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Endocrinology, doi:10.1210/en.2006-1728
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Endocrinology Vol. 148, No. 8 3685-3693
Copyright © 2007 by The Endocrine Society

Lipin1 Regulation by Estrogen in Uterus and Liver: Implications for Diabetes and Fertility

P. Mangala Gowri, Surojeet Sengupta, Suzanne Bertera and Benita S. Katzenellenbogen

Departments of Molecular and Integrative Physiology (P.M.G., S.S., B.S.K.) and Cell and Developmental Biology (B.S.K.), University of Illinois at Urbana-Champaign, Urbana, Illinois 61801; and Department of Pediatrics (S.B.), University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213

Address all correspondence and requests for reprints to: Dr. Benita S. Katzenellenbogen, University of Illinois, Department of Molecular and Integrative Physiology, 524 Burrill Hall, 407 South Goodwin Avenue, Urbana, Illinois 61801-3704. E-mail: katzenel{at}uiuc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogens are essential for fertility and also have important effects on regulation of adiposity and the euglycemic state. We report here that lipin1, a candidate gene for lipodystrophy and obesity that is a phosphatidic acid phosphatase critical in regulation of cellular levels of diacylglycerol and triacylglycerol and a key regulator of lipid utilization, is rapidly and robustly down-regulated in the uterus by estradiol via the estrogen receptor. Lipin1 is expressed predominantly in the uterine luminal and glandular epithelium, and during the estrous cycle, lipin1 is lowest when blood levels of estrogen are highest. Lipin1 is expressed throughout all cells in the liver of ovariectomized female mice, and a sustained down-regulation is observed at the mRNA, protein and immunohistochemical levels after estrogen administration. Because the coupling of proper energy use and availability is central to reproduction, we also investigated expression of lipin1 in the uterus and liver of several mouse models of diabetes. Nonobese diabetic (NOD) mice, which have high blood levels of estrogen and impaired fertility, were severely deficient in lipin1 in the uterus and liver, which, interestingly, could be restored by insulin treatment. By contrast, nonobese diabetic/severe combined immunodeficient (NOD-SCID) mice, which do not develop diabetes, showed normal levels of lipin1. Our findings of lipin1 regulation by estrogen in two key target organs suggest a new role for this lipid-regulating phosphatase not only in central metabolic regulation but also in uterine function and reproductive biology. Estrogen regulation of lipin1 may provide a mechanistic link between estrogens, lipid metabolism, and lipid signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGENS, WHICH ACT predominantly via their cognate estrogen receptors (ERs), closely control fertility and many aspects of reproduction, including the expression of important genes in the ovary and uterus (1, 2, 3). Estrogens also play roles in metabolic regulation and maintenance of the euglycemic status (4, 5, 6) and in body fat level and distribution (7, 8, 9, 10). Metabolic state and adiposity are also known to impact estrogen levels and reproductive status, underscoring the robust endocrine control of fertility. Metabolic disorders such as diabetes and lipodystrophy are associated with reduced fertility (11, 12, 13). In the nonobese diabetic (NOD) mouse model, circulating levels of estrogen are more than twice those of other strains of mice (14). Interestingly, the elevated level of estrogen in female NOD mice has been shown to play a critical permissive role in the events leading up to insulitis (15) and might explain the prevalence of diabetes in female compared with male NOD mice. In addition to impaired lipid metabolism in their livers (16), the onset of diabetes in these mice is associated with fertility impairment, resulting in smaller litter size (12, 16).

Lipin1, a candidate gene for lipodystrophy (17) and obesity (18) and a central metabolic regulator (19), is expressed in liver and adipose tissue, consistent with its role in regulation of adiposity and the diabetic state, yet little is known about the factors regulating lipin1 production and its possible roles in other tissues. Because diabetes is often associated with alterations in fertility (11, 12), we investigated uterine expression of lipin1, a phosphatidic acid phosphatase (PAP) critical in regulation of cellular levels of diacylglycerol and triacylglycerol (20), and its possible regulation by estrogen in the uterus of nondiabetic mice as well as in several mouse models of diabetes.

Variations in lipin levels are known to induce extreme states of adiposity (17, 18), with enhanced lipin expression promoting obesity and absence or reduced lipin levels resulting in lipodystrophy (adipose tissue deficiency). A transient increase in lipin1 is crucial for peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) activation before adipocyte differentiation (21), establishing a role for this protein in adipogenesis. Diabetic neuropathy has also been attributed to improper distribution of lipin1 in the sciatic nerve (22). The regulation of lipin1 itself, however, is not well understood. Lipin1 phosphorylation by insulin via the mammalian target of rapamycin (mTOR) pathway and dephosphorylation by epinephrine have been reported and, thus far, are the only studies available regarding hormonal regulation of this important protein (23, 24). In addition, a recent study reports (25) that lipin1 expression in liver is induced by PPAR{gamma} coactivator-1{alpha}, a transcriptional coactivator that controls several key hepatic metabolic pathways. Apart from severe abnormalities in body fat deposition, lipin1 deficiency due to the naturally occurring gene mutation in fld/fld (fatty liver dystrophy) mice is also associated with impaired fertility (13, 17).

Because of impaired fertility in lipin1-deficient mice, we have investigated in this report the expression of lipin1 in the uterus and its possible regulation by estrogen and compared the effects of estrogen on hepatic lipin1. We employed normal as well as diabetic NOD mice in this study, because NOD mice allowed comparison between effects of acute vs. prolonged exposures to estrogen. We also used the NOD-severe combined immunodeficient (SCID, recombination activating gene 1, or rag1 null mice) model (26) for comparative experiments, because these immune-compromised mice do not develop diabetes. Our findings reveal cross-talk between estrogen and insulin pathways in the regulation of lipin1. Lipin 1 is markedly down-regulated by estrogen, with reversal of the reduced lipin1 levels by insulin, implying that the effect of these hormones on uterine and liver functions might, at least in part, derive from their opposing regulation of lipin1. Furthermore, because lipin1 is a PAP (20), estrogen regulation of lipin1 may provide a mechanistic link between estrogens, lipid metabolism, and lipid signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal care and use
All procedures involving animals were in accordance with National Institutes of Health and Institutional Guidelines for the Care and Use of Laboratory Animals. C57BL6, CD-1, NOD, and NOD-SCID mice were used in this study. CD-1 and C57BL6 mice were purchased from Harlan and Co. (Indianapolis, IN) and used at approximately 10 wk of age. Mice were maintained at a controlled temperature (22 C) on a 12-h light, 12-h dark cycle (lights on 0700–1900) and provided water and standard rodent chow (Harlan Teklad, Madison, WI) ad libitum. Some C57BL6 mice were ovariectomized at 8 wk of age and were allowed to recover for 15–17 d. For studies, these mice were then injected sc every 24 h with vehicle (1:10, dimethylsulfoxide/corn oil), 17ß-estradiol (E2) at 2.5 µg/mouse, or ethinyl estradiol (EE) at 5 µg/mouse. For some experiments, mice were pretreated with ICI 182,780 (625 µg/mouse) or cycloheximide (CHX, 100 µg/mouse; Sigma Chemical Co., St. Louis, MO) for 30 min before injecting E2 for the times indicated. To avoid any possible alterations in gene expression due to diurnal variations (27), tissues were always harvested between 1000 and 1100 h. NOD and NOD-SCID mice were purchased from Jackson Laboratories (Bar Harbor, ME), and females were maintained in a pathogen-free environment. Blood glucose levels were monitored, and mice were determined to be hyperglycemic if their blood glucose was 300 mg/dl (16.7 mM) or above in three consecutive tests. Diabetic NOD females were injected ip with 1 U insulin (Humulin U Ultralente; Eli Lilly, Indianapolis, IN) per day for the times indicated before being killed to monitor the effect of insulin on lipin1 expression. Phases of the estrous cycle in mice were determined by examining vaginal smear cell types under the microscope.

Quantitative RT-PCR
Total RNA was extracted from tissues using TRIzol reagent (Invitrogen, Carlsbad, CA). Single-tube RT-PCR was performed with 10 ng template RNA using gene-specific primers and SYBR green master mix supplemented with MMLV reverse transcriptase and RNase inhibitor (Applied Biosystems Inc., Foster City, CA). Data were normalized to the expression of a housekeeping gene, 36B4. The sequences of primers used in this study were as follows: lipin1 forward, 5'-ACCC CAACCTCGTGGTCAA-3', and reverse, 5'TGCATCGCCAGAAGTAGAGGA-3'; 36B4 forward, 5'-GCAAAGGAAGAGTCGGAGGA-3', and reverse 5'-GCAGGCTGACTTGGTTGCTT-3'; complement C3 forward, 5'-TCATCCTCATTGAGACCCCC-3', and reverse 5'-CTGCCCCATGTTGACCAGTT-3'; lactoferrin (LF) forward, 5'-CCCTTGAGGAAGC- GGTATCC-3', and reverse 5'-ACACGAGCTACACAGGTTGGG-3'; apolipoprotein A-IV (ApoA4) forward, 5'-GGCTCTGGAAGACCTGAACA-3', and reverse 5'-ACCCAGCTGCTGTCTGAACT-3'; and glutathione S-transferase, {pi} 2(Gstp2) forward, 5'-TTGCCGATTACAACTTGCTG-3', and reverse 5'-GAGCCACATAGGCAGAGAGC-3'.

Antibodies against lipin1
Polyclonal antibodies were raised in rabbits against mouse lipin1 using peptide sequences, amino acids 285–304: SSSPHKMKESSPLGSRKTPD and 377–397: SKTDSPSRKKDKRSRHLGADG (Pocono Rabbit Farms and Laboratories Inc., Canadensis, PA). Both peptides were synthesized with an NH2-terminal Cys residue and were coupled to limpet hemocyanin before injection. Preimmune and postimmunization blood samples were analyzed for lipin1 titer using BSA-conjugated target peptide as bait in ELISA. Blood samples were collected every 28 d. Rabbits were killed by exsanguination, and the final bloods collected were aliquoted and stored at –80 C. The antibodies detect lipin1 {alpha} and ß protein isoforms (28) that differ by only 33 amino acids.

Western blots and immunohistochemistry
Total protein from tissues was extracted using lysis buffer(T-PER) from Pierce Inc. (Rockford, IL), in the presence of 1 mM phenylmethylsulfonyl fluoride plus complete Mini protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). Standard protocols were used for Western blots. Precast 4–20% gradient gels were purchased from Bio-Rad (Hercules, CA). Nitrocellulose and polyvinylidene difluoride membranes were used for transfer of uterine and liver proteins, respectively. The membranes were then incubated in blocking buffer [5% milk in Tris-buffered saline/0.05% Tween (TBS-T)] containing normal goat serum at a final concentration of 2% for 3 h at room temperature with constant rotation. A single wash was given to the membranes in TBS-T buffer before overnight incubation at 4 C with antibodies directed against lipin1 (1:1000) or ER{alpha} (mouse ER{alpha} antibody MC-20, 1:5000; Santa Cruz Biotechnology Inc., Santa Cruz, CA) in blocking buffer. All washings for lipin1 blot were performed in TBS-T buffer containing 0.2% Tween. The level of ß-actin was used as the internal indicator for equal sample loading. Bands in developed Western blots were quantified using IMAGEQUANT software.

For immunohistochemical studies, ovariectomized mice were treated with vehicle, E2, or EE for the times indicated. Tissue was fixed in paraformaldehyde at 4 C overnight, washed and suspended in 70% ethanol, and processed for sectioning. Sections were made to a thickness of 4 µm. Transverse sections of mouse uterus and liver were immunostained with polyclonal antibodies raised against lipin1 (1:1250) using the Zymed Histomouse kit (Zymed Laboratories, San Francisco, CA), following the manufacturer’s suggestions. Preimmune serum was used as a negative control.

Statistical analysis
At least three to six animals were used per group in each of the studies reported. Statistical analyses were conducted using the GLM procedure of SAS version 9.1.3 (SAS Institute Inc., Cary, NC). All data sets were subjected to Levene’s test (29) to ascertain whether individual variances were statistically equal. Dunnett’s test (30) was performed on data with homogeneous variances. For data sets with nonequal variances, multiple t tests were conducted, followed by adjustment of P values using the step-down Bonferroni method, an improved version of the sequentially rejective Bonferroni correction (31).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Lipin1 expression in the mouse uterus is down-regulated by E2 via the ER and appears to be a primary response to estrogen
E2 treatment of ovariectomized mice was found to rapidly and markedly down-regulate lipin1 mRNA in the uterus. Within 4 h of administration, lipin1 mRNA was reduced to 25% of the control level, and this effect was sustained at 24 h (Fig. 1Go). Treatment with the pure antiestrogen ICI 182,780 reversed the effect of E2, indicating the involvement of ER in regulation of lipin1 by E2. Furthermore, treatment with the protein synthesis inhibitor CHX did not prevent the down-regulation by E2 (Fig. 1Go). This suggests that lipin1 may be a primary target gene, where de novo protein synthesis is not required for lipin1 mRNA regulation by E2. In contrast to the down-regulation of lipin1 by E2, uterine complement C3 gene expression was stimulated by E2, as expected (32).


Figure 1
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  		FIG. 1. Regulation of lipin1 by estrogen in the mouse uterus. Ovariectomized C57BL6 mice were injected sc with vehicle or E2 (2.5 µg/mouse) for 4 h or 24 h before being killed. Where applicable, ICI (625 µg/mouse) or CHX (100 µg/mouse) was injected 30 min before E2 injections. Total RNA was extracted from the uteri using TRIzol and subjected to quantitative RT-PCR analysis using gene-specific primers to ascertain lipin1 mRNA expression. Levels of complement component C3 were also assayed to confirm E2 responsiveness of the uterus. Data were normalized using 36B4 expression as the internal control. Lipin1 and C3 mRNA levels were calculated relative to that of vehicle-treated control, which was set at 100%. Values are the mean ± SEM of three to six animals per group. *, P < 0.05; **, P < 0.01, statistically significant compared with its vehicle control, as determined by multiple t tests followed by step-down Bonferroni method, as described in Materials and Methods, because data sets had unequal variances.

 
Uterine lipin1 levels change during the estrous cycle and inversely correlate with blood levels of E2
We analyzed, as shown in Fig. 2Go, lipin1 levels in the uteri of inbred (C57BL6) and outbred (CD-1) mice at various phases of the estrous cycle. Lipin1 was lowest in the high-estrogen phases of the estrous cycle, i.e. at proestrous and estrous, when circulating levels of estrogen are known to be highest. Although the trend of reduction in lipin1 at estrus was statistically significant in CD-1 mice, this was not observed to be as robust in C57BL6 mice.


Figure 2
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  		FIG. 2. Expression of lipin1 in the uteri of mice during the estrous cycle. CD-1 and C57BL6 mice in different phases of the estrous cycle were used and total uterine RNA was extracted using TRIzol reagent. Lipin1 expression level was measured by real-time RT-PCR. Data were normalized against expression of an internal control, 36B4. Changes in lipin1 expression were calculated and are set relative to expression at the diestrous phase, which is considered 100%. Values are the mean ± SEM of three to six uteri per group. *, P < 0.05; **, P < 0.01, statistically significant compared with diestrous, as determined by multiple t tests followed by step-down Bonferroni method, as described in Materials and Methods.

 
To examine lipin1 protein in the uterus, antibodies (raised against two different peptides in two rabbits each) were made against lipin1. The four antibodies tested showed good detection of lipin1, but rabbit polyclonal antibodies against the peptide sequence 285–304 were used for all of the studies reported in this manuscript because of their very low background in both Western blots and immunostaining of mouse tissues.

Lipin1 protein was detected as a single band at approximately 140 kDa by Western blot, and the marked down-regulation of uterine lipin1 by E2 was evident (Fig. 3AGo), with lipin1 being barely detectable after 4 h of E2 treatment in ovariectomized mice. As expected, ICI 182,780 treatment before E2 exposure maintained lipin1 protein at control levels, and treatment with the protein synthesis inhibitor CHX maintained high lipin levels in the absence and presence of E2. ER{alpha} was reduced by E2 or ICI treatment (Fig. 3AGo), and CHX maintained the control uterine level of ER{alpha}, as expected (33).


Figure 3
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  		FIG. 3. Regulation of lipin1 and its localization in the mouse uterus. Rabbit polyclonal antibody raised against the peptide sequence SSSPHKMKESSPLGSRKTPD corresponding to amino acids 285–304 of mouse lipin1 was used to detect lipin1 protein. A, Western blot analysis of total protein from mouse uterus after the indicated treatments with control vehicle, E2, ICI 182,780, CHX alone or together for 4 h, as described in the legend of Fig. 1Go. Lipin1 protein appeared as a single band of approximately 140 kDa. ß-Actin was used as an internal control for sample loading. ER{alpha} protein was also monitored. B, Immunostaining of uterine sections from vehicle and 72-h E2-treated mice to show distribution of lipin1. Mice were treated with control vehicle or hormone as described in Materials and Methods. Uteri were harvested and immediately fixed in formalin. Sections were stained with either lipin1 antibody or with preimmune rabbit serum (negative control). Brown stain represents lipin1, and blue indicates hematoxylin staining of nuclei. Magnification, x20.

 
Lipin1 is present predominantly in the luminal and glandular epithelium of the uterus
Immunostaining of mouse uterine tissue (Fig. 3BGo) revealed lipin1 protein localization predominantly in the luminal and glandular epithelium. In the uteri of control vehicle-treated animals, the protein was uniformly distributed in the cytoplasm of these cells. However, upon E2 treatment, lipin1 staining was reduced and lipin1 appeared to become localized primarily to the apical region of the epithelium, so that the distribution became polarized (Fig. 3BGo, middle panel). Only low levels of lipin1 were observed in the uterine stroma (Fig. 3BGo) and myometrium (data not shown) before or after E2 exposure.

Lipin1 in the uterus and liver of nondiabetic and diabetic mice
We next compared lipin1 status in wild-type mice with that in prediabetic and diabetic NOD mice, a mouse model of insulin-dependent type 1 diabetes mellitus (IDDM). These mice were studied because they have elevated circulating levels of estrogen (14), and hence are useful for studying effects of prolonged elevated estrogen exposure, and also because NOD mice are insulin-deficient and known to have reduced fertility with poor implantation and low viability of embryos (12, 16). The prediabetic NOD (Pd-NOD) mouse uterus appeared to be compromised in its lipin1 mRNA level (P = 0.07) compared with other common laboratory strains of mice (C57BL6 and CD-1) (Fig. 4AGo). In these mice, insulitis begins at 8–10 wk of age, and at 16 wk, the majority of the animals are diabetic, based on glucose levels and other metabolic parameters. In diabetic mice at 20 wk (NOD mice), lipin1 levels were greatly reduced (Fig. 4AGo). This reduction in lipin1 may be associated with loss of the hormone insulin, or changes in blood glucose or other metabolic parameters associated with insulin status, because we found that insulin injection restored lipin1 to levels similar to that of wild-type C57BL6 and CD-1 mice (Fig. 4AGo). We also examined NOD-SCID mice because these immunodeficient mice do not develop diabetes. In NOD-SCID mice, uterine lipin1 levels were high and similar to other wild-type nondiabetic mouse strains (Fig. 4AGo). Although lipin1 levels were only marginally reduced in the liver of Pd-NOD mice, they were significantly reduced in diabetic NOD mice (Fig. 4BGo). As was observed in the uterus, insulin treatment of NOD mice elevated lipin1 expression in the liver, and the up-regulation of lipin1 by insulin was even more pronounced in the liver of NOD mice than in the uterus (Fig. 4Go, B vs. A). And, as observed in the uterus, lipin1 was not compromised in the liver of NOD-SCID mice (Fig. 4BGo).


Figure 4
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  		FIG. 4. Lipin1 expression in the uterus and liver of wild-type, Pd-NOD and diabetic NOD mice. Commonly used nondiabetic C57BL6 and/or CD-1 strains of mice (10 wk of age) were compared with Pd-NOD and diabetic NOD mice for uterine (A) and liver (B) lipin1 expression. Total RNA was extracted using TRIzol reagent and was subjected to real-time RT-PCR. Lipin1 levels are expressed relative to that in the C57BL6 mouse, which is set at 100%. Note that Pd-NOD mice of two different ages were examined in A. The effect of insulin on lipin1 was examined in diabetic female NOD mice that were injected with one unit of insulin per day for 5 d before harvesting tissues. NOD-SCID mice, which do not develop diabetes, were also used for comparison. Values are the mean ± SEM of four to six animals per group. *, P < 0.05; **, P < 0.01, statistically significant compared with C57BL6 control, as determined by multiple t tests followed by step-down Bonferroni method, as described in Materials and Methods.

 
Sections of the uterus from intact (i.e. not ovariectomized) Pd-NOD and diabetic NOD mice (20 wk of age) were immunostained for detection of lipin1. Lipin1 distribution in the Pd-NOD uterus was similar to that observed in the uterus of estrogen-treated ovariectomized wild-type mice (Fig. 5Go left, compare with Fig. 3BGo, middle). Lipin1 was predominantly present in the epithelial cells, at the apical surface in the prediabetic mouse uterus (Pd-NOD), and in the uterus of the diabetic animals, lipin1 protein staining was greatly reduced (Fig. 5Go, NOD).


Figure 5
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  		FIG. 5. Immunohistochemical analysis of lipin1 localization and expression in the Pd-NOD and NOD mouse uterus. Rabbit polyclonal antibody raised against mouse lipin1 amino acid sequence 285–304 was used for immunostaining uterine sections from 20-wk-old Pd-NOD or NOD mice. Brown stain represents lipin1, and blue stain indicates hematoxylin staining of nuclei. Magnification, x20.

 
Estrogen regulation of lipin1 in liver in comparison with that in uterus
To determine whether the effect of estrogen regulation on lipin1 was specific to the uterus or was also observed in another tissue known to express lipin1 and contain ERs, we examined the effect of estrogen on lipin1 RNA and protein in the liver. In addition to expressing ER{alpha}, liver is one of the major nonreproductive target tissues of estrogen. For these studies, we used a less readily metabolized form of E2, EE, because the liver is a major site of E2 inactivation, and previous studies by us and others have shown that EE effectively stimulates estrogen-regulated gene expression in the liver (34, 35).

As shown in Fig. 6AGo, treatment with EE reduced lipin1 levels in the uteri of ovariectomized mice while increasing expression of two well known uterine estrogen-induced genes, complement C3 and lactoferrin. In the liver, lipin1 mRNA was also robustly down-regulated after EE treatment and two estrogen-up-regulated hepatic genes, ApoA4 and Gstp2, were up-regulated over time, as expected (34) (Fig. 6BGo). Reduced levels of lipin1 protein were also observed by Western blot in uterus at 24 and 72 h, and in liver after EE exposure, particularly after 72 h of treatment (Fig. 6Go, C and D). Hence, lipin1 is down-regulated at the mRNA and protein level by estrogen not only in uterus but also in the liver, a metabolically important estrogen target tissue.


Figure 6
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  		FIG. 6. Effect of EE treatment on gene expression in the uterus and liver of ovariectomized mice. Quantitative RT-PCR was performed on total RNA from uterus (A) and liver (B) to determine lipin1 expression at various times of EE treatment. Positive markers of estrogen action in the tissues, namely complement C3 and lactoferrin in the uterus and ApoA4 and Gstp2 in the liver, were also determined. These data sets showed homogeneous variances and therefore were subjected to Dunnett’s test for statistical analysis. Values are the mean ± SEM of three to six animals per group. *, P < 0.05; **, P < 0.01, statistically significant compared with zero time control. C, Western blots to detect lipin1 protein in the uteri and livers of ovariectomized mice treated with control vehicle or EE for the indicated times. Expression of ß-actin was used as loading control. Lanes 1–3 indicate proteins from three individual animals for each treatment group. D, Densitometric analysis of Western blots from C depicted as lipin1 expression normalized to ß-actin. Because of unequal variances in the densitometric values as determined by Levene’s test, adjustment of P values determined by multiple t tests was done using the step-down Bonferroni method. *, P < 0.05 compared with control.

 
Immunohistochemical analysis of lipin1 expression in female mouse liver
We next examined the distribution of lipin1 in the liver of female ovariectomized mice that were either treated with control vehicle or EE by immunohistochemical analysis. Immunostaining with lipin1 antibody shows that lipin1 is robustly expressed in the control liver, and its localization is chiefly cytoplasmic (Fig. 7Go). With EE treatment, there was an overall decrease in lipin1 in the EE-treated liver, and a somewhat uneven distribution of lipin1 is observed in which the reduction in lipin 1 is less in cells closest to and surrounding the blood vessels (Fig. 7Go). In contrast to the uterus where we observed lipin1 relocalization to the cell periphery (Fig. 3BGo), liver cells expressing lipin1 still exhibited a chiefly cytoplasmic localization of lipin1 after estrogen treatment. That estrogen down-regulates lipin1 in the liver of female ovariectomized mice is therefore confirmed at the mRNA (Fig. 6BGo) and protein (Figs. 6Go, C and D, and 7Go) levels.


Figure 7
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  		FIG. 7. Effect of EE on lipin1 expression and distribution in mouse liver. Rabbit polyclonal antibody raised against mouse lipin1 amino acid sequence 285–304 was used for immunostaining sections of liver from ovariectomized mice treated with vehicle or 5 µg EE for 72 h. Liver tissue was harvested and immediately suspended in formalin and left at 4 C overnight. The tissue was then washed and suspended in 70% ethanol before processing for sectioning. Brown stain represents lipin1, and blue indicates hematoxylin staining of nuclei. Magnification, x10 (top) and x20 (bottom).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our findings reveal that lipin1 is rapidly and markedly down-regulated by E2 in the uterus and liver and that lipin1 levels are compromised and low in a mouse model of diabetes and reduced fertility. We investigated and compared lipin1 status in wild-type mice and in NOD mice, a mouse model of IDDM for several reasons: 1) these mice have elevated circulating levels of estrogen (14) and are thus useful for studying effects of prolonged elevated estrogen exposure; 2) NOD mice are insulin-deficient due to autoimmune pancreatic ß-cell loss and show fertility impairment with poor implantation and low viability of embryos (12, 16); and 3) these mice exhibit low body fat and dyslipidemia (36). In addition, because lipin1-deficient mice (fld/fld) are deficient in body fat, show diabetes-like symptoms, and are known to have impaired fertility (13, 17, 18), we hypothesized that these changes might reflect altered expression or regulation of lipin1 in the uterus. Furthermore, apart from regulating metabolism in the liver (37), estrogen has long been known to regulate lipid metabolism in the uterus (38).

Estrogen mainly acts through its receptor subtypes ER{alpha} and ERß and, in the uterus and liver, ER{alpha} is the predominant receptor. Estrogen loss upon ovariectomy is well known to be associated with increased adiposity and body weight gain, and ER{alpha} knockout (ERKO) mice as well as aromatase-deficient mice show increased adiposity and glucose intolerance (7, 8, 9, 10). A recent study with ERKO mice reveals that estrogen modulates insulin sensitivity, perhaps by up-regulating lipogenic genes via suppression of leptin receptor gene expression (37). Our observed down-regulation of lipin1 by E2 and EE in both uterus and liver correlates well with other reports showing estrogen to down-regulate important adiposity-determining genes like PPAR{gamma} (7, 10, 39) and lipoprotein lipase (7, 10) and also with findings that ERKO mice show dysregulation of fat metabolism (8).

Lipin1 has been recently identified as a Mg2+-dependent phosphatidate phosphatase (20), or PAP-1, an enzyme involved in the dephosphorylation of phosphatidic acid (PA). PA is situated at a critical juncture in the Kennedy pathway for the biosynthesis of phospholipids and triacylglycerol and plays crucial roles in cellular lipid signaling and in lipid synthesis and storage (40). The dephosphorylation of PA by lipin1 (see Fig. 8Go) is known to attenuate the activity of PA and generates diacylglycerol, which acts as a substrate for triacylglycerol (TAG), a fat storage form, and a precursor for the important phospholipid membrane components phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine (41). Mutation of the lipin1 homolog in yeast, PAH1, leads to accumulation of PA and reduced TAG; the latter effect is shown to be specific to the lack of phosphatase activity encoded by the yeast lipin1 homolog PAH1 (20). Additionally, PAP-1 has been shown to be highly sensitive to changes in diet and nutrient status (42). PA, a lipid second messenger, interacts with the rapamycin interacting domain of mTOR (43), revealing a central role for mTOR in integrating nutrient and mitogen signals. Estrogen is a major regulator of cell proliferation in the uterus, and in ER-containing human breast cancer MCF7 cells, estrogen-induced cell proliferation is dependent on mTOR (44). In mice, it has recently been demonstrated that lipin1 is a major PAP in vivo (24). Lipin1, by regulating the cellular levels of PA, thus emerges as a key protein, integrating several important cellular pathways (Fig. 8Go) involving lipid flux (through triglycerides), lipid signaling, and de novo synthesis of phospholipids for cellular membranes (42).


Figure 8
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  		FIG. 8. A model depicting pathway integrations involving lipin1 and highlighting the effects of estrogen. The phospholipid PA plays a central role in biosynthetic and signaling events. Absolute levels of PA are regulated by lipin1 (PAP-1), which itself is regulated by at least two hormones, estrogen and insulin. Reduced lipin1 levels by estrogen will maintain high PA levels, presumably reducing diacylglycerol (DAG) and TAG and enhancing signaling via intracellular and membrane PA effectors such as Raf-1, protein kinase C (PKC), and the Ras/MEK/ERK signaling complex and via PI lipids. Estrogen not only reduces lipin1 but also alters its intracellular localization in uterine cells that may have important effects on these biosynthetic and cell signaling events. Insulin, most likely through the involvement of mTOR, increases cellular lipin1, although the mechanism is not fully known. See text for details. CDP, Cytidine-5'-diphosphate.

 
Treatment with E2 brought about an overall reduction in lipin1 in the liver, whereas in the uterus, two events appeared to be occurring: a reduction in the level of lipin1 and relocalization/movement to the apical periphery of the epithelial cells. This suggests a high degree of refinement in estrogen action: on the one hand, down-regulating lipin1 to possibly facilitate proliferation (by maintaining cellular PA levels) and on the other, enriching membrane-associated lipin1 and possible association of PA with proteins such as raf-1, protein kinase C, and the Ras/MEK/ERK signaling complex (45, 46, 47, 48), thus directing this important protein to several different tasks. In fact, PAP-1 translocation to membranes to facilitate de novo synthesis of phospholipids has been reported in type-2 lung cells (42). Estrogen is known to increase Pi incorporation into uterine lipids (38) and to enhance phosphatidylinositol (PI) lipid turnover (49). Indeed the resultant maintenance of enhanced levels of PA by estrogen that would be associated with the down-regulation of lipin1 might also stimulate elevated PI and PI lipid metabolism (Fig. 8Go). Thus, lipin1 regulation by E2 might be responsible for the previous observations of estrogen stimulation of phosphatidylinositol lipid metabolism in the mouse uterus and the generation of PI lipids important for signal transduction and estrogen-induced mitogenesis (49). Also, because lipin1 is highly conserved from yeast to mammalian species (19), and lipin1 homologs are known to be peripheral membrane proteins (19, 41), capable of translocating to membranes and interacting with integral membrane proteins (50), it is conceivable that uterine lipin1 might function in this manner to affect cell signaling pathways. Lipin1 might also play a role in regulation of the uterine cell cycle, because lipin1 has been found to have anti-proliferative effects in murine pro B cells (51). Because estrogen is known to be mitogenic, enhancing proliferation of uterine cells, it is possible that the E2-elicited down regulation of lipin1 might contribute to the uterotrophic stimulation by estrogen. These are aspects that appear worthy of future analysis.

In conclusion, lipin1, an important PAP, is down-regulated by estrogen in the uterus and liver and is compromised in these tissues of animals with type-1 diabetes. Thus far, lipin1 deficiency has been shown to be correlated with insulin-resistant type-2 diabetes (17). It is therefore significant that we report lipin1 dysregulation in the uterus and liver of an autoimmune model of IDDM. Lipin1 deficiency is associated with impaired fertility (13, 17), and here we have shown that a diabetic mouse model with impaired fertility has reduced lipin1 levels in the uterus and liver, which can be restored by insulin treatment. The current investigation, in combination with other studies (17, 20, 22, 23, 24), implies a more broad role for lipin1 beyond adiposity and glucose regulation, to include uterine function and reproductive biology.

The large nuclear receptor superfamily encompasses proteins that regulate transcriptional networks in target tissues, and these receptors appear to divide along two physiological paradigms involving 1) reproduction, development, and growth and 2) nutrient uptake, metabolism, and excretion (52). Our findings support previous observations that hormones and their receptors involved in reproduction, such as estrogens and ER, can also exert important actions on metabolism (37, 38, 49). Lipin1 regulation by estrogen may serve as a critical link between reproduction and growth, and metabolic paradigms, and it may provide a mechanistic link between estrogens, lipid metabolism, and lipid signaling.


   Footnotes
 
This work was supported by National Institutes of Health Grants CA18119 and P01AG024387.

Disclosure Statement: All authors declare no conflicts of interest.

First Published Online April 26, 2007

Abbreviations: CHX, Cycloheximide; E2, estradiol; EE, ethinyl estradiol; ER, estrogen receptor; ERKO, ER knockout; IDDM, insulin-dependent type 1 diabetes mellitus; mTOR, mammalian target of rapamycin; NOD, nonobese diabetic; PA, phosphatidic acid; PAP, PA phosphatase; Pd-NOD, prediabetic NOD; PI, phosphatidylinositol; PPAR{gamma}, peroxisome proliferator-activated receptor-{gamma}; SCID, severe combined immunodeficient; TAG, triacylglycerol; TBS-T, Tris-buffered saline/0.05% Tween.

Received December 22, 2006.

Accepted for publication April 18, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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