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Altered Gene Expression Pattern in the Fatty Liver Dystrophy Mouse Reveals Impaired Insulin-mediated Cytoskeleton Dynamics*

  • Martin Klingenspor
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  • Ping Xu
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  • Robert D. Cohen
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  • Carrie Welch
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  • Karen Reue
    Correspondence
    Recipient of an American Heart Association Established Investigator Award. To whom correspondence should be addressed: West Los Angeles VA Medical Center, 11301 Wilshire Blvd., Bldg. 113, Rm. 312, Los Angeles, CA 90073
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grant HL28481.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.The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) (Ifld 1), (Ifld 2), (Ifld 3), (F23), and (F24).
    § Supported by Research Fellowship Kl 973/3-1 from the Deutsche Forschungsgemeinschaft. Present address: Dept. of Biology, Philipps-University, D-35043, Marburg, Germany.
    ‖ Present address: Dept. of Medicine, Columbia University College of Physicians and Surgeons, New York, NY 10032.
Open AccessPublished:August 13, 1999DOI:https://doi.org/10.1074/jbc.274.33.23078
      The mouse fatty liver dystrophy (fld ) mutation is characterized by transient hypertriglyceridemia and fatty liver during the neonatal period, followed by development of a peripheral neuropathy. To uncover the metabolic pathway that is disrupted by the fld mutation, we analyzed the altered pattern of gene expression in the fatty liver of fld neonates by representational difference analysis of cDNA. Differentially expressed genes detected include a novel member of the Ras superfamily of small GTP-binding proteins, a novel Ser/Thr kinase, and several actin cytoskeleton-associated proteins including actin, profilin, α-actinin, and myosin light chain. Because these proteins have a potential functional link in the propagation of hormone signals, we investigated cytoskeleton dynamics in fld cells in response to hormone treatment. These studies revealed that preadipocytes from fld mice exhibit impaired formation of actin membrane ruffles in response to insulin treatment. These findings suggest that the altered mRNA expression levels detected infld tissue represent a compensatory response for the nonfunctional fld gene and that the fld gene product may be required for development of normal insulin response.
      fld
      fatty liver dystrophy
      RDA
      representational difference analysis
      Chr
      chromosome
      PCR
      polymerase chain reaction
      EPPS
      4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid
      Fatty liver dystrophy (fld )1 is a recessive mutation that arose spontaneously in an inbred mouse strain and is named for the hallmark fatty liver present in neonatal mice and impaired nerve function apparent in adult animals (
      • Langner C.A.
      • Birkenmeier E.H.
      • Ben-Zeev O.
      • Schotz M.C.
      • Sweet H.O.
      • Davisson M.T.
      • Gordon J.I.
      ). The fatty liver develops as the newborn mice begin to suckle milk and is accompanied by elevated plasma triglyceride levels (1000 mg/dl). However, the fatty liver and hypertriglyceridemia resolve spontaneously when mice reach 2–3 weeks of age, at which time the mutant animals begin to develop a peripheral neuropathy that persists throughout their lifetime. The neuropathy is associated with abnormal myelin formation and axonal degeneration in the peripheral nerve (
      • Langner C.A.
      • Birkenmeier E.H.
      • Roth K.A.
      • Bronson R.T.
      • Gordon J.I.
      ). The fld gene has been mapped to mouse chromosome (Chr) 12 (
      • Rowe L.B.
      • Sweet H.O.
      • Gordon J.I.
      • Birkenmeier E.H.
      ), but neither the mutant gene nor the biochemical basis for the lipid and nerve defects have been identified (reviewed in Ref.
      • Reue K.
      • Doolittle M.H.
      ).
      Previous studies of the fld fatty liver have revealed altered mRNA levels for proteins involved in lipid metabolism, including hepatic lipase (60% reduction), apolipoprotein A-IV (100-fold induction), and apolipoprotein C-II (6-fold induction) (
      • Langner C.A.
      • Birkenmeier E.H.
      • Ben-Zeev O.
      • Schotz M.C.
      • Sweet H.O.
      • Davisson M.T.
      • Gordon J.I.
      ). Using quantitative two-dimensional gel electrophoresis, we have detected approximately 25 additional proteins that show significantly altered levels in the fatty liver of neonatal fld mice; the majority of these proteins could not be identified on the basis of current two-dimensional protein data base information (
      • Rehnmark S.
      • Giometti C.S.
      • Slavin B.G.
      • Doolittle M.H.
      • Reue K.
      ). The goal of the present study was to identify genes with altered expression levels in the fatty liver of fld mice, with the intent of uncovering a specific metabolic pathway affected by the mutation. Toward this end, we have employed representational difference analysis (RDA) to isolate cDNA tags corresponding to mRNAs with altered expression in the fld fatty liver.
      The RDA technique was originally developed and applied to isolate differences between complex genomes, such as genetic lesions in tumors that result from DNA deletion, insertion, or rearrangement (
      • Lisitsyn N.A.
      • Lisitsyn N.M.
      • Wigler M.H.
      ,
      • Lisitsyn N.A.
      • Lisitsina N.M.
      • Dalbagni G.
      • Barker P.
      • Sanchez C.A.
      • Gnarra J.
      • Linehan W.M.
      • Reid B.J.
      • Wigler M.H.
      ). Subsequently, RDA has been adapted for use at the cDNA level and employed to isolate sequence tags corresponding to mRNA species that are differentially expressed between two cell populations (
      • Braun B.S.
      • Frieden R.
      • Lessnick S.L.
      • May W.A.
      • Denny C.T.
      ,
      • Chu C.C.
      • Paul W.E.
      ,
      • Edman C.F.
      • Prigent S.A.
      • Schipper A.
      • Feramisco J.R.
      ,
      • Hubank M.
      • Schatz D.G.
      ). In cDNA-RDA, representations generated by PCR from two cDNA populations of interest are compared in successive rounds of subtraction-hybridization, kinetic enrichment, and selective amplification, resulting in sequences corresponding to mRNA species that are expressed at different levels in the two populations.
      Using RDA we have isolated 22 mRNA species with altered expression levels in the fatty liver of fld neonates compared with their wild type counterparts. These RDA sequences include several that encode proteins associated with the actin cytoskeleton, a putative novel Ser/Thr protein kinase, and a putative novel member of the Ras superfamily of small GTP-binding proteins. Because members of the Ras and kinase protein families are functionally linked to components of the actin cytoskeleton in the process of hormone signal propagation, we investigated whether hormone-induced changes in the cytoskeleton were impaired in fld cells. Indeed, it was found that preadipocytes isolated from fld mice fail to form actin membrane ruffles in response to insulin. These results establish thatfld mouse tissues exhibit altered expression levels of cytoskeleton-associated and putative signal transduction proteins, which is associated with impaired cytoskeleton response to hormones such as insulin.

      EXPERIMENTAL PROCEDURES

      Animals

      Nontested breeding pairs of the mouse strain BALB/cByJ-fld were obtained from the Mouse Mutant Resource Colony at the Jackson Laboratory (Bar Harbor, ME) and bred to producefld/fld offspring used in these studies. Thefld/fld pups were recognized at 3–5 days of age by their smaller body size, swollen abdomen, pale liver, and delayed onset of fur growth. Adult fld/fld mice were recognized by the characteristic whole body tremor and clenching of hind legs when handled. Throughout the text, the term “fld ” is used to indicate mice of the fld/fld genotype, and “wild type” is used to indicate mice of genotypes +/+ and+/fld , which both appear phenotypically normal.

      Representational Difference Analysis

      Representational difference analysis was performed as described below, based on the protocol by Hubank and Schatz (
      • Hubank M.
      • Schatz D.G.
      ).

      Amplicon Generation

      RNA was prepared from liver of wild type and fld littermates at 6 days of age by the acid phenol-guanidine method (Trizol; Life Technologies, Inc.). Total RNA (400–500 μg) obtained from two isogenic littermates was combined for poly(A)+ RNA isolation (Poly(A)-Tract IV, Promega, Madison, WI), and 5 μg of the resulting poly(A)+ RNA was used for cDNA synthesis (Superscript Choice System, Life Technologies, Inc.). 1 μg of cDNA was digested with Dpn II (New England Biolabs, Beverly, MA), extracted with phenol-chloroform, and precipitated in ethanol. cDNA fragments were ligated to R-Bgl24 oligomers in the presence of R-Bgl12 oligomers (Operon Technologies, Alameda, CA; see Ref.
      • Hubank M.
      • Schatz D.G.
      for sequences of oligonucleotides used in RDA). Ligated cDNA fragments were diluted and amplified for 19 cycles with R-Bgl24 as a primer. The resulting amplicon preparation was extracted with phenol-chloroform, and R-Bgl24 adaptors were removed byDpn II digestion. All manipulations were performed in parallel for wild type and fld samples.

      Tester Preparation

      Amplicons prepared from wild type andfld RNA were each used as tester in independent reactions. For tester preparation, 10–20 μg of amplicon was subjected to gel electrophoresis in 1.2% low melting agarose. The size range of 100–1500 base pairs was excised and gel purified using Qiaex II resin (Qiagen, Valencia, CA). 1 μg of gel purified tester was ligated to the new oligomer, J-Bgl24. To permit precise adjustment of driver/tester ratios in subtractive hybridization steps, DNA concentrations were measured fluorometrically using Hoechst dye and calf thymus DNA as a standard (
      • Labarca C.
      • Paigen K.
      ). The accuracy of DNA quantitation was verified on agarose gels.

      Subtractive Hybridization

      In the first round of subtractive hybridization, 40 μg of driver and 0.4 μg of tester were mixed, extracted with phenol-chloroform, and precipitated with ethanol. The resulting pellet was washed twice in 70% ethanol, dried, and thoroughly dissolved in 4 μl of hybridization buffer (30 mm EPPS, pH 8.0, 3 mm Na2EDTA). The DNA droplet was overlaid with mineral oil and denatured at 98 °C for 5 min in a PCR machine. The reaction tube was immediately transferred to a 67 °C water bath, and 1 μl 5 m NaCl was added. The tester-driver mixture was incubated at 67 °C for 20 h.

      Amplification of Difference Products

      After hybridization for 20 h, the hybridization mixture was diluted step-wise in TE (10 mm Tris-Cl, 1 mm EDTA, pH 8.0) in the presence of 40 μg of yeast tRNA to a final volume of 400 μl. The diluted hybridization mix was subjected to 10 cycles of PCR amplification using J-Bgl24 as a primer. The resulting PCR product was extracted with phenol-choroform, precipitated with ethanol in the presence of glycogen, and treated with Mung Bean nuclease (New England Biolabs, Beverly, MA) for 30 min at 30 °C to remove single-stranded DNA, which consisted primarily of unhybridized driver sequences. The Mung Bean nuclease reaction mixture was denatured at 95 °C for 8 min and chilled on ice. The first difference product was generated from the denatured Mung Bean nuclease mixture by 19 cycles of PCR amplification using J-Bgl24 as a primer. In total, three or four rounds of subtractive hybridization and selective amplification of tester-tester hybrids were performed. In each new round of subtraction the difference product of the previous subtraction was used as the tester in combination with a fresh aliquot of the initial amplicon from the opposite source as driver (40 μg). Two RDA experiments were performed with different tester/driver ratios: in one experiment, tester/driver ratios were 1:100, 1:400, 1:80,000, and 1:800,000 for rounds 1–4 (
      • Hubank M.
      • Schatz D.G.
      ); the second experiment used a constant ratio of 1:100 for a total of three rounds. Difference products resulting from the final round of subtraction from each experiment were cloned into pBlueScript SK+ (Stratagene, La Jolla, CA).

      Northern Blot and DNA Sequence Analysis of RDA Clones

      Northern blot analysis was performed using 10 μg of total RNA from liver of 6-day-old and 3-month-old wild type and fld mice as described (
      • Cohen R.D.
      • Castellani L.W.
      • Qiao J.-H.
      • Van Lenten B.J.
      • Lusis A.J.
      • Reue K.
      ). RDA plasmid inserts were sequenced by the dideoxy method, and sequences were compared with those in nucleotide data bases using the BLAST algorithm (
      • Altschul S.F.
      • Gish W.
      • Miller W.
      • Myers E.W.
      • Lipman D.J.
      ).

      Reverse Transcriptase-PCR

      Total RNA from liver, adipose, and sciatic nerve was treated with DNase I (Ambion, Austin, TX) at 37 °C for 30 min to remove any potential contaminating genomic DNA. cDNA was synthesized from 2 μg of DNase-treated RNA using avian myeloblastosis virus reverse transcriptase and oligo(dT) primer (cDNA Cycle Kit, Invitrogen, Carlsbad, CA). PCR amplification was performed using one-tenth of the resulting cDNA in a total volume of 50 μl containing 50 mm KCl, 10 mm Tris-HCl, pH 8.3, 2 mm MgCl2, 0.001% gelatin, 200 μmdNTPs, 0.01 μg/μl forward and reverse primers, and 1 unit of AmpliTaq (Perkin-Elmer, Foster City, CA). PCR cycling conditions consisted of a hot start “touchdown” protocol in which the annealing temperature was gradually decreased over a 10 °C range (from 63 to 53 °C) to reduce nonspecific priming (
      • Don R.H.
      • Cox P.T.
      • Wainwright B.J.
      • Baker K.
      • Mattick J.S.
      ), and products were analyzed by agarose gel electrophoresis. The initial reaction conditions were denaturation at 94 °C for 1 min, annealing at 63 °C for 1 min, and extension at 72 °C for 2 min. The annealing temperature was decreased 0.5 °C at each cycle for 20 cycles and maintained at 53 °C for an additional 12 cycles. Products were analyzed by agarose gel electrophoresis. The gene specific primers employed were as follows: Ifld-1, forward, 5′-TCGGGGAACCACTTGATG-3′, and reverse, 5′-ACTACGCCCCGACGGTGTTTGA-3′; Ifld-2, forward, 5′-ATGACGCGCTGCGGGACAAG-3′, and reverse, 5′-CTGCCAGCCAGCATGGTGAAGAG-3′; HPRT, forward, 5′-CACAGGACTAGAACACCTGC-3′, and reverse, 5′-GCTGGTGAAAAGGACCTCT-3′; and PMP-22, forward, 5′-ACACTGCTACTCCTCATCAGTGAG-3′, and reverse, 5′-CAGGATCACATAGATGATACCACTG-3′.

      Chromosomal Localization of RDA Sequences

      Selected RDA sequences were mapped in the mouse genome using a [(C57BL/6J × Mus spretus )F1 × C57BL/6J] interspecific backcross (
      • Welch C.L.
      • Xia Y.-R.
      • Schecter I.
      • Farese R.
      • Mehrabian M.
      • Mehdizadeh S.
      • Warden C.H.
      • Lusis A.J.
      ). Isolated inserts were prepared from RDA plasmids, radiolabeled, and hybridized to restriction-digested genomic DNA from C57BL/6J and M. spretus mice to identify restriction fragment length variants. Distinct variants were then scored in 67 backcross mice that had been typed for more than 350 genetic markers. Linkage was detected with Map Manager version 2.6.5 (
      • Manly K.F.
      ).

      Primary Cell Culture and Actin Cytoskeleton Staining

      Preadipocytes were released from inguinal fat pads of 4-week-old wild type and fld mice by collagenase digestion followed by filtration through 62-μm nylon mesh (
      • Briquet-Laugier V.
      • Dugail I.
      • Ardouin B.
      • Le Liepvre X.
      • Lavau M.
      • Quignard-Boulange A.
      ). Preadipocytes were recovered by centrifugation at 800 × g for 5 min at room temperature and plated onto sterile coverslips at a density of 4 × 104 cells/coverslip in 6-well culture dishes and maintained for 24 h in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 15 nm insulin. After 24 h, cells were approximately 50% confluent.
      For hormone stimulation studies, cells were incubated in Dulbecco's modified Eagle's medium devoid of serum and insulin for 18 h to bring them to a quiescent state. Cells were subsequently treated for 10 min at 37 °C with phosphate-buffered saline alone, with 1% fetal calf serum, or with 40 ng/ml insulin and then fixed and stained with tetramethylrhodamine isothiocyanate-phalloidin (Sigma) as described (
      • Ridley A.J.
      ). Stained cells were observed with a Zeiss Axioskop microscope at an excitation wavelength of 540 nm and an emission wavelength of 590 nm.

      DISCUSSION

      The phenotype of an organism or tissue can ultimately be traced back to the set of genes expressed in individual cells. Thus, the characterization of differences in mRNA expression between cell types provides a window into the fundamental differences in cellular function that may exist between them. Using the RDA technique, we have isolated several mRNAs with differential expression patterns in the fatty liver of fld mutant mice compared with their wild type counterparts. RDA has previously been utilized for comparison of expression patterns in cultured cells undergoing different treatments (
      • Braun B.S.
      • Frieden R.
      • Lessnick S.L.
      • May W.A.
      • Denny C.T.
      ,
      • Chu C.C.
      • Paul W.E.
      ,
      • Edman C.F.
      • Prigent S.A.
      • Schipper A.
      • Feramisco J.R.
      ,
      • Hubank M.
      • Schatz D.G.
      ); the current study further establishes RDA as a reliable technique to isolate differentially expressed sequences from animal tissues as a means to compare wild type and mutant phenotypes.
      The expression levels for mRNAs isolated by RDA included species of high, moderate, and low abundance and both large and modest differences in hepatic expression levels between fld and wild type mice (summarized in Table I). Overall, differential mRNA expression levels were confirmed for ∼70% of the RDA clones isolated here, indicating a success rate for RDA that compares favorably to related techniques for differential mRNA cloning (
      • Sunday M.E.
      ,
      • Wan J.S.
      • Sharp S.J.
      • Poirier G.M.-C.
      • Wagaman P.C.
      • Chambers J.
      • Pyati J.
      • Hom Y.-L.
      • Galindo J.E.
      • Huvar A.
      • Peterson P.A.
      • Jackson M.R.
      • Erlander M.G.
      ). Most of the remaining clones isolated corresponded to mRNAs that could not be detected on Northern blots containing total RNA, indicating that they may be expressed at low levels. Preliminary experiments performed with primers designed to two of these sequences have allowed detection using reverse transcriptase-coupled PCR (data not shown). Most striking in terms of relative mRNA levels in fld versus wild type were the three sequences Ifld1 , Ifld2 , and Ifld3 , which exhibited vastly elevated expression levels in fld in contrast to barely detectable expression in wild type tissue.
      A potential limitation of the RDA technique is that it does not reveal whether differential expression at the mRNA level is ultimately reflected in the corresponding protein levels. Clearly it is not feasible to directly examine protein levels for the majority of RDA products, many of which are novel, but we have confirmed that protein levels for two of the RDA products do indeed increase in parallel with the elevated mRNA levels. Using quantitative two-dimensional gel electrophoresis, we previously demonstrated that apo A-IV and actin protein levels are increased 7- and 2-fold, respectively, in liver from neonatal fld compared with wild type mice (
      • Rehnmark S.
      • Giometti C.S.
      • Slavin B.G.
      • Doolittle M.H.
      • Reue K.
      ). In these two-dimensional electrophoresis studies, we detected nearly two dozen additional unidentified proteins with altered expression levels in thefld fatty liver; it is possible that some of these correspond to additional RDA products isolated in the current studies.
      Of primary significance in our analysis of differential gene expression in fld cells is the novel insight it has provided into the nature of the metabolic defect resulting from the fld gene mutation. Our studies revealed altered gene expression levels for several proteins that may be implicated in insulin signaling and cytoskeleton dynamics. For example, Ifld1 , which is dramatically induced in the fld fatty liver, shares approximately 60% amino acid identity to a mouse Rac protein, including conservation of GTP-binding and effector motifs, andIfld2 was identified as a putative novel Ser/Thr protein kinase based on 75% identity to members of this family (Fig. 2). Both Rac and Ser/Thr protein kinases are involved in insulin signal transduction and the resulting cytoskeletal rearrangement; Rac is absolutely required for membrane ruffle formation (
      • Ridley A.J.
      • Paterson H.F.
      • Johnston C.L.
      • Diekmann D.
      • Hall A.
      ), and its effect may be mediated by Ser/Thr kinases such as p21-activated protein kinases (
      • Dharmawardhane S.
      • Sanders L.C.
      • Martin S.S.
      • Daniels R.H.
      • Bokoch G.M.
      ). Furthermore, a number of additional RDA clones encode proteins associated with the actin cytoskeleton. These include several proteins (i.e. actin, profilin, α-actinin, and myosin) that are known to be localized to membrane ruffles (
      • Machesky L.M.
      • Pollard T.D.
      ,
      • Bretscher A.
      • Lynch W.
      ,
      • Conrad P.A.
      • Giuliano K.A.
      • Fisher G.
      • Collins K.
      • Matsudaira P.T.
      • Taylor L.D.
      ). Although our genetic mapping results excluded Ifld1 ,Ifld2 , and these cytoskeletal components as thefld gene, the altered mRNA expression levels detected infld cells may represent a compensatory response for the nonfunctional fld gene, indicating that the fld gene product may be required for development of normal insulin response. Consistent with this interpretation is our recent finding that fld mice are hyperinsulinemic and glucose intolerant, indicating impaired response of fld tissues to insulin.
      K. Reue, manuscript in preparation.
      The RDA clones characterized in this study were all isolated from liver, but two of the novel sequences that were isolated (Ifld1 and Ifld2 ) were also found to be expressed in two other tissues that are clearly affected by the fld mutation, adipose tissue, and peripheral nerve. In both adipocytes and peripheral nerve Schwann cells, signaling pathways triggered by insulin and/or insulin-like growth factors are crucial for the regulation of differentiation and metabolism (
      • Spritz N.
      • Singh H.
      • Marinan B.
      ,
      • Cheng H.L.
      • Feldman E.L.
      ,
      • Ong J.M.
      • Kirchgessner T.G.
      • Schotz M.C.
      • Kern P.A.
      ,
      • Semenkovich C.F.
      • Wims M.
      • Noe L.
      • Etienne J.
      • Chan L.
      ,
      • Raynolds M.V.
      • Awald P.D.
      • Gordon D.F.
      • Gutierrez-Hartmann A.
      • Rule D.C.
      • Wood W.M.
      • Eckel R.H.
      ). Our novel finding of impaired insulin response in fld cells may therefore represent a direct causal link to the phenotype of fld adipocytes and Schwann cells. The signaling pathways involved in insulin-mediated cytoskeleton dynamics and membrane ruffle formation and the gene products involved in this biochemical cascade have not been fully elucidated. The identification of the fld gene defect may therefore shed light on molecular events involved in development of normal insulin response.

      Acknowledgments

      We thank Yu-Rong Xia and Jake Lusis (UCLA) for assistance in genomic mapping of RDA clones. We thank David Schatz (Yale) for providing a detailed protocol for cDNA-RDA.

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