Skip to main content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Arterioscler Thromb Vasc Biol. Author manuscript; available in PMC 2009 Apr 1.
Published in final edited form as:
Arterioscler Thromb Vasc Biol. 2008 Oct; 28(10): 1738–1744.
Published online 2008 Jul 31. doi: 10.1161/ATVBAHA.108.171538
PMCID: PMC2655237
NIHMSID: NIHMS70223
PMID: 18669885

Alterations in Hepatic Metabolism in fld Mice Reveal a Role for Lipin 1 in Regulating VLDL-Triacylglyceride Secretion

Zhouji Chen, Matthew C. Gropler, Jin Norris, John C. Lawrence, Jr, Thurl E. Harris, and Brian N. Finck
Author information Copyright and License information PMC Disclaimer

Associated Data

Supplementary Materials

Abstract

Objective

Lipin 1 controls fatty acid metabolism in the nucleus as a transcriptional regulator and in the cytosol as an enzyme catalyzing the penultimate step in phosphoglycerol triacylglyceride (TAG) synthesis. We sought to evaluate the effects of lipin 1 on hepatic TAG synthesis and secretion by gain-of-function and loss-of-function approaches.

Methods and Results

Rates of TAG synthesis were not impaired in hepatocytes isolated from adult lipin 1—deficient (fld) mice and were actually increased in 14-day-old fld mice. Additionally, compared to littermate controls, VLDL-TAG secretion rates were markedly increased in fld mice of both ages. Lipin 1 overexpression did not alter TAG synthesis rates but significantly suppressed VLDL-TAG secretion. The lipin 1-mediated suppression of VLDL-TAG secretion was linked to the peptide motif mediating its transcriptional-regulatory effects. However, the expression of candidate genes required for VLDL assembly and secretion was unaltered by lipin 1 activation or deficiency. Finally, the hepatic expression of lipin 1 was diminished in obese insulin-resistant mice, whereas adenoviral-mediated overexpression of lipin 1 in liver of these mice inhibits VLDL-TAG secretion and improves hepatic insulin signaling.

Conclusions

Collectively, these studies reveal new and unexpected effects of lipin 1 on hepatic TAG metabolism and obesity-related hepatic insulin resistance.

Keywords: lipin 1, liver, triglyceride, VLDL secretion, dyslipidemia

Obesity, insulin resistance, and diabetes are linked to increased rates of triacylglyceride (TAG)-rich very low—density lipoprotein (VLDL) secretion.1 Dyslipidemia is a primary risk factor for cardiovascular disease and is linked to a poor prognosis after an ischemic event.2,3 With the increased prevalence of obesity and related metabolic diseases, dyslipidemias are emerging as a major public health concern. However, the regulatory mechanisms governing VLDL synthesis, secretion, and degradation are incompletely understood, and factors that play important roles in these processes continue to emerge.

Lipin 1 is an intracellular protein that has emerged as a critical regulator of lipid metabolism.4 Spontaneous loss-of-function mutations in the gene encoding lipin 1 in fatty liver dystrophic (fld) mice cause a spectrum of metabolic abnormalities, including hepatic steatosis, hyperlipidemia, lipodystrophy, and insulin resistance.5–7 Interestingly, dyslipidemia and hepatic steatosis in fld mice spontaneously resolves just before weaning.5 However, adult fld mice remain lipodystrophic and exhibit insulin resistance and increased susceptibility to atherosclerotic lesion formation.6

Recent work has determined that lipin 1 regulates metabolism via activity in both the cytoplasmic and nuclear compartments. Lipin 1 exhibits activity as a Mg2+-dependent phosphatidic acid phosphohydrolase (PAP-1) enzyme,8–10 which catalyzes the dephosphorylation of phosphatidic acid (PA) to generate diacylglycerol (DAG; Figure 1). This constitutes the penultimate step in the phosphoglycerol TAG synthesis pathway. However, whether lipin 1—associated PAP-1 activity is necessary and sufficient to drive TAG synthesis in liver cells has not been determined. Unlike other enzymes in this TAG synthetic pathway that are integral membrane proteins, lipin 1 is soluble and transiently associates with membranes.11 Moreover, lipin 1 rapidly translocates within the cell in response to phosphorylation by the insulin signaling pathway.8 Interestingly, lipin 1 also contains a putative nuclear localization signal and can be nuclear-localized.7,12 Lipin 1 has been shown to act as a transcriptional regulator by associating with DNA-bound transcription factors to modulate their activity.13,14 For example, lipin 1 interacts with and coactivates the peroxisome proliferator-activated receptor α (PPARα) and PPARγ coactivator 1α (PGC-1α) and enhances the expression of genes involved in fatty acid oxidation.13 Collectively, these studies suggest that lipin 1 could play important roles in regulating TAG metabolism through its activity as a PAP-1 enzyme and its transcriptional effects on fatty acid metabolic gene programs.

An external file that holds a picture, illustration, etc. Object name is nihms-70223-f0001.jpg

Lipin 1—deficient hepatocytes from 14-day-old mice exhibit high rates of TAG synthesis and secretion. The schematic depicts the terminal steps of the phosphoglycerol TAG synthesis pathway. Lipin proteins catalyze the PAP-1 (Mg2+-dependent) reaction. PA indicates phosphatidic acid; PAP, PA phosphohydrolase; DAG, diacylglycerol; DGAT, diacylglycerol acyltransferase; TAG, triacylglycerol. The graph depicts mean rates of TAG synthesis and secretion by primary hepatocytes isolated from 14-day-old fld or wild-type littermate mice. At 6 to 8 hours after isolation, hepatocytes were labeled with 2-3H-glyceol for 3 hours, and 3H-TAG in cell (synthesis) or medium (secretion) were determined. From these values, the secretion efficiency (ie, the percentage of total TAG that was secreted) was also calculated. The mean and SD of 3 independent experiments each done in triplicate are shown. **P<0.01 vs WT hepatocytes. The gel images depict the synthesis and secretion of 35S-labeled apoB48 proteins as described in Methods. Inset values represent mean normalized intensity of the synthesized and secreted 35S-labeled apoB48 proteins.

Availability of neutral lipids, especially TAG, is a driving force for VLDL biogenesis. By promoting TAG synthesis through its PAP-1 activity, lipin 1 might be expected to stimulate hepatic VLDL-TAG secretion. However, overexpression of lipin 1 in liver lowers the circulating TAG concentration in vivo13 and neonatal lipin 1—deficient fld mice are hyperlipidemic,5 suggesting that lipin 1 inhibits rather than promotes hepatic VLDL-TAG secretion. To address this issue, we used both loss-of-function and gain-of-function models to evaluate the effects of lipin 1 on hepatic TAG synthesis and secretion. We also used lipin 1 proteins with site-directed mutations in critical domains to distinguish the role of its PAP-1 and transcriptional coactivator activity. Our data indicate that lipin 1 is not required for robust hepatic TAG synthesis but plays a significant role in regulating hepatic VLDL-TAG secretion.

Experimental Procedures

Mouse Studies

All genetically altered or mutant mice were compared to appropriate strain-matched or littermate controls, and only male mice were studied. In studies using fld mice,5 homozygous fld (fld/fld) mice were compared to heterozygous (fld/+) or wild-type (+/+) littermate control mice (Balb-c strain). With regards to TAG metabolism, we found no difference between heterozygous and wild-type mice. Hereon, both will be considered “wild-type”. PPARα null mice15 were extensively backcrossed into the C57BL/6 strain. Uncoupling protein 1 diphtheria toxin A (UCP-DTA) mice16 were compared to nontransgenic littermate controls (FVB strain). All mice, including C57BL/6 controls, were generated from breeding pairs established in the Washington University mouse facility.

Triacylglyceride secretion rates were determined by using Triton WR-1339 injection to inhibit lipoprotein lipolysis, as previously described.17 Plasma TAG concentrations were determined at 0, 60, and 120 minutes after injection of Triton WR-1339, and rates of TAG secretion were expressed as mg/kg body weight per h.

To examine insulin-stimulated phosphorylation of Akt, mice were fasted overnight and injected i.p. with human insulin (10 mU/g body weight). Mice were killed 5 minutes later and liver was dissected and snap frozen for protein isolation.

All animal experiments were approved by the Animal Studies Committee of Washington University School of Medicine.

Mammalian Cell Culture

Primary cultures of mouse hepatocytes were prepared as described18 from fld or littermate control mice, and metabolic studies were performed 6 to 8 hours later. For studies using adenovirus, hepatocytes were isolated from fld or littermate control mice, and after a 2-hour attachment period they were infected with adenovirus to drive overexpression of proteins defined below, then studied after 40 to 48 hours of infection. For studies using lipin 1 mutant constructs, C57BL6J mice were used.

Adenovirus Constructs

The adenoviral constructs driving expression of HA-tagged lipin-1β or GFP have been previously described.8,13 HA-lipin 1β(D712E) and HA-lipin 1β(LXXFF) were subcloned into the Ad-track CMV vector and recombined into Ad-EASY to generate adenovirus. For studies using adenovirus in vivo, adenoviral vectors were injected intravenously as previously described.13 Mice were used in TAG secretion studies or euthanized for tissue collection 7 days after adenovirus administration.

Adenoviral transductions were carried out by incubating hepatocytes with adenoviruses in a 5% FBS/DMEM media overnight in 24-well plates (0.5 mL/well) or 6-well plates (2 mL/well) at an M.O.I. of 8. Experiments were carried out 40 to 48 hours after adenoviral transduction.

Metabolic Studies on Hepatocytes

To conform to space requirements, Methods for the hepatocyte metabolic studies can be found in the supplemental information.

Gene Expression Analyses

For quantitative polymerase chain reaction (PCR) studies, first-strand cDNA was generated by reverse transcription using total RNA. Real-time RT-PCR was performed using the ABI PRISM 7500 sequence detection system (Applied Biosystems) and the SYBR green kit. Primer sets (available on request) were designed to span exon splice borders. Arbitrary units of target mRNA were corrected by measuring the levels of 36B4 RNA.

Western Blotting Analyses

Western blotting analyses were performed with antibodies directed against the HA epitope tag (Covance), total Akt (Cell Signaling), and phoshpoAkt (Ser473 or Thr308; Cell Signaling).

Statistical Analyses

Statistical comparisons were made using analysis of variance (ANOVA) coupled to Scheffe test. All data are presented as means±SEM, with a statistically significant difference defined as a probability value <0.05.

Results

Increased Rates of TAG Synthesis and Secretion in Hepatocytes From 14-Day-Old Lipin 1—Deficient Mice

We sought to evaluate the effects of lipin 1 deficiency on rates of hepatic TAG synthesis and to probe the possibility that impaired VLDL-TAG secretion leads to hepatic steatosis in mice lacking lipin 1. Rates of TAG synthesis and secretion were actually increased quite significantly in hepatocytes isolated from 14-day-old (P14) fld mice compared with littermate controls (Figure 1). Importantly, the percentage of newly-synthesized TAG that was secreted was also increased, suggesting that this effect on secretion is not entirely explained by increased TAG synthesis. Pulse-chase studies in isolated hepatocytes demonstrated that synthesis and secretion of apoB proteins was significantly increased in fld hepatocytes (Figure 1). Together, these results demonstrate that a lack of lipin 1 does not impair hepatic TAG synthesis and secretion.

Increased Hepatic VLDL-TAG Secretion by Adult fld Mice and Its Reversal With Lipin 1 Overexpression

We also evaluated TAG synthesis and secretion in adult (P42) fld mice, which do not exhibit hepatic steatosis and have normal circulating lipid levels.5 Rates of TAG synthesis were not altered in adult fld mice compared to littermate controls, but rates of TAG secretion and the percentage of newly-synthesized TAG that was secreted were increased in P42 fld mice (Figure 2A). Adult fld hepatocytes showed no changes in rates of apoB synthesis or secretion (Figure 2B). Moreover, as demonstrated by using density-gradient ultracentrifugation analysis of the secreted 35S-labeled apoB-containing lipoproteins, fld hepatocytes secreted more VLDL-sized apoB48-containing particles than hepatocytes from littermate controls (Figure 2C). The hepatocytes of both genotypes secreted very small amounts of apoB100, which distributed only in the VLDL fractions (data not shown). Together, these results indicate that increased particle size of apoB48-containing lipoproteins account for the increased VLDL-TAG secretion by the fld hepatocytes.

An external file that holds a picture, illustration, etc. Object name is nihms-70223-f0002.jpg

Hepatocytes from adult fld mice exhibit normal rates of TAG synthesis and increased VLDL-TAG secretion. A, The graph depicts mean rates of TAG synthesis and secretion by primary hepatocytes isolated from 42-day-old fld or wild-type littermate mice as described in Figure 1 and Methods. The mean and SD of 3 independent experiments each done in triplicate are shown. **P<0.01 vs WT hepatocytes. B, The gel images depict the synthesis and secretion of 35S-labeled apoB proteins as described in Methods. Inset values represent mean normalized intensity of the synthesized and secreted 35S-labeled apoB48 proteins. C, Density distribution of apoB48-containing lipoprotein particles secreted by hepatocytes isolated from adult fld mice (fld) and littermate control (WT). Hepatocytes were continuously labeled with 35S-methinonine for 3 hours in the presence of 0.3 mmol/L oleate, and 35S-labeled apoB-containing lipoproteins in media were fractionated and quantified by density-gradient ultracentrifugation analysis and immuno-precipitation as described in Methods. Each data point represents mean of 2 independent experiments.

Because fld mice lack lipin 1 in all tissues, the studies above fail to distinguish between the hepatic effects of lipin 1 deficiency and its effects in other peripheral tissues (eg, lack of adipose tissue depots). To evaluate the direct hepatic effects of lipin 1 on VLDL assembly and secretion, lipin 1β was overexpressed in hepatocytes isolated from fld mice. Lipin 1β overexpression did not affect rates of TAG synthesis, but markedly suppressed TAG secretion, leading to a significant decrease in the efficiency of secretion of newly synthesized TAG (Figure 3A). Synthesis and secretion of apoB was not significantly affected by lipin 1 overexpression (Figure 3B). Lipin 1 overexpression markedly decreased the VLDL-sized, while increasing the HDL-like particles, in the secreted apoB-containing lipoproteins (data not shown). These results suggest that lipin 1 exerts its effects mainly by modulating the particle size of secreted apoB48-containing lipoproteins.

An external file that holds a picture, illustration, etc. Object name is nihms-70223-f0003.jpg

Lipin 1 overexpression suppresses hepatic VLDL-TAG secretion in fld mice. A, Hepatocytes isolated from livers of adult fld mice were infected with adenoviruses expressing GFP alone (GFP) or lipin 1β plus GFP (lipin 1) and studied 48 hours later. Synthesis and secretion of TAG and apoB were determined in the presence of exogenous oleate as described in Figure 1 and Methods at 48 hours after viral transduction. The mean and SD of 3 independent experiments each done in triplicate are shown. **P<0.01 vs GFP control. B, The graph depicts mean and SD (n=4) of rates of hepatic VLDL-TAG secretion in vivo determined using Triton WR-1339 from 4 adult male WT or fld mice infected with Ad-GFP or Ad-lipin 1β.

To establish the physiological relevance of the above findings in vivo, we examined the effect of liver-specific complementation of lipin 1 in fld mice. Lipin 1β was overexpressed in liver of fld mice and their littermate controls by adenovirus injection and VLDL secretion was assessed after injection with Triton-1339, which inhibits VLDL lipolysis. Under basal conditions (infection with Ad-GFP), there was a 2-fold increase in VLDL secretion in the fld mice compared to the littermate controls (Figure 3C). Overexpression of lipin 1 in the liver significantly decreased rates of VLDL-TAG secretion in both fld and control mice, with a more pronounced effect in fld mice. Altogether, these findings suggest that lipin 1 functions directly in the liver to suppress VLDL-TAG secretion.

The Lipin 1 LXXIL Motif Is Required for its Effects on TAG Metabolism

The two known molecular functions of lipin 1 relate to its activity as a PAP-1 enzyme and its role as a transcriptional regulator. Two previously described13 lipin 1 proteins with site-directed mutations in key domains were used to determine which molecular function was involved in this biological effect. Lipin 1 (D712E) lacks PAP-1 activity but retains the ability to interact with and coactivate PPARα, whereas lipin 1 (LXXFF) lacks both the ability to interact with PPARα and PAP activity.13 We found that the overexpressed D712E mutant retained the ability to suppress TAG secretion in WT mice, indicating that PAP activity is dispensable for these effects (Figure 4A). In contrast, overexpression of the LXXFF allele failed to suppress TAG secretion, implying a critical role of the transcriptional regulatory function of lipin 1 in mediating its inhibitory effects on VLDL-TAG secretion.

An external file that holds a picture, illustration, etc. Object name is nihms-70223-f0004.jpg

The LXXIL motif of lipin 1 is required to suppress VLDL-TAG secretion in mouse hepatocytes. A, The amino acid sequence of C-terminal lipin 1 motifs required for PAP-1 activity (DXDXT) and nuclear receptor interaction (LXXIL) are shown. Asterisks denote amino acid residues mutated in D712E and LXXFF mutant lipin 1 alleles. The autoradiograph of representative Western blotting analyses for the HA tag of lipin 1 demonstrates that wild-type, D712E, and LXXFF lipin 1 proteins are robustly expressed at a similar level in hepatocytes by their respective adenoviral vectors. Primary hepatocytes were isolated from 6-week-old C57BL/6 mice and infected with adenovirus driving expression of wild-type, D712E, or LXXFF lipin 1β or GFP (control). The graphs depict mean rates of TAG synthesis and secretion. The mean and SD of 3 independent experiments each done in triplicate are shown. *P<0.05 vs GFP control and lipin 1(LXXFF). B, The graphs depict mean rates of TAG synthesis and secretion by primary hepatocytes isolated from PPARα null mice. The mean and SD of 3 independent experiments done in triplicate are shown. **P<0.01 vs GFP control.

Given the large body of work indicating that PPARα ligands suppress hepatic TAG secretion,19 we also tested whether lipin 1 was exerting its effects via activation of PPARα. However, lipin 1β retained the ability to suppress TAG secretion in hepatocytes from PPARα null mice (Figure 4B), indicating that these effects are independent of PPARα.

Lipin 1 Suppresses Scd1 Expression but Does Not Regulate Expression of Apob and Mttp

The maintenance of normal rates of hepatic TAG synthesis in fld mice perhaps suggested that other components of the TAG synthesis pathway, including other lipin family members, might be upregulated. However, quantitative RT-PCR analyses demonstrated that the expression of Lpin2 and Lpin3 was unchanged in fld mice or with lipin 1 overexpression (Table). We also observed no change in the expression of other enzymes in this pathway (Dgat1, Dgat2, and Gpam; data not shown). The expression of steroyl-coenzyme A (CoA) desaturase (Scd1), which may promote TAG synthesis and VLDL, was markedly increased in neonatal and adult fld mice (Table) yet suppressed by lipin 1 overexpression.

Table

Expression of Genes Involved in TAG Metabolism With Deactivation or Overexpression of Lipin 1

WT Hepatocytes
fld Hepatocytes
P8 fldP42 fldAd-GFPAd-Lipin 1bAd-GFPAd-Lipin 1b
Lpin21.1±0.381.3±0.151.0±0.101.1±0.181.1±0.271.1±0.20
Lpin31.0±0.291.3±0.241.0±0.251.3±0.091.4±0.241.1±0.28
Scd18.4±3.04*6.7±2.54*1.0±0.180.2±0.31**4.2±1.29*1.6±1.17**
Apoa425.1±4.42*3.4±0.21*1.0±0.180.5±0.16**2.1±0.16*0.9±0.15**
Apob1.9±0.36*1.1±0.311.0±0.250.9±0.111.2±0.350.9±0.23
Apoc30.9±0.370.8±0.421.0±0.070.6±0.14**1.4±0.160.7±0.14**
Apoe0.9±0.350.9±0.171.0±0.020.9±0.050.9±0.110.7±0.10
Mttp1.4±0.331.1±0.261.0±0.101.0±0.090.9±0.090.8±0.11

Left, Values represent mean (±SEM) hepatic expression of the indicated genes expressed as arbitrary units (AU) normalized (=1.0) to the mean expression in WT control mice (n=5 for P8 mice and n=6 for P42 mice).

*P<0.05 compared to age-matched WT littermate mice.

Right, Hepatocytes were isolated from P42 WT or fld mice, pooled within genotypes, and then infected with adenovirus overexpressing lipin 1β or GFP at time of plating. RNA was isolated 48 h later. Values represent mean (±SEM) expression of the indicated genes normalized (=1.0) to the mean expression in WT GFP hepatocytes. Results represent 6 independent wells of hepatocytes pooled from 3 mice of each genotype.

*P<0.05 compared to WT hepatocytes.
**P<0.05 vs Ad-GFP controls of the same genotype.

We next evaluated the expression of genes involved in VLDL metabolism. Consistent with increased rates of apoB synthesis by neonatal fld hepatocytes, the expression of apoB (Apob) was strongly induced in 10-day-old fld mice compared to controls (Table), though the relevance of transcriptional regulation of Apob is unclear given the high level of posttranslational regulation of apoB secretion. The expression of the gene encoding apoA4 (Apoa4) was also markedly increased in fld mice. In contrast, the expression of the genes encoding apoE (Apoe), apoC3 (Apoc3), and microsomal TAG transfer protein (Mttp) were not changed. Among other genes evaluated in adult fld mice (P42), only the expression of Apoa4 was altered (Table).

We next quantified the expression of genes involved in VLDL metabolism in response to lipin 1 overexpression in hepatocytes from wild-type and fld mice. Lipin 1 overexpression did not affect the expression of Apob, Apoe, or Mttp. However, the expression of Apoa4 and Apoc3 was markedly suppressed by lipin 1β overexpression in hepatocytes of both fld and littermate control mice. Consistent with our metabolic studies, the transcriptional effects of lipin 1 were also observed with overexpression of lipin 1(D712E) but not the LXXFF allele (supplemental Figure I). Collectively, these data indicate that the effects of lipin 1 are not associated with altered expression of Apob and Mttp; the two obligatory components of the VLDL-TAG exporting machinery.

Lipin 1 Suppresses VLDL-TAG Secretion and Increased Hepatic Insulin Sensitivity in UCP-DTA Mice

To explore a potential link between lipin 1 expression and increased VLDL-TAG production associated with metabolic syndrome, we used UCP-DTA mice,16 which are obese, insulin-resistant, and hyperlipidemic because of transgenic ablation of most of the brown adipose tissue. As previously noted,16 UCP-DTA mice were hypertriglyceridemic (Figure 5A). We also found that rates of TAG secretion were dramatically increased in insulin-resistant UCP-DTA mice versus NTG littermates (Figure 5A). Interestingly, hepatic expression of lipin 1 was significantly diminished in UCP-DTA mice (Figure 5A).

An external file that holds a picture, illustration, etc. Object name is nihms-70223-f0005.jpg

Lipin 1 overexpression suppresses hepatic VLDL-TAG secretion and increases insulin signaling in UCP-DTA mice. A, Left, The graph depicts plasma TAG levels in nontransgenic (NTG) or uncoupling protein 1 diphtheria toxin A (UCP-DTA) mice after a 4-hour fast. *P<0.05 vs NTG mice (n=5). Middle, The graph depicts mean rates of hepatic VLDL-TAG secretion in NTG or UCP-DTA mice determined using Triton WR-1339 as described in Methods. *P<0.01 vs NTG (n=4). Right, The graph depicts the expression of lipin 1β in liver of NTG and UCP-DTA mice as quantified by quantitative RT-PCR. **P<0.01 vs NTG control (n=6) B, Left, The graph depicts mean and SD (n=4) of plasma TAG levels in NTG or UCP-DTA mice infected with Ad-GFP or Ad-lipin 1β after a 4-hour fast. Right, The graph depicts mean and SD (n=5) of in vivo rates of hepatic TAG secretion in NTG or UCP-DTA mice infected with Ad-GFP or Ad-lipin 1β. C, The autoradiographs depict representative results of Western blotting analyses using hepatic protein lysates from NTG and UCP-DTA mice injected with insulin or saline. Duplicate blots were probed with antibodies against Akt phosphorylated at serine 473 (ser473), threonine 308 (thr308), or total Akt protein.

To determine whether restoration of lipin 1 expression corrected the metabolic derangements in VLDL metabolism in UCP-DTA mice, we overexpressed lipin 1 in the liver of UCP-DTA mice using an adenovirus. Liver-specific lipin 1 overexpression lowered the fasting plasma TAG concentration (Figure 5B) and reversed TAG hyper-secretion in UCP-DTA mice (Figure 5B). Lipin 1 overexpression also markedly increased insulin-mediated phosphorylation of Akt at serine 473 and threonine 308 in the liver of UCP-DTA mice (Figure 5C) despite having no effect on hepatic TAG levels (data not shown). Collectively, these data indicate that overexpression of lipin 1 in liver of UCP-DTA mice is sufficient to improve hepatic insulin signaling and suppress VLDL-TAG secretion in this obese mouse model.

Discussion

Lipin 1 is emerging as an important regulator of lipid homeostasis via its function as an PAP-1 enzyme in the phosphoglycerol TAG synthesis pathway and in the nucleus as a transcriptional regulator. Herein, we found that lipin 1 was neither required for nor sufficient to induce hepatic TAG synthesis, but instead, lipin 1 markedly suppressed hepatic VLDL-TAG secretion. The repressive effects of lipin 1 were independent of its PAP activity but required the domain that mediates its interaction with nuclear receptor transcription factors. Finally, we found that hepatic Lpin1 expression was markedly diminished in obese, insulin-resistant UCP-DTA mice and that hepatic overexpression of lipin 1 in these mice suppressed VLDL-TAG secretion and increased insulin-stimulated phosphorylation of Akt.

The physiological and cellular mechanisms that cause hepatic steatosis in neonatal fld mice is incompletely understood. Hepatic steatosis is common in lipodystrophic mouse models20,21 and in humans with genetic or acquired forms of lipodystrophy.22,23 This may relate to insufficient capacity to store fatty acids in adipose depots leading to hepatic TAG accretion, which would be an overt problem in fld mice exposed to TAG-rich maternal milk. In addition, neonatal fld mice exhibit a significant defect in fatty acid oxidation,24 consistent with hepatic steatosis in other models of fatty acid oxidation deficiencies.25 The present study excludes defects in VLDL-TAG secretion, which can also cause fatty liver,17,18 as contributing to the steatotic phenotype of fld mice. Although fld mice almost completely lack PAP-1 activity in most extrahepatic tissues, previous work has shown that at least residual PAP-1 activity remains in liver,8,10 probably because of high hepatic expression of another lipin family protein, lipin 2.10 However, we also show that overexpression of lipin 1β is not sufficient to drive increased TAG synthesis. Together, these data likely suggest that the PAP-1 reaction is not rate-limiting for phosphoglycerol TAG synthesis.

Although lipin 1 activity does not influence rates of TAG synthesis in hepatocytes, our gain-of-function and loss-of-function studies uniformly revealed that lipin 1 suppresses VLDL-TAG secretion. This is consistent with the original report demonstrating that neonatal fld mice are hypertriglyceridemic and lipoproteinemic.5 We found that VLDL-TAG secretion was also increased in hepatocytes isolated from adult fld mice, which exhibit normal lipemia.5,6 Increased catabolism of VLDL-TAG by peripheral tissues may explain the normal plasma lipid levels in adult fld mice.26,27 It is also tempting to speculate that an increased capacity for VLDL-TAG secretion is one compensatory mechanism leading to the temporal resolution of the neonatal fatty liver observed in fld mice.5 Although increased VLDL-TAG secretion by fld hepatocytes could be an adaptive response to lack of adipose tissue, our overexpression data indicate that lipin 1 also plays a direct role in regulating hepatic VLDL-TAG secretion.

Although lipin mutation studies indicate that lipin 1 acts via a transcriptional mechanism, lipin 1 did not suppress the expression of Apob and Mttp genes, the two obligatory factors for VLDL assembly and secretion. Although genetic variations in the Apoc3/Apoa4 gene cluster are linked to altered lipemia and incidence of cardiovascular disease,28,29 based on the data from overexpression and knockout models,30–32 it is unlikely that regulation of Apoc3/Apoa4 expression per se can explain the observed effects of lipin 1 on hepatic VLDL-TAG secretion. Scd1 was also found to be increased in fld mice of both ages and was repressed by lipin 1 overexpression. Scd1 null mice exhibit diminished hepatic TAG stores and VLDL secretion,33 potentially suggesting that it could play a role in the observed biological effects.

Finally, we recently reported that hepatic lipin 1 gene expression is inversely related to body mass index and plasma insulin concentration in extremely obese human volunteers.34 Lipin 1 gene expression was also reactivated by marked weight loss after gastric bypass surgery. The increase in lipin 1 expression in the liver was coincident with a significant decline in hepatic TAG content and rates of hepatic VLDL-TAG secretion after gastric bypass surgery in these subjects.35 Consistent with this, we find that lipin 1 gene expression is diminished in obese insulin-resistant UCP-DTA mice. More importantly, we show that lipin 1 overexpression reduces hepatic TAG secretion rates in these mice and improves insulin-mediated phosphorylation of Akt. Given the data presented herein, future strategies for modulating lipin 1 activity in liver might prove useful in the treatment of insulin resistance and dyslipidemia. Obviously, a better understanding of the full gamut of lipin 1 effects on lipid metabolism is required before it can be seriously considered as a therapeutic target.

Supplementary Material

supplemental i

Acknowledgments

Sources of Funding

This work was also supported by NIH grants R01 DK078187 (to B.N.F.), R01 HL73939 (to Z.C.), and R01s DK52753 and DK28312 (to J.C.L.). This work was also supported, in part, by Pilot and Feasibility awards from the Digestive Diseases Research Core Center (P30 DK52574) and the Clinical Nutrition Research Unit Core Center (P30 DK56341) to B.N.F.

Footnotes

Disclosures: None.

References

1. Mittendorfer B, Patterson BW, Klein S, Sidossis LS. VLDL-triglyceride kinetics during hyperglycemia-hyperinsulinemia: effects of sex and obesity. Am J Physiol Endocrinol Metab. 2003;284:E708–E715. [PubMed] [Google Scholar]
2. Ginsberg HN, Zhang YL, Hernandez-Ono A. Regulation of plasma triglycerides in insulin resistance and diabetes. Arch Med Res. 2005;36:232–240. [PubMed] [Google Scholar]
3. Rader DJ, Rosas S. Management of selected lipid abnormalities. Hyper-triglyceridemia, low HDL cholesterol, lipoprotein(a), in thyroid and renal diseases, and post-transplantation. Med Clin North Am. 2000;84:43–61. [PubMed] [Google Scholar]
4. Reue K, Zhang P. The lipin protein family: dual roles in lipid biosynthesis and gene expression. FEBS Lett. 2008;582:90–96. [PMC free article] [PubMed] [Google Scholar]
5. Langner CA, Birkenmeier EH, Ben-Zeev O, Schotz MC, Sweet HO, Davisson MT, Gordon JI. The fatty liver dystrophy (fld) mutation. A new mutant mouse with a developmental abnormality in triglyceride metabolism and associated tissue-specific defects in lipoprotein lipase and hepatic lipase activities. J Biol Chem. 1989;264:7994–8003. [PubMed] [Google Scholar]
6. Reue K, Xu P, Wang XP, Slavin BG. Adipose tissue deficiency, glucose intolerance, and increased atherosclerosis result from mutation in the mouse fatty liver dystrophy (fld) gene. J Lipid Res. 2000;41:1067–1076. [PubMed] [Google Scholar]
7. Peterfy M, Phan J, Xu P, Reue K. Lipodystrophy in the fld mouse results from mutation of a new gene encoding a nuclear protein, lipin. Nat Genet. 2001;27:121–124. [PubMed] [Google Scholar]
8. Harris TE, Huffman TA, Chi A, Shabanowitz J, Hunt DF, Kumar A, Lawrence JC., Jr. Insulin controls subcellular localization and multisite phosphorylation of the phosphatidic acid phosphatase, lipin 1. J Biol Chem. 2007;282:277–286. [PubMed] [Google Scholar]
9. Han GS, Wu WI, Carman GM. The Saccharomyces cerevisiae Lipin homolog is a Mg2+-dependent phosphatidate phosphatase enzyme. J Biol Chem. 2006;281:9210–9218. [PMC free article] [PubMed] [Google Scholar]
10. Donkor J, Sariahmetoglu M, Dewald J, Brindley DN, Reue K. Three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns. J Biol Chem. 2007;282:3450–3457. [PubMed] [Google Scholar]
11. Coleman RA. How do I fatten thee? Let me count the ways. Cell Metab. 2007;5:87–89. [PMC free article] [PubMed] [Google Scholar]
12. Peterfy M, Phan J, Reue K. Alternatively spliced lipin isoforms exhibit distinct expression pattern, subcellular localization, and role in adipogenesis. J Biol Chem. 2005;280:32883–32889. [PubMed] [Google Scholar]
13. Finck BN, Gropler MC, Chen Z, Leone TC, Croce MA, Harris TE, Lawrence JC, Jr, Kelly DP. Lipin 1 is an inducible amplifier of the hepatic PGC-1alpha/PPARalpha regulatory pathway. Cell Metabol. 2006;4:199–210. [PubMed] [Google Scholar]
14. Santos-Rosa H, Leung J, Grimsey N, Peak-Chew S, Siniossoglou S. The yeast lipin Smp2 couples phospholipid biosynthesis to nuclear membrane growth. EMBO J. 2005;24:1931–1941. [PMC free article] [PubMed] [Google Scholar]
15. Lee SS, Pineau T, Drago J, Lee EJ, Owens JW, Kroetz DL, Fernandez-Salguero PM, Westphal H, Gonzalez FJ. Targeted disruption of the alpha isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators. Mol Cell Biol. 1995;15:3012–3022. [PMC free article] [PubMed] [Google Scholar]
16. Lowell BB, V SS, Hamann A, Lawitts JA, Himms-Hagen J, Boyer BB, Kozak LP, Flier JS. Development of obesity in transgenic mice after genetic ablation of brown adipose tissue. Nature. 1993;366:740–742. [PubMed] [Google Scholar]
17. Chen Z, Fitzgerald RL, Schonfeld G. Hypobetalipoproteinemic mice with a targeted apolipoprotein (Apo) B-27.6-specifying mutation: in vivo evidence for an important role of amino acids 1254-1744 of ApoB in lipid transport and metabolism of the apoB-containing lipoprotein. J Biol Chem. 2002;277:14135–14145. [PubMed] [Google Scholar]
18. Chen Z, Fitzgerald RL, Averna MR, Schonfeld G. A targeted apolipoprotein B-38.9-producing mutation causes fatty livers in mice due to the reduced ability of apolipoprotein B-38.9 to transport triglycerides. J Biol Chem. 2000;275:32807–32815. [PubMed] [Google Scholar]
19. Staels B, Dallongeville J, Auwerx J, Schoonjans K, Leitersdorf E, Fruchart J-C. Mechanism of action of fibrates on lipid and lipoprotein metabolism. Circulation. 1998;98:2088–2093. [PubMed] [Google Scholar]
20. Shimomura I, Hammer RE, Richardson JA, Ikemoto S, Bashmakov Y, Goldstein JL, Brown MS. Insulin resistance and diabetes mellitus in transgenic mice expressing nuclear SREBP-1c in adipose tissue: model for congenital generalized lipodystrophy. Genes Dev. 1998;12:3182–3194. [PMC free article] [PubMed] [Google Scholar]
21. Moitra J, Mason MM, Olive M, Krylov D, Gavrilova O, Marcus-Samuels B, Feigenbaum L, Lee E, Aoyama T, Eckhaus M, Reitman ML, Vinson C. Life without white fat: a transgenic mouse. Genes Dev. 1998;12:3168–3181. [PMC free article] [PubMed] [Google Scholar]
22. Ristig M, Drechsler H, Powderly WG. Hepatic steatosis and HIV infection. AIDS Pat Care STDs. 2005;19:356–365. [PubMed] [Google Scholar]
23. de Craemer D, Van Maldergem L, Roels F. Hepatic ultrastructure in congenital total lipodystrophy with special reference to peroxisomes. Ultrastructural Pathol. 1992;16:307–316. [PubMed] [Google Scholar]
24. Rehnmark S, Giometti CS, Slavin BG, Doolittle MH, Reue K. The fatty liver dystrophy mutant mouse: microvesicular steatosis associated with altered expression levels of peroxisome proliferator-regulated proteins. J Lipid Res. 1998;39:2209–2217. [PubMed] [Google Scholar]
25. Leone TC, Weinheimer CJ, Kelly DP. A critical role for the peroxisome proliferator-activated receptor alpha (PPARalpha) in the cellular fasting response: the PPARalpha-null mouse as a model of fatty acid oxidation disorders. Proc Natl Acad Sci U S A. 1999;96:7473–7478. [PMC free article] [PubMed] [Google Scholar]
26. Xu J, Lee WN, Phan J, Saad MF, Reue K, Kurland IJ. Lipin deficiency impairs diurnal metabolic fuel switching. Diabetes. 2006;55:3429–3438. [PubMed] [Google Scholar]
27. Phan J, Reue K. Lipin, a lipodystrophy and obesity gene. Cell Metab. 2005;1:73–83. [PubMed] [Google Scholar]
28. Lai CQ, Parnell LD, Ordovas JM. The APOA1/C3/A4/A5 gene cluster, lipid metabolism and cardiovascular disease risk. Curr Opin Lipidol. 2005;16:153–166. [PubMed] [Google Scholar]
29. Lopez-Miranda J, Perez-Martinez P, Marin C, Moreno JA, Gomez P, Perez-Jimenez F. Postprandial lipoprotein metabolism, genes and risk of cardiovascular disease. Curr Opin Lipidol. 2006;17:132–138. [PubMed] [Google Scholar]
30. Ebara T, Ramakrishnan R, Steiner G, Shachter NS. Chylomicronemia due to apolipoprotein CIII overexpression in apolipoprotein E-null mice. Apolipoprotein CIII-induced hypertriglyceridemia is not mediated by effects on apolipoprotein E. J Clin Invest. 1997;99:2672–2681. [PMC free article] [PubMed] [Google Scholar]
31. Hirano T, Takahashi T, Saito S, Tajima H, Ebara T, Adachi M. Apoprotein C-III deficiency markedly stimulates triglyceride secretion in vivo: comparison with apoprotein E. Am J Physiol Endocrinol Metab. 2001;281:E665–E669. [PubMed] [Google Scholar]
32. Duverger N, Tremp G, Caillaud JM, Emmanuel F, Castro G, Fruchart JC, Steinmetz A, Denefle P. Protection against atherogenesis in mice mediated by human apolipoprotein A-IV. Science. 1996;273:966–968. [PubMed] [Google Scholar]
33. Miyazaki M, Kim YC, Gray-Keller MP, Attie AD, Ntambi JM. The biosynthesis of hepatic cholesterol esters and triglycerides is impaired in mice with a disruption of the gene for stearoyl-CoA desaturase 1. J Biol Chem. 2000;275:30132–30138. [PubMed] [Google Scholar]
34. Croce MA, Eagon JC, LaRiviere LL, Korenblat KM, Klein S, Finck BN. Hepatic lipin 1beta expression is diminished in insulin-resistant obese subjects and is reactivated by marked weight loss. Diabetes. 2007;56:2395–2399. [PubMed] [Google Scholar]
35. Klein S, Mittendorfer B, Eagon JC, Patterson B, Grant L, Feirt N, Seki E, Brenner D, Korenblat K, McCrea J. Gastric bypass surgery improves metabolic and hepatic abnormalities associated with nonalcoholic Fatty liver disease. Gastroenterology. 2006;130:1564–1572. [PubMed] [Google Scholar]
-