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Am J Physiol Endocrinol Metab 293: E587-E594, 2007. First published June 5, 2007; doi:10.1152/ajpendo.00278.2007
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LCAT-null mice develop improved hepatic insulin sensitivity through altered regulation of transcription factors and suppressors of cytokine signaling

Lixin Li,1,* Mark Naples,2,* Hui Song,1,* Ronghua Yuan,1 Feilu Ye,1 Sharmi Shafi,1 Khosrow Adeli,2 and Dominic S. Ng1

1Department of Medicine, St. Michael's Hospital; and 2Division of Clinical Biochemistry, Department of Laboratory Medicine and Pathobiology, Hospital for Sick Children, Toronto, Ontario, Canada

Submitted 3 May 2007 ; accepted in final form 30 May 2007


   ABSTRACT
TOP
 ABSTRACT
RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We previously reported that LCAT-deficient mice develop not only low HDL-cholesterol but also hypertriglyceridemia, hepatic triglyceride (TG) overproduction, and, unexpectedly, improved hepatic insulin sensitivity and reduced hepatic TG content. Here, we examined the mechanistic links underlying this apparent paradox. The LDL receptor-deficient (Ldlr)–/–xLcat–/– mouse model and age- and sex-matched Ldlr–/–xLcat+/+ littermates, both in C57Bl/6 background, were employed. Studies of hepatic insulin signal transduction showed an upregulation of hepatic Irs2 mRNA level (5.3-fold, P = 0.02), IRS-2 protein mass level (1.5-fold, P = 0.009) and pIRS-2 (1.8-fold. P = 0.02) in the Ldlr–/–xLcat–/– mice. There was a 1.2-fold increase in pAkt (P = 0.03) with a nonsignificant change in total Akt. We observed a significant shift in its downstream transcription factor FoxO-1 to the cytosolic compartment (2.3-fold increase in cytosolic/nuclear ratio, P = 0.04). We also observed a significant 3.1-fold increase in nuclear abundance of FoxA-2 mass (P = 0.017) and a 1.5-fold upregulation of its coactivator PGC-1beta (P = 0.002), the coordinated actions of which promotes hepatic TG production and beta-oxidation. Increased hepatic insulin signaling in the Ldlr–/–xLcat–/– mice was associated with an upregulation of the Tcfe3 gene (1.7-fold, P = 0.024), a selective downregulation of the Socs-1 gene by 60% (P = 0.01), and no change in PTP-1B protein mass. These data suggest that LCAT deficiency induces complex alterations in hepatic signal transduction cascades, which explain, at least in part, the observed enhanced insulin signaling in association with hepatic TG overproduction and reduced hepatic TG content.

lecithin:cholesterol acyltransferase; insulin signaling; forkhead proteins; suppressors of cytokine signaling; TFE3

LECITHIN:CHOLESTEROL ACYLTRANSFERASE (LCAT) plays a central role in reverse cholesterol transport by mediating the esterification of cell-derived cholesterol primarily in high-density lipoprotein (HDL) particles and is a major modulator of plasma HDL-cholesterol (HDL-C) levels. The phenotypes in complete LCAT deficiency include profound low HDL-C, elevated plasma free cholesterol-to-esterified cholesterol ratio (FC/CE), modest hypertriglyceridemia (HTG), and, in many incidences, anemia and glomerulopathy (16, 19).

Our group recently reported (20) that the HTG seen in LCAT-deficient mice was in part attributable to hepatic overproduction of TG. Furthermore, we also observed an associated hepatic upregulation of Srebp1 and its downstream lipogenic genes, a metabolic profile frequently observed in rodent models of insulin resistance. On the other hand, our study also revealed that the LCAT-deficient mice in the LDL receptor-null background had reduced fasting insulin and glucose levels in conjunction with a downregulation of hepatic expression of Pepck, a metabolic profile consistent with improved hepatic insulin sensitivity. The mechanism for the link between hepatic TG overproduction and improved insulin sensitivity in LCAT-deficient mice has not been explored.

Downregulation of hepatic Pepck gene in LCAT-deficient mice could be the result of direct inhibitory effect of the increased expression of sterol regulatory element-binding protein-1 (SREBP-1) (34) Pepck is also known to be the direct target of insulin signaling, primarily through inactivation of forkhead box "other"-1 (FoxO-1) (1, 22). Thus, enhanced activities of hepatic insulin signaling cascade may contribute to the suppression of Pepck through phosphorylation and nuclear exclusion of FoxO-1. However, a recent study by Ide et al. suggested that chronic upregulation of SREBP-1 activity suppresses the expression of insulin receptor substrate-2 (IRS-2) (12), attenuating the insulin-signaling cascade. In previous studies, we observed a nearly twofold upregulation of hepatic Srebp1 mRNA level in the LCAT-deficient mice (20, 24). To test the possibility that enhancing the insulin-signaling cascade may contribute to the observed downregulation of Pepck, additional regulatory factors of the expression of Irs2 that might override the SREBP-1 effect need to be explored.

The leucine zipper-containing basic helix-loop-helix protein TFE3 (encoded by Tcfe3), has recently been shown to potently transactivate the Irs2 gene through binding to the E-box in the promoter (18). Increased expression of Tcfe3 in mouse liver promotes insulin signaling through increase in phosphorylation of IRS-2, Akt, and FoxO-1. Other effects include increased phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) and induction of Insig, the latter being expected to attenuate the nuclear abundance of SREBP-1. In normal mice, overexpression of hepatic Tcfe3 gene resulted in a significant reduction of fasting levels of insulin and glucose in association with a reduction of hepatic TG and cholesterol but increased glycogen contents. Overexpression of Tcfe3 in db/db mice resulted in marked improvement in blood glucose, putatively through the restoration of the expressions of Irs2, Akt, Hk2, and Insig (18). Thus TFE3 plays an important role in modulating hepatic insulin and glucose homeostasis.

The suppressor of cytokine signaling (SOCS) family comprises eight members (3). Several members of this family, most notably SOCS-1, -3, and -6, have been shown to play important roles in insulin signaling and glucose homeostasis. Recent studies revealed that Socs1 and Socs3 are upregulated in insulin-resistant obese mouse models (27). Transgenic overexpression of Socs1 or -3 in the liver causes systemic insulin resistance, upregulation of Pepck transcription, increased nuclear abundance of SREBP-1c, and hepatic steatosis (28). SOCS upregulation inhibits insulin signaling in part through attenuation of IRS phosphorylation and expression (5, 27). On the other hand, suppression of hepatic SOCS-1 through gene-targeted deletion has been shown to enhance hepatic insulin signaling (13). Ueki et al. (28) demonstrated that partial inhibition of SOCS-1 and SOCS-3, alone or together, is sufficient to result in improvement of insulin sensitivity, hepatic steatosis, and hyperlipidemia in ob/ob mice.

The forkhead protein FoxA-2 has recently been shown to play critical roles in glucose and lipoprotein metabolism (31, 32). Binding of insulin to its receptor phosphorylates FoxA-2 by activating Akt, resulting in the nuclear exclusion of the transcription factor. In mice, insulin-deficient states, e.g., fasting or in models of type 1 diabetes, are associated with marked nuclear localization of FoxA-2 and chronic activation of its transcriptional programs of lipid metabolism and ketogenesis (31). On the other hand, in mouse models of insulin resistance, chronic hyperinsulinemia is associated with increased localization of FoxA-2 in the cytoplasm and remains inactive. It was suggested that the level of nuclear localization of FoxA-2 correlates inversely with the plasma level of insulin (31). Adenovirus-mediated overexpression of the phosphorylation-defective FoxA-2 mutant T156 developed lower plasma levels of glucose and insulin and was associated with increase in hepatic IRS-2 protein and phosphorylated Akt (31). Chronic FoxA-2 activation also results in upregulation of Dgat2 and Mttp, increased plasma TG and ketone bodies, and reduction in hepatic TG content (32). Therefore, increased nuclear abundance of FoxA-2 in the liver may be associated with improved insulin sensitivity, increased beta-oxidation, and increased production and secretion of TG-rich lipoproteins. The inductive effects of FoxA-2 are further augmented in the presence of its co-activator PGC-1beta (32).

In this paper, we characterize the hepatic insulin-signaling cascade in its regulatory role in Pepck expression in the LCAT-deficient mice. We further test the hypothesis that altered regulation of Tcfe3, Socs, and PTP1B are involved in the modulation of the insulin signaling. We also studied the role of FoxA-2 in the observed increase in hepatic TG secretion and improved insulin sensitivity in these mice.


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Lcat–/– mice were backcrossed with C57Bl/6 females (Jackson Laboratories, Bar Harbor, ME) for more than seven generations in our laboratory, as previously reported (24). Ldlr–/– mice in C57Bl/6 background were purchased from Jackson Laboratories. Ldlr–/–xLcat–/– double-knockout (DKO) mice were generated by crossing Ldlr–/– mice with Lcat–/– mice. Double heterozygous offspring were cross-bred to generate Ldlr–/–xLcat+/–, which were used to generate Ldlr–/–xLcat–/– mice, with Ldlr–/–xLcat+/+ littermates used as controls. Control mice for each genotype were age and sex matched. All experimental procedures were approved by the Animal Care Committee at St. Michael's Hospital.

Antibodies. Rabbit polyclonal anti-FoxO-1 antibody was purchased from Chemicon International (Temecula, CA). Rabbit polyclonal anti-HNF-3beta/FoxA-2, anti-IRS-2, and anti-PTP-1B were purchased from Upstate Cell Signaling solutions (Cedarlane; Hornby, ON, Canada). Polyclonal TATA box-binding protein (TBP) antibody was purchased from GenWay (San Diego, CA). Rabbit polyclonal anti-IR (beta-subunit) and mouse monoclonal anti-phosphotyrosine (pTyr) antibodies were purchased from Santa Cruz Biotechnology, (Santa Cruz, CA). Rabbit anti-Akt, anti-phospho-Akt (Ser473), and anti-phospho-Akt (Thr308) were purchased from Cell Signaling Technology (Beverly, MA).

Quantitation of hepatic gene mRNAs. Study mice were fasted overnight prior to being killed. Hepatic mRNA levels of Socs1, -3, -6, insulin receptor substrate-2 (Irs2), TFE3 (Tcfe3), PPAR{gamma} coactivator-beta (Pgc1beta), and GAPDH (Gapdh) were analyzed by semiquantitative RT-PCR as previously described (24). Briefly, total RNA was extracted using TRIzol (Invitrogen) per the manufacturer's suggested protocol. RT-PCR was performed using the Superscript One-step RT-PCR kit (Invitrogen). Intensity of the PCR product for each gene of interest was determined using the Bio-Rad GS800 densitometer normalized to that of Gapdh under identical conditions. Primer pairs used to amplify these genes were as follows (forward and reverse, respectively): Irs1 5'-cagtatggtgggtgggaaac, 3'-gttgccacccctagacaaaa; Irs2 5'-tttcaacacctccctctgct, 3'-tcaggataacctgccagacc; Socs1 5'-cctttgacaagcggactctc, 3'-agctcaccagcctcatctgt; Socs3 5'-cctttgacaagcggactctc, 3'-agctcaccagccccatctgt; Socs6 5'-accattgctacctccaatgc, 3'-ccaataccacccctgttttg; Tcfe3 5'-cctgaaggcatctgtggatt, 3'-tgtaggtccagaagggcatc; Pgc1{alpha} 5'-aaacttgctagcggtcctca, 3'-tttctgtgggtttggtgtga; Pgc1beta 5'-cctacccacaaggacagcat, 3'-tcctcttcctcctcctcctc; Vlcad 5'-cactcaggcagttctggaca, 3'-tcccagggtaacgctaacac; Mcad 5'-ggcaaatgcctgtgattctt, 3'-acccattgcgatcttgaaac; Gapdh 5'-gggtgtgaaccacgagaaat, 3'-cctgcttcaccaccttcttg.

Western blot analyses. After overnight fasting, experimental mouse livers were isolated and snap-frozen at –86°C until use. Total protein was extracted from tissue lysates as previously described (24). Cytosolic and nuclear components of whole liver were isolated using the Compartment Protein Extraction Kit (cat. no.2145; Chemicon, Temecula, CA). Protein concentrations were determined using the MicroBCA Protein Assay Kit (Pierce, Rockford, IL). FoxO-1 protein expression levels in cytosolic and nuclear fractions were analyzed individually using Western blot technique as previously described (24). FoxA-2 protein expression levels were analyzed in the nuclear fractions with TBP as a loading control.

In vivo insulin treatment. Following an overnight fast, anesthetized mice were injected with Humulin R (5 U/kg body wt) via the inferior vena cava. The liver was resected 1 min after the bolus injection and immediately snap-frozen in liquid nitrogen.

To assess insulin-mediated phosphorylation of Akt, IR, and IRS-2, frozen liver tissue was lysed in the presence of phosphatase inhibitors, and protein concentrations were measured as described above. Equivalent amounts of total protein from Ldlr–/– and Ldlr–/–xLcat–/– mice (0.5–1.5 mg) were subjected to overnight immunoprecipitation at 4°C using polyclonal antibodies specific for either insulin receptor (IR) or IRS-2. Following immunoprecipitation, samples were denatured and resolved by SDS-PAGE and then transferred to PVDF membranes, which were subsequently probed with a monoclonal anti-phosphotyrosine antibody (phosphorylation of Akt in response to insulin was assessed directly using an anti-phospho-Akt antibody, thereby circumventing this immunoprecipitation step). Membranes were later stripped and reprobed for substrate mass using the appropriate antibodies. Detection of immunoreactive bands was achieved by exposing film to PVDF membranes preincubated in ECL reagents (Western Lightning, PerkinElmer) per the manufacturer's instructions. Relative band intensity was quantified using an imaging densitometer aided by FluorChem software (Alpha Innotech).

Insulin tolerance test. Mice were fasted for 4 h starting at 9 AM before receiving an intraperitoneal bolus of insulin (Humulin R; 0.75 U/kg body wt). Blood glucose was measured prior to the insulin injection (time 0 min) and at 20, 60, and 120 min after the injection. Glucose values at each time point were expressed as percent reduction in glucose from baseline.

Plasma ketone bodies. Plasma samples were taken from mice after an overnight fast. With the ketone meter method, plasma level of beta-hydroxybutyrate was performed on Abbott's Precision Xtra meter using Abbott's Blood Beta-Ketone test strips (Abbott, Mississauga, ON, Canada). With the D-3-hydroxybutyrate method, plasma level of D-3-hydroxybutyrate was measured using the Ranbut assay kit (Randox International, Antrim, UK).

Statistical analyses. Comparison of group means ± SD was by Student's t-test, and a two-tailed P value of <0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Evidence for enhanced insulin sensitivity in LCAT-deficient mice. We (20) have previously documented improved fasting glucose and insulin, as well as a trend toward improved glucose tolerance in the LCAT-deficient mice. Here, we performed intraperitoneal insulin tolerance tests (ITT) to further evaluate the insulin sensitivity status of the LCAT-deficient mice. Following insulin injection, we observed a marked and significant lowering of plasma glucose at 20 and 60 min postinjection in the LCAT-deficient mice compared with control littermates (Fig. 1).


Figure 1
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  		Fig. 1. Assessment of insulin sensitivity using the insulin tolerance test. Serial glucose levels post-insulin injection at 0.75 U/kg body wt after a 5-h fast starting at 9 AM. Results are expressed as %reduction vs. baseline. LCAT, lecithin:cholesterol acyltransferase; LDLR, low-density lipoprotein receptor. Dotted line, Ldlr–/–xLcat–/– (n = 10); solid line, Ldlr–/–xLcat+/+ controls (n = 9).

 
Perturbations in hepatic insulin-signaling cascades. LCAT-deficient mice were previously shown to exhibit significant upregulation of Srebp1, which may underlie the observed hepatic TG overproduction. To investigate whether the chronic upregulation of Srebp1 in the LCAT-deficient mice may result in the suppression of Irs2 mRNA expression, we measured the hepatic Irs2 mRNA expression by RT-RCR. Our study revealed a 5.3-fold upregulation (P = 0.024) of hepatic Irs2 gene in the Ldlr–/–xLcat–/– mice compared with the Ldlr–/– control (Fig. 2A). This finding corroborates with a 1.5-fold increase (P = 0.009) in IRS-2 protein mass by Western blot analysis (Fig. 2B). We also observed a 1.84-fold increase in pIRS-2 (Fig. 2C). On the other hand, there was no difference in mRNA levels of Irs1 (data not shown).


Figure 2
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  		Fig. 2. A: hepatic expression and phosphorylation of IRS-2 in Ldlr–/–xLcat–/– mice vs. Ldlr–/–xLcat+/+ control mRNA levels by semiquantitative RT-PCR after normalization to that of Gapdh gene (n = 4 for each genotype). B: total IRS-2 protein mass by Western blot (n = 6 for each genotype). C: phosporylated (p)IRS-2 (n = 5 for LCAT-deficient mice; n = 3 for controls).

 
To determine whether the increased pIRS-2 seen in the Ldlr–/–xLcat–/– mice is associated with upstream and downsteam alterations in the insulin-signaling cascade, we evaluated the insulin-induced phosphorylation of IR and Akt by Western blot analysis. We observed a 1.27-fold increase in pIR level in the LCAT-deficient mice (P = 0.027). On the other hand, the IR mass was unchanged between the two groups (0.96-fold, P = 0.81; Fig. 3). We also observed a 1.23-fold increase in pAkt protein level 1 min after insulin injection (P = 0.026), consistent with a net increase in insulin-induced phosphorylation of the active form of Akt (Fig. 4).


Figure 3
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  		Fig. 3. Insulin-simulated hepatic expression of phospho-insulin receptor (pIR) (A) and total IR protein mass (B) Ldlr–/–xLcat–/– (n = 8) vs. Ldlr–/–xLcat+/+ (n = 6) controls.

 

Figure 4
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  		Fig. 4. Insulin-stimulated hepatic expression of (a) pAkt (Ser473, n = 10; A) and total Akt protein mass (n = 8; B) Ldlr–/–xLcat–/– mice vs. Ldlr–/–xLcat+/+ controls.

 
Subcellular distribution of FoxO-1 protein in liver. FoxO-1 is a downstream substrate for activated Akt. Increased phosphorylation of FoxO-1 by pAkt would result in redistribution of pFoxO-1 to the cytoplasm, reducing the transactivation of its target genes, which include Pepck and Irs2. To address whether the observed increase in pAkt impacts on FoxO-1 phosphorylation, we quantified the relative abundance of FoxO-1 between nuclear and cytoplasmic fractions by Western blot. We observed a 2.3-fold increase (P = 0.04) in cytosolic/nuclear FoxO-1 protein ratio in the Ldlr–/–xLcat–/– mice compared with their Ldlr–/–xLcat+/+ controls, a finding consistent with enhanced insulin signaling upstream of FoxO-1 (Fig. 5A).


Figure 5
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  		Fig. 5. Subcellular distribution of forkhead proteins in livers of Ldlr–/–xLcat–/– mice vs. Ldlr–/–xLcat+/+ control mice. A: cytoplasmic/nuclear ratio of FoxO-1 (n = 6 for each genotype). B: nuclear abundance of FoxA-2, normalized to TBP (n = 6 for each genotype). C: hepatic mRNA levels of Pgc1beta by semiquantitative RT-PCR in Ldlr–/–xLcat–/– mice vs. Ldlr–/–xLcat+/+ controls.

 
Nuclear abundance of FoxA-2 in liver. Recently, FoxA-2 in the liver, a downstream substrate for activated Akt phosphorylation, has been shown to play important roles in glucose homeostasis, very low-density lipoprotein (VLDL) production, and ketogenesis differentially through its multitude of target genes. By performing Western blot on the nuclear subfraction, we observed a 3.1-fold increase (P = 0.017) in the nuclear abundance of FoxA-2 in the Ldlr–/–xLcat–/– mice compared with their Ldlr–/–xLcat+/+ controls. This finding is consistent with the notion of increased transactivation of the Foxa-2 target genes (Fig. 5B).

Expression of FoxA-2 coactivators and target genes in beta-oxidation. Increased nuclear abundance of FoxA-2 is associated with increased transactivation of its target genes. In addition to its effects in upregulating at least two of the genes involved in VLDL assembly, FoxA-2 has also been shown to induce a number of genes involved in mitochondrial beta-oxidation. Furthermore, PGC-1beta has been shown to be a coactivator for FoxA-2, and its recruitment results in enhanced transactivation of the target genes. Since PGC-1beta has been shown to be inducible at the transcriptional level by a number of metabolic changes, including high-fat diet, we investigated the expression level of PGC-1beta in the study mice. We observed a 1.5-fold upregulation of Pgc1beta in the Ldlr–/–xLcat–/– mice (P = 0.002) (Fig. 5C). We investigated the expression of two key FoxA-2-regulated genes in mitochondrial beta-oxidation, Vlcad and Mcad. We observed a 1.7-fold increase in the mRNA level of Vlcad (P = 0.0002). However, the RT-PCR of Mcad gene showed no difference between the two groups (1.11-fold change with P = 0.50; Fig. 6). To investigate the impact of FoxA-2 activation of the genes for beta-oxidation, we measured the fasting level of beta-hydroxybutyrate by using two independent methods. We observed a 1.23-fold increase in plasma beta-hydroxybutyrate with both methods, with P = 0.07 for the ketone meter method and P = 0.12 for the Ranbut assay (Fig. 7).


Figure 6
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  		Fig. 6. Hepatic mRNA levels of genes in mitochondrial beta-oxidation by semiquantitative RT-PCR after both normalized to Gapdh. A: Vclad (n = 6 for each genotype). B: Mcad (n = 6 for each genotype).

 

Figure 7
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  		Fig. 7. Serum beta-hydroxybutyrate levels by Ketone Meter method and D-3-hydroxybutyrate method with Ranbut assay (n = 10 for each genotype).

 
Hepatic expression of Socs, Tcfe3, and PTP1B. A number of intracellular proteins have been shown to play important roles in modulating the insulin-signaling cascade, particularly in their roles in regulating the IRS. It was recently discovered that hepatic overexpression of TFE3 in mice significantly enhances insulin-signaling pathways, especially through transcriptional upregulation of Irs2. We investigated whether expression of the Tcfe3 gene might be altered in LCAT-deficient mice, which would potentially contribute to the hepatic insulin sensitivity phenotypes. We observed a 1.7-fold upregulation of Tcfe3 in the Ldlr–/–xLcat–/– mice (P = 0.034; Fig. 8). To explore putative roles for some of the members of the SOCS family known to modulate insulin signaling, we determined the levels of gene expression of Socs1, -3, and -6 in liver of the Ldlr–/–xLcat–/– mice compared with that in the Ldlr–/–xLcat+/+ controls. We observed a significant 60% reduction (P = 0.01) in hepatic mRNA expression of Socs1. On the other hand, neither Socs3 nor Socs6 mRNA level was significantly altered (Fig. 8). PTP-1B has been shown to modulate insulin signaling through dephosphorylation of both IR and IRS-1/2, and deletion of the PTP1B gene has been shown to enhance insulin signaling and improve insulin sensitivity. We performed Western blot analysis on PTP-1B protein mass and observed no change in abundance of PTP-1B protein in the liver (results not shown).


Figure 8
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  		Fig. 8. Hepatic mRNA levels of Socs and Tcfe3 by semiquantitative RT-PCR after normalization to Gapdh. Open bar, Ldlr–/–xLcat+/+ control; filled bar, Ldlr–/–xLcat–/–. *P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this paper, we report that, in LDLR knockout background, deletion of the Lcat gene confers improved hepatic insulin signaling and insulin sensitivity. In addition to our previous observation of a concomitant reduction in fasting glucose and insulin levels in the LCAT-deficient mice, our ITT data provide additional in vivo evidence for improved insulin sensitivity, a metabolic alteration likely not restricted to the liver. We observed enhancement of the insulin-signaling cascade in the liver, which is linked to our previous observation of suppressed Pepck expression. This enhancement of hepatic insulin signaling is mediated through the coordinated modulation of a number of transcription-modulatory factors pertinent to insulin signaling acting in synergy. Concomitantly, the enhanced insulin signaling was further linked to an increase in FoxA-2 activation, in part explaining the simultaneous presence of increased TG secretion, reduced hepatic TG content, and modest increase in beta-oxidation. In our previous report, LDL receptor knockout mice made LCAT-deficient were linked to hypertriglyceridemia with associated upregulation of Srebp1 and its lipogenic target genes Acc1, Fas, and Scd1 but was also associated with downregulation of Pepck and reduced fasting levels of glucose and insulin (20). The concomitant upregulation of hepatic Srebp1 and the target lipogenic genes are commonly observed in murine models of insulin resistance, including the ob/ob mouse model and the lipodystrophy model. However, in these insulin-resistant mice, the chronic upregulation of SREBP-1 was also found to be associated with a marked suppression of the expression of hepatic Irs2, contributing to the observed hepatic insulin resistance. Ide et al. (12) have recently shown that the suppressive effect of SREBP-1 is likely mediated through binding of nuclear SREBP-1 to the sterol response element (SRE) in the IRS-2 promoter. Unexpectedly, Irs2 gene expression, IRS-2 protein mass, and phosphorylated IRS-2 were all found to be upregulated in the Ldlr–/–xLcat–/– mice compared with the Ldlr–/–xLcat+/+ controls, a combination of findings more in line with the observed overall improved insulin sensitivity.

To examine the impact of the increased expression of IRS-2 and its insulin-stimulated phosphorylation in the LCAT-deficient mice, we observed a modest increase in insulin-stimulated phosphorylation of Akt. FoxO-1 has recently been shown to be a direct substrate of pAkt and the major mediator of insulin-induced inhibition of gluconeogenesis. It acts through direct binding to the promoters of gluconeogenesis genes, e.g., PEPCK and glucose 6-phosphate and activating the process of glucose production (22). We observed a shift in the relative abundance of FoxO-1 between the cytoplasmic and the nuclear fractions, consistent with the notion of enhanced insulin signaling, and it may in part explain our previously observed reduced mRNA level of Pepck (20) (Fig. 9).


Figure 9
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  		Fig. 9. Integrative metabolic regulatory network in the present study. See text for definitions.

 
To explain the upregulation of Irs2 gene in the presence of chronic upregulation of Srebp1, additional counteracting regulatory factors need to be sought. Recently, the novel transcription factor TFE3, a member of the bHLH protein family, has been shown to regulate a number of metabolic genes through binding to the E box in their promoters (18). Tcfe3, the gene that encodes TFE3, is highly expressed in organs involved in energy metabolism. Overexpression of Tcfe3 resulted not only in upregulation of Irs2 mRNA but also in IRS-2 mass and pIRS-2. In addition, Akt mass and insulin-stimulated phosphorylation of Akt, GSK-3beta, and FoxO-1 were also significantly increased in conjunction with increased liver glycogen, reduced TG and cholesterol contents, and reduced fasting levels of insulin and glucose. In db/db, a mouse model of insulin resistance, adenovirus-mediated overexpression of Tcfe3 reversed the reduced basal levels of Irs2, Akt, Gsk3beta, and Insig (18). In the same study, the authors postulated that the proximity of the E box with the IRE and SRE in the IRS-2 promoter permits interactions between the transactivation of TFE3 and inhibitory effect of SREBP-1. In LCAT-deficient mice, our observation of a 1.7-fold upregulation of Tcfe3 gene in the liver may account, at least in part, for the absence of suppression of Irs2 gene and IRS-2 protein mass. Furthermore, Tcfe3 upregulation is also consistent with our observed upregulation of Irs2 mRNA levels, IRS-2 protein mass, and increased phosphorylation of IRS-2, FoxO-1, and Akt in conjunction with reduced hepatic TG and cholesterol contents and in vivo improvement of hepatic insulin sensitivity (20, 24).

Our observation of the increase in nuclear abundance of FoxA-2 and a modest upregulation of its coactivator PGC-1beta in the Ldlr–/–xLcat–/– mice may also explain, in part, the unexpected association between hepatic TG overproduction with improved insulin sensitivity and beta-oxidation. Previous studies revealed that constitutive nuclear localization of FoxA-2 through overexpresseion of the phosphorylation-defective FoxA-2 mutant in mice led to the induction of the expression of Dgat2 and Mttp. This results in a concomitant increase in endogenous TG synthesis and lipidation of apolipoprotein B, leading to enhanced VLDL synthesis and secretion (32). In this study, FoxA-2 appeared to have a stronger inductive effect on DGAT-2 than that of MTP (30). Furthermore, increased nuclear abundance of FoxA-2 also increases hepatic expression of genes involved in mitochondrial beta-oxidation, including Mcad, Vlcad, and Cpt1. These transcriptional inductive effects were further augmented by concomitant expression of its coactivator PGC-1beta. In the Ldlr–/–xLcat–/– mice, we previously reported a nearly twofold increase in mRNA levels of Dgat2 but no significant change in Mttp (24). The differential regulation of these two putative FoxA-2 target genes could be a result of the differential inductive potency of FoxA-2 on MTP and DGAT-2 (32). On the other hand, as it has been shown that Mttp is negatively regulated by SREBP-1 (23), the observed upregulation of Srebp1 in the Ldlr–/–xLcat–/– mice could have partially antagonized the effect of FoxA-2 on MTP. Likewise, our observation of a modest upregulation of Vlcad mRNA, but a lack of significant change in the expressions of other genes in beta-oxidation like Mcad could again be attributable to either a variable response of target genes to a modest inductive signal, or the observed change in Mcad mRNA levels may be the result of competing transcriptional signals. Our observation of a trend toward an increase in plasma level of ketone bodies in LCAT-deficient mice is consistent with the notion of increased beta-oxidation and ketogenesis as a direct effect of increased FoxA-2 activation through Vlcad. Putting it all together, the increased nuclear abundance of FoxA-2 in the LCAT-deficient mice could account for the combined observations of increase in TG synthesis, enhanced beta-oxidation, and insulin sensitivity. In turn, these data may, at least in part, account for the observed significant reduction in hepatic TG content in the Ldlr–/–xLcat–/– mice (24).

Our observation of a selective downregulation of Socs1 gene in the liver of Ldlr–/–xLcat–/– mice may also contribute to the observed phenotype of enhanced hepatic insulin signaling and improved insulin sensitivity in these mice. Several members of the SOCS family, most notably SOCS-1, -3, and -6, are involved in insulin signaling and glucose homeostasis (5). Obese mice are associated with increased expression of Socs1 and Socs3 in the liver and muscle (28), partially impairing insulin-induced phosphorylation of IRS. Transgenic overexpression of Socs1 or Socs3 in liver causes systemic insulin resistance, upregulation of Pepck1 mRNA, increased nuclear abundance of Srebp1c, and hepatic steatosis (28). Ueki et al. (28) demonstrated that partial inhibition of SOCS-1 and SOCS-3, alone or together, is sufficient to result in improvement of insulin sensitivity, hepatic steatosis, and hyperlipidemia in ob/ob mice. Socs1 knockout mice bred into the interferon-{gamma} knockout background, the latter to ameliorate the fatal inflammation seen in the Socs1 single-knockout mice, developed enhanced insulin sensitivity selectively in the liver in associaton with upregulation of hepatic IRS-2 mass, insulin-induced phosphorylation of IRS-2, and upregulation of phosphorylated Akt (13). The expressions of gluconeogenic enzymes PEPCK and glucose-6-phosphatase in response to insulin were both reduced, consistent with the observations of reduced fasting glucose and insulin levels and their upstream enhancement in insulin signaling. Recent data suggest that SOCS-1 preferentially binds to the kinase domain of the IR which is critical for the recognition of IRS-2 rather than IRS-1, providing an explanation for the selective upregulation of IRS-2 mass and its phosphorylation in SOCS-1-deficient mice (13, 28). On the basis of these studies, the reduction of Socs1 expression in LCAT-deficient mice could be a significant contributor to the overall enhancement in insulin sensitivity. Unlike TFE3 and SOCS-1, expression of PTP-1B, a well-established negative modulator of insulin signaling, did not seem to be altered in the LCAT-deficient mice.

The mechanism for the observed altered levels of gene expression of both SOCS-1 and TFE3 in the liver of LCAT-deficient mice is unclear. SOCS expression is regulated at both the transcriptional and translational levels. Although a large array of cytokines have been shown to induce expression of Socs (5), only a limited number of factors are known to mediate the suppression at the mRNA level. Promoter analysis revealed that STAT6 and Ets synergistically suppress Socs1 expression (26). Protooncoprotein GFI-1B has also been shown to suppress both Socs1 and Socs3 expressions (14). Socs1 expression has been shown to be silenced by hypermethylation in a variety of cancer cells. The significance of these factors in the observed downregulation of Socs1 in the liver of Ldlr–/–xLcat–/– mice is not known.

The mechanistic links between the primary defect, namely LCAT deficiency, and the observed altered TG and polyunsaturated fatty acid (PUFA) metabolism and enhancement in insulin sensitivity are likely multifactorial. We postulate that alterations in hepatic cholesterol flux and in PUFA metabolism may underlie parts of the observed metabolic perturbations.

In rodents, increased dietary cholesterol raises hepatic cholesterol content and promotes hepatic steatosis, hypertriglyceridemia (9, 10), and insulin resistance (17), putatively through induction of endoplasmic reticulum stress (21) and mitochondrial dysfunction (15). Hepatic cholesterol content is significantly reduced in LCAT-deficient mice despite compensatory upregulation of Hmgcr gene (24), minimizing the metabolic stress. On the other hand, cellular cholesterol depletion may induce inactivation of protein phosphatase-2A (PP2A) (30), leading to sustained phosphorylation of a variety of signaling molecules and transcription factors, including ERK (30), Akt (29), AMP-activated protein kinase (AMPK) (33), carbohydrate response element-binding protein (ChREBP) (6), etc. Activation of Akt and AMPK with their known downstream insulin-enhancing effects (25, 29, 33) thus represents candidate-contributing links.

Dietary PUFA reduces insulin resistance through a variety of mechanisms, but the effect of a reduced PUFA pool on insulin sensitivity is not well studied. Phosphorylation of ChREBP by protein kinase A and AMPK produces the inactive form, which remains cytosolic, and its activation involves dephosphorylation by PP2A and nuclear translocation (6). Nuclear abundance of ChREBP is elevated in mouse models of insulin resistance, and gene silencing reversed many features of insulin resistance (7). PUFA inhibits ChREBP activities primarily by blocking its nuclear translocation and slowing the mRNA decay (6). Putatively LCAT-deficient mice (11, 24) develop a reduced PUFA pool, which would imply predisposition to insulin resistance. However, a concomitant low PP2A and high activated AMPK activities as discussed may counteract the disinhibition of ChREBP by maintaining the inactive phosphorylated form.

PUFA also strongly induces the expression of adipose differentiation-related protein (ADRP), a key lipid droplet-associated protein and an important modulator of hepatic TG metabolism (4, 8). Adrp knockout mice developed marked reduction in hepatic TG content, but the rate of TG secretion was preserved (2), an observation rather similar to the TG overproduction/low hepatic TG phenotype seen in LCAT-deficient mice (20, 24). It is conceivable that the putative low PUFA pool may contribute, at least in part, to the TG overproduction/low hepatic TG phenotype through altered Adrp metabolism. Additional studies are required to further elucidate these links.

In summary, although hepatic TG overproduction and low HDL-C are cardinal features of insulin resistance, we reported that the hepatic TG overproduction and profound low HDL in LCAT-deficient mice are associated with enhanced hepatic insulin sensitivity. Mechanistically, we observed alterations in the expressions of a number of transcription factors, and SOCS recently was shown to be important in insulin signaling and glucose homeostasis, which coordinately promote improvement in hepatic insulin sensitivity. These phenotypes are distinctly different from other murine models of TG overproduction frequently seen in models of insulin resistance, e.g., ob/ob and db/db mice. The increased nuclear abundance of FoxA-2 may also provide the important metabolic link between the observed hepatic TG overproduction and a metabolic milieu of improved insulin signaling along with favorable changes in the markers of hepatic beta-oxidation. Our findings have provided novel insights into the impact of how a primary defect in LCAT action affecting high-density lipoprotein metabolism may result in significant improvement in insulin sensitivity.


   GRANTS
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 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported in part by a Canadian Institute of Health Research operating grant (MOP 77527) to D. S. Ng and a Grant-in-aid by the Heart and Stroke Foundation of Ontario (T5323) to K. Adeli.


   ACKNOWLEDGMENTS
 
We acknowledge the excellent technical support of Man khun Chan and Jianzhong Zhao. We also acknowledge the Banting and Best Diabetes Center at the University of Toronto for a summer studentship to F. Ye and to St. Michael's Hospital for a summer studentship to S. Shafi.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. S. Ng, St. Michael's Hospital Shuter Wing 3-041, 30 Bond St., Toronto, ON, Canada M5B 1W8 (e-mail: ngd{at}smh.toronto.on.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* L. Li, M. Naples, and H. Song contributed equally to the manuscript. Back


   REFERENCES
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 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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