains normal glucose homeostasis and insulin sensitivity, as demonstrated by normal fasting glucose levels (Table 1 and Fig. 6B) and by better tolerance to exogenous glucose (Fig.  6B, AUC 19,124 Ϯ 1457 versus 27,290 Ϯ 1113; p Ͻ 0.002) and insulin (Fig. 6C, AUC 9,206 Ϯ 559 versus 13,938 Ϯ 403, p Ͻ 0.0001) in Wy14,643-fed by comparison to RD-fed wild-type mice (dashed versus solid lines). In contrast, Wy14,643 treat-ment failed to affect insulin secretion (Fig. 6A), glucose tolerance (Fig. 6B, AUC 16,577 Ϯ 858 versus 16,115 Ϯ 784) and insulin tolerance in Ppar␣ Ϫ/Ϫ mice (Fig. 6C, AUC 11,738 Ϯ 1,197 versus 11,528 Ϯ 1,425).

Discussion
The current studies demonstrate that hepatic CEACAM1 expression is lower at fasting than refeeding. The rise in CEACAM1 expression occurs in parallel to insulin surges in the early hours of refeeding, consistent with the ability of insulin to induce Ceacam1 promoter activity (7). Whereas it is possible that minimal basal insulin contributes to low hepatic CEACAM1 levels at fasting, the current studies provide evidence that this is largely mediated by a Ppar␣-dependent mechanism. That Ppar␣ activation down-regulates CEACAM1 expression at fasting is demonstrated by 1) reduction in the promoter activity of rat and mouse Ceacam1 in response to Wy14,643 (Ppar␣ agonist) in cultured hepatoma cells, 2) rapid decline of Ceacam1 mRNA and CEACAM1 protein content upon treatment of hepatoma cells with Wy14,643, 3) reduction of Ceacam1 mRNA and CEACAM1 protein levels by supplementing the diet with Wy14,643 in Ppar␣ ϩ/ϩ but not Ppar␣ Ϫ/Ϫ mice, and 4) binding of liganded Ppar␣ to Ceacam1 promoter in liver lysates derived from Ppar␣ ϩ/ϩ but not Ppar␣ Ϫ/Ϫ mice. Moreover, the rapid down-regulation of

on biochemical parameters
Male mice (n Ͼ 8) were fed either RD alone or supplemented with Wy14,643 (Wy) for 7 days. Blood was drawn from overnight-fasted mice to measure blood glucose as well as plasma C-peptide/insulin molar ratio (insulin clearance). Values are expressed as the mean Ϯ S.E.   Ceacam1 mRNA and CEACAM1 protein content by Wy14,643 in hepatoma cells suggests that the effect of Ppar␣ activation on Ceacam1 expression in murine liver can occur independently of confounding metabolic factors. Further studies are needed to delineate the mechanisms and identify co-repressors/co-regulators of Ceacam1 expression by Ppar␣, but ChIP analysis suggests that liganded Ppar␣ directly regulates Ceacam1 expression. Moreover, our observations are consistent with the reported decrease of Ceacam1 mRNA in mice treated with Ppar␣-selective piperidine agonists that are potent Ppar␣ activators (30). Although Ppar␣ is more commonly known to increase expression of genes, such as those involved in fatty acid catabolism, it has also been shown to repress the expression of many liver-specific genes involved in glucose metabolism, cell adhesion, the CYP2C family of steroid hydroxylases, and positive acute-phase response genes induced during inflammation (30,31).

RD
The opposing effect of Ppar␣ and insulin on hepatic CEACAM1 expression is probably related to the well characterized role of CEACAM1 in regulating insulin action by promoting insulin clearance (5,19,32). Fasting promotes a shift from glycolytic to lipolytic metabolism and robust Ppar␣ activation (16,33). In the early hours of refeeding, Ppar␣ maintains fatty acid ␤-oxidation during the stepwise metabolic recovery by insulin surges until glycogen stores are replenished (17) and glycolysis resumes (18). As summarized in Fig. 7 and reviewed in Hue and Taegtmeyer (34), long chain fatty acyl CoA is transported to the mitochondria at fasting to undergo fatty acid ␤-oxidation to produce acetyl-CoA followed by citrate. Inhibition of pyruvate dehydrogenase by acetyl-CoA leads to accumulation of citrate in the cytoplasm and its gradual inhibition of 6-phosphofructo-1-kinase. This elevates glucose 6-phosphate (G-6-P) levels and, subsequently, causes inhibition of hexokinase and rerouting of G-6-P to the glycogen synthesis pathways until glycogen is replenished, at which point fatty acid oxidation stops, mediated largely by the gradual increase in malonyl-CoA levels and its inhibition of carnitine palmitoyltransferase I-mediated transport of LCFAcyl-CoA (long chain fatty acyl-CoA) into the mitochondria. Contributing to the regulation of malonyl-CoA content is the activity of fatty acid synthase that catalyzes malonyl-CoA conversion to palmitic acid. Down-regulation of this step by insulin phosphorylation of CEACAM1 (10) leads to the gradual recovery of malonyl-CoA and, subsequently, gradual inhibition of carnitine palmitoyltransferase I (CPT1; Fig. 7, dashed lines). This paradigm positions CEACAM1 at the crossroads of the coordinated regulation of fatty acid oxidation by PPAR␣ and insulin in the fasting-refeeding transition (35).
Global Ceacam1 null mice and L-SACC1 mutants with liverspecific overexpression of the dominant negative phosphorylation-defective CEACAM1 mutant develop secondary insulin resistance due to chronic hyperinsulinemia (5,19,32). Moreover, reduced hepatic CEACAM1 expression by a high fat diet results in impaired insulin clearance and insulin insensitivity (21). This demonstrates that lowering CEACAM1 levels can cause insulin resistance. Yet activation of Ppar␣ by synthetic ligands, such as fibrates, improves insulin sensitivity and reverses dyslipidemia. Hence, the reduction in hepatic CEACAM1 levels (and consequently, insulin extraction) by Ppar␣ activators may appear contradictory to the well characterized negative role of impairing CEACAM1-dependent insulin clearance pathways on insulin sensitivity. But Wy14,643 activation of Ppar␣ blunts, almost completely, acute-phase insulin secretion in response to glucose and markedly reduces plasma C-peptide levels in Ppar␣ ϩ/ϩ mice, suggesting a negative effect of Ppar␣ on insulin secretion. However, this occurs in the absence of insulin insufficiency and any adverse effect on glucose homeostasis, and Wy14,643 activation of Ppar␣ increases tolerance to exogenous glucose and insulin in Ppar␣ ϩ/ϩ wild-type but not in Ppar␣ Ϫ/Ϫ mutants. Thus, we postulate that reduction in CEACAM1-dependent hepatic insulin clearance pathways by Ppar␣ activation mediates a compensatory mechanism for the decline in insulin secretion caused by Ppar␣ activation in pancreatic ␤-cells to limit insulin insufficiency and prevent systemic insulin resistance.
By demonstrating changes in CEACAM1 expression during the fasting-refeeding transition, with hepatic CEACAM1 levels being lower during fasting via a Ppar␣-dependent mechanism, the current studies provide in vivo evidence for the physiologic regulation of hepatic CEACAM1. Moreover, they lend further credence to the critical role of CEACAM1 in promoting normal metabolism. The relevance of this regulation can be exploited . Acetyl-CoA strongly inhibits pyruvate dehydrogenase (PDH; thick red line) to prevent glycolysis (right arm) and reroute pyruvate to gluconeogenesis. This also leads to accumulation of citrate in the cytoplasm and, subsequently, inhibition of 6-phosphofructo-1-kinase (PFK1) and the rise in glucose-6-phosphate (G-6-P), which in turn inhibits hexokinase (HK) and ultimately mediates glycogen repletion (green arrows). At the end of the completion of glycogen replenishment (within ϳ8 h of refeeding), ␤-oxidation stops, mediated largely by the gradual recovery of malonyl-CoA levels and its inhibition of carnitine palmitoyltransferase I activity. The current studies propose that the pulsatile release of insulin in the early hours of refeeding elevates CEACAM1 expression and its tyrosine phosphorylation (pY) by the insulin receptor (IR) to cause its binding to fatty acid synthase (FAS) and reduction of its activity, thus, contributing to the gradual increase in malonyl-CoA levels and inhibition of carnitine palmitoyltransferase I (dashed lines). In this manner, reduction of CEACAM1 transcription by PPAR␣ activation at fasting and its stimulation by insulin positions CEACAM1 to contribute to the regulation of fatty acid ␤-oxidation in the fasting-refeeding transition, as mediated by the coordinated action of PPAR␣ and pulsatile insulin surges in the early hours of refeeding. LCFAcyl-CoA, long chain fatty acyl CoA.