Abstract

   Increasing evidence indicates that parental diet affects the metabolism
   and health of offspring. It is reported that paternal low-protein diet
   (pLPD) induces glucose intolerance and the expression of genes involved
   in cholesterol biosynthesis in mouse offspring liver. The aim of the
   present study was to determine the effect of a pLPD on gene expression
   in offspring white adipose tissue (WAT), another important tissue for
   the regulation of metabolism. RNA-seq analysis indicated that pLPD up-
   and down-regulated 54 and 274 genes, respectively, in offspring WAT.
   The mRNA expression of many genes involved in lipogenesis was
   down-regulated by pLPD feeding, which may contribute to metabolic
   disorder. The expression of carbohydrate response element-binding
   protein β (ChREBP-β), an important lipogenic transcription factor, was
   also significantly lower in the WAT of pLPD offspring, which may have
   mediated the down-regulation of the lipogenic genes. By contrast, the
   LPD did not affect the expression of lipogenic genes in the WAT of the
   male progenitor, but increased the expression of lipid oxidation genes,
   suggesting that a LPD may reduce lipogenesis using different mechanisms
   in parents and offspring. These findings add to our understanding of
   how paternal diet can regulate metabolism in their offspring.

   Keywords: Paternal diet, Low-protein diet, Adipocytes, ChREBP
     __________________________________________________________________

   Maternal undernutrition increases the risk of lifestyle-related
   diseases, such as cardiovascular disease and type II diabetes, in
   children ([34]Black et al. 2008), as originally reported by Barker et
   al. ([35]Barker et al. 1993) and also documented in the case of the
   Dutch famine ([36]de Rooij et al. 2006). Studies using mice have
   demonstrated that in utero undernutrition of F1 embryos alters the
   germline DNA methylome of F1 adult males and induces glucose
   intolerance in the F2 generation through the paternal line ([37]Radford
   et al. 2014). Thus, although the effect of the maternal environment has
   been thoroughly investigated, an increasing number of studies also
   indicate that there are associations between paternal nutrition and
   offspring health. In addition, an epidemiological study conducted in
   Sweden showed that grandpaternal famine is associated with obesity and
   cardiovascular disease in grandchildren ([38]Kaati et al. 2002).

   A number of mouse models have demonstrated the effects of paternal
   dietary manipulation on offspring metabolism and health. High-fat diet
   feeding of male rats induces pancreatic β cell dysfunction and glucose
   intolerance in female offspring ([39]Ng et al. 2010). In addition, the
   offspring of male mice fed a low-protein diet (LPD) exhibit higher
   expression of genes involved in lipid and cholesterol biosynthesis in
   neonatal liver ([40]Carone et al. 2010). This paternal LPD (pLPD) also
   induces greater adiposity, glucose intolerance, and cardiovascular
   dysfunction, and increases serum tumor necrosis factor α
   concentrations, in offspring mice ([41]Watkins and Sinclair 2014).
   Finally, fasting of male mice before mating leads to changes in serum
   glucose, insulin-like growth factor 1 (IGF1), and corticosterone
   concentrations in their offspring ([42]Anderson et al. 2006). Although
   these studies demonstrate that paternal undernutrition
   transgenerationally induces metabolic disorders, the tissue-specific
   effects on gene expression in the offspring have not been evaluated.
   Therefore, the aim of the current study was to analyze the effect of a
   pLPD on gene expression in offspring adipocytes, which contribute to
   whole-body metabolic regulation.

   Lipid metabolism in white adipose tissue (WAT) plays an important role
   in metabolic homeostasis ([43]Rosen and Spiegelman 2006; [44]Virtue and
   Vidal-Puig 2010). De novo lipogenesis in WAT, which converts non-lipid
   precursors to fatty acids, is down-regulated in obesity, and the
   reinstatement of lipogenesis in WAT prevents obesity-dependent insulin
   resistance ([45]Cao et al. 2008; [46]Huo et al. 2012). Palmitoleate, a
   fatty acid produced by lipogenesis, appears to mediate the insulin
   resistance caused by the lower lipogenic rate in WAT ([47]Yang et al.
   2011; [48]Huo et al. 2012). The transcription factor carbohydrate
   response element-binding protein (ChREBP, also known as MLXIPL) is an
   important regulator of lipogenesis in WAT ([49]Uyeda & Repa 2006;
   [50]Herman et al. 2012; [51]Baraille et al. 2015). It is a member of
   the basic-helix-loop-helix leucine zipper transcription factor family,
   and binds to glucose or carbohydrate response elements (ChOREs), which
   are composed of two E-boxes (CACGTG) or E-box-like sequences, after
   heterodimerizing with max-like protein X (MLX) ([52]Yamashita et al.
   2001; [53]Ma et al. 2006). Two isoforms of ChREBP, ChREBP-α and the
   N-terminally truncated ChREBP-β, which is expressed from an alternative
   promoter, have been identified ([54]Herman et al. 2012). In response to
   high concentrations of glucose in WAT, phosphatase 2A is activated by
   metabolites and dephosphorylates ChREBP-α, leading to the translocation
   of ChREBP-α into the nucleus and the induction of ChREBP-β gene
   transcription following binding of ChREBP-α to the upstream promoter
   ([55]Kabashima et al. 2003; [56]Herman et al. 2012; [57]Baraille et al.
   2015; [58]Abdul-Wahed et al. 2017). ChREBP-β more potently induces the
   expression of lipogenic genes than the more abundant ChREBP-α
   ([59]Herman et al. 2012). Furthermore, adipose-specific ChREBP knockout
   mice exhibit systemic insulin resistance ([60]Vijayakumar et al. 2017).
   In contrast to the situation in WAT, lipogenesis in the liver is
   enhanced in obesity, which also promotes insulin resistance and
   lipotoxicity ([61]Beaven et al. 2013). This may be, at least in part,
   due to greater synthesis of ceramides from palmitate, which potently
   induce insulin resistance ([62]Chavez & Summers 2012), and activation
   of the innate immune system by saturated fatty acids ([63]Fessler et
   al. 2009). Thus, lipogenesis in the WAT and liver has opposite effects
   on insulin sensitivity ([64]Eissing et al. 2013).

   To determine the mechanism underlying the pLPD-induced metabolic
   disorder in mouse offspring, we investigated the effect of pLPD on gene
   expression in the WAT of these animals. We found that pLPD caused lower
   expression of a number of genes involved in lipogenesis. In particular,
   ChREBP-β expression was lower, whereas insulin-like growth
   factor-binding protein 2 (Igfbp2) expression was higher, suggesting
   that an impairment in insulin signaling induced by IGFBP2 may suppress
   ChREBP-α-dependent ChREBP-β expression. By contrast, LPD did not affect
   the expression of lipogenic genes in WAT of the male progenitor, but
   increased the expression of lipid oxidation genes, suggesting that LPD
   may reduce lipid storage by activating lipid oxidation.

Materials and Methods

Mice and diets

   All animal care and experiments were conducted in accordance with the
   guidelines of the Animal Care and Use Committee of the RIKEN Institute.
   C57BL/6 mice (C57BL6/JJmsSlc background) were obtained from Japan SLC
   (Shizuoka, Japan), and diets were obtained from Oriental Yeast Co
   (Shiga, Japan). To compare gene expression in inguinal WAT (iWAT) in
   the offspring of mice fed a control diet (CD) or a pLPD with that
   previously reported in liver ([65]Carone et al. 2010), all procedures
   and the composition of the diet were matched to those described by
   Carone et al. ([66]Carone et al. 2010). Mice were fed with the CD for
   two generations to minimize the transgenerational effect of the chow
   fed by the animal supplier. After weaning at 3 weeks of age, male mice
   were fed with the CD or LPD, while female mice were fed with the CD. At
   10–12 weeks of age, male and virgin female mice were placed in the same
   cage for mating, in which they consumed CD, for 2 days only, to avoid a
   significant reduction in the effect of the LPD in male mice. After
   this, the males were removed and the pregnant females were fed the CD
   until their offspring reached 3 weeks of age, when they were weaned.
   From this time, the offspring were fed the CD and killed at 5 weeks of
   age, when their iWAT was rapidly dissected and flash-frozen in liquid
   N[2].

RNA sequencing (RNA-seq) analysis

   Total RNA was isolated from iWAT using TRIZOL reagent (Thermo Fisher
   Scientific, Waltham, MA). RNA quality was evaluated using an RNA 6000
   Pico kit (Agilent, Santa Clara, CA). Fifty nanograms of total RNA was
   used for RNA sequencing (RNA-seq) library preparation with a NEBNext
   rRNA Depletion Kit and a NEBNext Ultra Directional RNA Library
   Preparation Kit (New England Biolabs, Ipswich, MA), then 2 × 36 base
   pair-end sequencing was performed using NextSeq500 (Illumina, San
   Diego, CA) by Tsukuba i-Laboratory LLP (Tsukuba, Ibaraki, Japan).

Bioinformatics and statistics for RNA-seq data

   RNA-seq data were analyzed using CLC Genomics Workbench version 9.5.1
   (QIAGEN) ([67]Nielsen et al. 2010). Sequences were mapped to the mouse
   mm10 genome. Expression values were calculated as “reads per kilobase
   per million reads” using CLC Genomics Workbench (Qiagen). Genes were
   considered to be differentially expressed when false discovery rate
   (FDR) adjusted P-values were < 0.05. Pathway enrichment analysis for
   differentially expressed genes (DEGs) was conducted using the Kyoto
   Encyclopedia of Genes and Genomes (KEGG) database ([68]Kanehisa et al.
   2014) and the Database for Annotation, Visualization, and Integrated
   Discovery (DAVID) ([69]Huang da et al. 2009a; [70]Huang da et al.
   2009b).

Quantitative RT-PCR (qRT-PCR) analysis

   RT-PCR analysis was performed using the OneStep SYBR Green PCR mix
   (Takara, Shiga, Japan), following the manufacturer’s instructions. The
   quantitative RT-PCR (qRT-PCR) was performed using a 7500 Fast Real-time
   PCR System (Applied Biosystems, Foster City, CA). Oligonucleotide
   primers were designed to produce 70–150 base amplification products.
   The primers used are shown in Table S1. 36B4, which encodes Rplp0
   ribosomal protein, large, P0, was used as a reference gene, and the
   relative expression levels of each target gene were quantified using
   the 2^−ΔΔCt method ([71]Livak and Schmittgen 2001).

Quantitative chromatin immunoprecipitation (qChIP)

   Chromatin immunoprecipitation (ChIP) assays were performed as described
   previously, with some modifications ([72]Haim et al. 2013; [73]Yoshida
   et al. 2015). Fresh iWAT from 5-week-old offspring was rinsed once with
   1× phosphate-buffered saline (PBS) supplemented with protease
   inhibitors (PI), then minced into small pieces. Minced tissues were
   fixed with 1% formaldehyde for 20 min at room temperature with moderate
   shaking, then washed twice to remove lipid. Small adipose tissue pieces
   were resuspended in 1 ml of adipocyte lysis buffer 1 (50 mM HEPES, pH
   7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 1% NP-40, and 0.25% Triton
   X-100) supplemented with PI, rotated for 5 min, then homogenized using
   a Dounce homogenizer (30 strokes). Samples were rotated at 4° for 15
   min (vortexed every 5 min), then 20 additional strokes were performed.
   The samples were centrifuged (5 min, 3,000 rpm, 4°), and the pellet was
   washed three times in lysis buffer 2 (10 mM Tris-HCl, pH 8.0, 200 mM
   NaCl, 0.5 mM EGTA, 1 mM EDTA, and PI) to remove lipid. For DNA
   shearing, the nuclear pellet was resuspended in 200 µl of sodium
   dodecyl sulfate (SDS) lysis buffer (1% SDS, 10 mM EDTA, pH 8.1, with 50
   mM Tris-HCl, pH 8.1) supplemented with PI, and incubated on ice for 20
   min prior to sonication. Sonication was optimized for a DNA fragment
   size of ∼200–600 bases. After sonication, lysis buffer 3 (10 mM
   Tris-HCl, pH 8.0, 100 mM NaCl, 0.5 mM EGTA, 1 mM EDTA, 1% Triton X-100,
   0.1% sodium deoxycholate, and PI) was added, and the final SDS
   concentration was adjusted to 0.1%. Dynabead Protein A was prepared by
   washing twice with PBS containing bovine serum albumin. ChREBP antibody
   (NBP2-44307, Novus, Littleton, CO) and rabbit IgG control (sc-2017,
   Santa Cruz, Santa Cruz, CA) were bound to the beads by incubation for 3
   h at 4°, then the beads were washed three times. A mixture of beads and
   lysate was rotated for 14 h at 4°. Next, the beads were washed six
   times, before elution in elution buffer. The eluted samples were
   reverse-crosslinked overnight at 65° and then treated with RNase and
   Protease K at 37°. Finally, DNA was purified using a QIAgen Kit prior
   to qRT-PCR. The ChIP-qPCR primers used are listed in Table S1.

Statistics for non-RNA-seq data

   Data are presented as mean ± SD, and group comparisons were performed
   using independent sample t-tests. The significance level was set at P <
   0.05.

Data availability

   Figure S1 contains the volcano plot and GO analysis for RNA-seq data of
   F0 generation under LPD. Table S1 contains the information on primer
   sequences used for qRT-PCR and qChIP. File S1 contains the list of up-
   and down-regulated genes induce by pLPD in male offspring iWAT. File S2
   contains the list of up- and down-regulated genes induce by LPD in
   father iWAT. The raw data and processed data files were deposited in
   the gene expression omnibus (GEO) database of NCBI with the accession
   number [74]GSE121774. Supplemental material available at FigShare:
   [75]https://doi.org/10.25387/g3.7996325.

Results

Down-regulation of lipogenic gene expression in the iWAT of the offspring of
mice fed a pLPD

   To determine the effects of a pLPD on gene expression in offspring
   iWAT, we used essentially the same feeding regimen reported by Carone
   et al. ([76]Carone et al. 2010). Mice were first fed with the CD for
   two generations to minimize the transgenerational effect of the chow
   provided by the animal supplier. After weaning at 3 weeks of age, male
   F0 mice were fed the CD or LPD, while female F0 mice were fed the CD.
   Mating was performed within a 2 day period to avoid any significant
   reduction in the effects of the LPD on male F0 mice. The male F1
   offspring obtained were killed at 5 weeks of age, and total RNA was
   prepared from their iWAT, which was used for transcriptomic analysis by
   RNA-seq. This analysis identified 54 genes that were up- and 274 genes
   that were down-regulated in iWAT from mice that were the offspring of
   male progenitors fed with the LPD compared with those from CD fathers
   ([77]Figure 1A).

Figure 1.

   [78]Figure 1
   [79]Open in a new tab

   The effects of a pLPD on gene expression in offspring iWAT. (A) F0 male
   mice (n = 3) were fed with a CD or a LPD and mated with female mice fed
   a CD to generate F1 offspring. Using total RNA extracted from the
   inguinal white adipose tissue (iWAT) of F1 male offspring (three mice
   with three different mothers), gene expression was analyzed using
   RNA-seq. A volcano plot depicting the differentially expressed genes (n
   = 328) induced by pLPD feeding in male offspring iWAT. pLPD
   up-regulated 54 genes (purple spots) and down-regulated 274 genes
   (green spots) (FDR < 0.05). Four representative down-regulated
   lipogenic genes are shown. (B and C) Gene ontology analysis for the
   biological processes (GO-BP) represented among the up- and
   down-regulated genes, respectively. The P-value for the enrichment was
   obtained using a modified Fisher’s exact test. Y-axis: name of the BP
   term; x-axis: −log10 (P-value). Seven GO terms (P < 0.05) were
   over-represented among the up-regulated genes and are mainly associated
   with the immune response (B). For the down-regulated genes (FDR <
   0.05), only the top 20 GO-BP terms are shown. The down-regulated genes
   included many GO-BP terms involved in metabolism, such as lipid
   metabolism and glycolysis (orange bar) (C).

   To identify the pathways associated with the DEGs, lists of up- and
   down-regulated genes were analyzed for KEGG pathway enrichment using
   the DAVID database. Seven biological processes (BPs) were significantly
   over-represented among the up-regulated genes, which were mainly
   involved in immunity, such as T cell proliferation and activation,
   immune system processes, and the response to lipopolysaccharide
   ([80]Figure 1B and File S1). The down-regulated genes contained
   numerous representatives from metabolic pathways, such as lipid
   metabolism, fatty acid biosynthesis, and glycolysis. Another major
   enriched group is involved in adipocyte development and function,
   including collagen fibril organization, positive regulation of
   phagocytosis, extracellular fibril organization, and related
   annotations ([81]Figure 1C and File S1). Thus, the results of
   genome-wide profiling indicate metabolic and developmental programming
   of the WAT of male offspring in response to a pLPD.

   Because lipogenesis in WAT is down-regulated in obesity and the
   reinstatement of lipogenesis in WAT prevents obesity-dependent insulin
   resistance ([82]Cao et al. 2008; [83]Huo et al. 2012), we focused on
   the genes involved in lipogenesis. The genes that were down-regulated
   by a pLPD were significantly enriched for BPs related to lipid
   biosynthesis ([84]Figure 1C). Many genes enriched in this pathway are
   key enzymes responsible for the regulation of triglyceride
   biosynthesis, including ATP citrate lyase (Acly), acetyl-CoA
   carboxylase 2 (Acacb), elongation of very-long-chain fatty acids
   protein 5 (Elovl5), elongation of very-long-chain fatty acids protein 6
   (Elovl6), thromboxane A synthase 1 (Tbxas1), and trans-2, 3-enoyl-CoA
   reductase (Tecr). To attempt to corroborate the RNA-seq data, we
   measured the relative expression levels of these genes by qRT-PCR,
   using 36B4 as a reference gene. The results were consistent with the
   RNA-seq data and indicated that the expression levels of lipogenic
   genes were significantly reduced by pLPD feeding ([85]Figure 2A). In
   addition, the mRNA expression of ChREBP, which up-regulates the
   expression of multiple lipogenic genes ([86]Uyeda & Repa 2006;
   [87]Herman et al. 2012; [88]Baraille et al. 2015), was also was also
   significantly reduced by pLPD feeding (log[2]FC = −1.48, FDR = 0.03)
   ([89]Figure 1A). We examined the expression of the two ChREBP isoforms
   by qRT-PCR. Interestingly, expression of ChREBP-β, which more potently
   induces lipogenesis genes compared to more abundant ChREBP-α, was
   markedly reduced in LPD offspring, whereas, ChREBP-α expression
   remained unchanged ([90]Figure 2A).

Figure 2.

   [91]Figure 2
   [92]Open in a new tab

   pLPD-induced down-regulation of lipogenic genes in F1 offspring iWAT.
   (A) Differential mRNA expression of selected lipogenic genes shown in
   [93]Figure 1A was corroborated using qRT-PCR (n = 3). Gene expression
   was normalized to the reference gene 36B4, and actin was used as a
   negative control. Bars represent the mean ± SD *, P < 0.05; **, P <
   0.01; ns, not significant; CD, control diet; LPD, low-protein diet. (B
   and C) The down-regulation of Pfkl and Ppp2r5b, and up-regulation of
   Igfbp2, in F1 iWAT was confirmed by qRT-PCR. (D) pLPD reduced the
   amount of ChREBP bound to the ChREBP-β gene promoter in F1 offspring
   iWAT, demonstrated using qChIP of F1 offspring iWAT and an anti-ChREBP
   antibody. Primers that amplified the region containing the ChREBP-β
   gene promoter were used for qPCR. Mean values relative to the input ±
   SD are shown (n = 3). *, P < 0.05; **, P < 0.01; ns, not significant.
   (E) A speculative scheme for the down-regulation of the lipogenic genes
   in F1 iWAT.

Possible mechanism underlying the pLPD-induced down-regulation of lipogenic
genes

   It was previously shown that glycolysis is tightly linked to the
   transcription of the ChREBP-β gene from the upstream promoter following
   dephosphorylation of ChREBP-α, which leads to its nuclear translocation
   ([94]Kabashima et al. 2003; [95]Uyeda & Repa 2006; [96]Herman et al.
   2012; [97]Baraille et al. 2015). In offspring WAT, mRNA expression of
   phosphofructokinase (Pfkl), one of the key glycolytic enzymes, and
   protein phosphatase 2 regulatory subunit B’β (Ppp2rb5), which
   dephosphorylates ChREBP-α ([98]Uyeda & Repa 2006; [99]Kabashima et al.
   2003), was also reduced by pLPD feeding ([100]Figure 2B). These results
   are consistent with pLPD reducing glycolytic rate and the
   dephosphorylation of ChREBP-α, which would suppress ChREBP-β gene
   transcription in offspring iWAT.

   This finding prompted us to look for altered expression of upstream
   regulators of glycolysis. Interestingly, we found that the mRNA
   expression of Igfbp2, a regulator of IGF1 signaling, which affects
   glucose metabolism, was up-regulated by pLPD feeding ([101]Figure 2C).
   IGF1 signaling substantially overlaps with insulin signaling, sharing
   intermediates such as insulin receptor substrate 1, and the
   phosphoinositide 3-kinase/AKT pathway, activation of which increases
   glucose uptake by up-regulating the expression of glucose transporter
   4, regulating its intracellular trafficking, and by stimulating the
   activity of some glycolytic enzymes ([102]Ward and Thompson 2012).
   IGFBP2 inhibits IGF1 activity by binding to the IGF1 receptor and
   preventing access of IGF1, resulting in the suppression of IGF1
   signaling glycolytic enzymes ([103]Ward and Thompson 2012). Therefore,
   up-regulation of IGFBP2 may inhibit glycolysis, leading to suppression
   of the nuclear translocation of ChREBP-α.

   Previous studies have identified two putative ChOREs, the first of
   which is an E-box region upstream of exon 1b, and the second of which
   is downstream of exon 1b ([104]Herman et al. 2012; [105]Zhang et al.
   2015). We therefore performed a ChIP-qPCR experiment using an
   anti-ChREBP antibody that recognizes both ChREBP isoforms to analyze
   the binding of ChREBP to these two sites. More ChREBP was found to bind
   to the second putative ChORE, but the amount bound was 60% lower in F1
   mice with a pLPD-fed parent than in those with a CD-fed parent
   ([106]Figure 2D). Thus, pLPD reduced the amount of ChREBP, presumably
   ChREBP-α, binding to the promoter of ChREBP-β, leading to a reduction
   in the expression of ChREBP-β. Taken together, these data suggest that
   down-regulation of lipogenic genes can be explained, at least in part,
   by an increase in Igfbp2 expression, which suppresses IGF1 signaling,
   ChREBP-β expression, and therefore lipogenic gene expression
   ([107]Figure 2E).

Comparison of gene expression patterns in iWAT From F1 offspring and male
progenitors

   We next asked whether the alteration in gene expression pattern in F1
   offspring iWAT mimicked that in their male parents. For this purpose,
   we performed transcriptome profiling of F0 iWAT in 10–12-week-old male
   mice fed the LPD or CD. RNA-seq analysis identified 269 genes up- and
   334 genes down-regulated in F0 iWAT from mice fed the LPD compared with
   those fed the CD (Figure S1A). The pathways associated with DEGs were
   analyzed for KEGG pathway enrichment using the DAVID database. The
   up-regulated genes were significantly enriched for genes involved in
   lipid metabolism, oxidation-reduction processes, and the tricarboxylic
   acid (TCA) cycle, whereas the down-regulated genes are involved in
   muscle and cardiac function and development (Figure S1B and S1C, and
   File S2). Many genes implicated in lipid metabolism were induced in F0
   iWAT by the LPD, including peroxisome proliferator-activated receptor α
   (Ppara), carnitine palmitoyltransferase 1B (Cpt1b), carnitine
   palmitoyltransferase II (Cpt2), hydroxyacyl-CoA dehydrogenase
   trifunctional multienzyme complex subunit α (Hadha), acetyl-CoA
   acyltransferase 2 (Acaa2), and acyl-CoA dehydrogenase very-long-chain
   (Acadvl). The results of qRT-PCR indicated that the mRNA expression of
   Ppara, Hadha, Cpt1b, Acaa2, and Acadvl was significantly higher in
   LPD-fed mouse iWAT, and that of Cpt2 tended to be higher ([108]Figure
   3A). Because PPARα is a transcription factor that up-regulates the
   transcription of multiple genes involved in lipid oxidation ([109]Leone
   et al. 1999), higher expression of PPARα may cause higher expression of
   lipid oxidation genes.

Figure 3.

   [110]Figure 3
   [111]Open in a new tab

   Comparison of the gene expression changes induced by LPD feeding in F0
   male progenitor and F1 offspring iWAT. (A) F0 male mice were fed a CD
   or a LPD, then total RNA was prepared from the iWAT of F0 mice at 10–12
   weeks of age, and used for RNA-seq analysis. The results shown in
   Figure S1 suggested the down-regulation of genes involved in fatty acid
   oxidation. The down-regulation of key genes involved in fatty acid
   oxidation was confirmed by qRT-PCR (n = 4). Target gene expression was
   normalized to that of the reference gene 36B4. *, P < 0.05; ns, not
   significant. Bars represent the mean ± SD (B) A comparison of the genes
   affected by LPD in male F0 and F1 offspring iWAT. The Venn diagram
   shows the numbers of genes that were up- or down-regulated by LPD
   feeding in male F0 and F1 offspring iWAT. CD, control diet; LPD,
   low-protein diet. (C) A speculative schematic view of the effect of a
   LPD on metabolism in F0 iWAT.

   Next, we compared the overlap between sets of DEGs in F1 and F0 iWAT.
   Among the 54 up-regulated genes in the F1 mice and the 269 up-regulated
   genes in the F0 mice, there were only four common genes ([112]Figure
   3B). Furthermore, only 12 genes showed overlapping differential
   expression among the 274 and 334 down-regulated genes in the F1 and F0
   iWAT, respectively. These results indicate that the transcriptomic
   changes in offspring iWAT were dissimilar from those in F0 iWAT.
   Analysis of the DEGs suggested that lipogenesis is down-regulated in F1
   iWAT, whereas lipolysis is up-regulated in F0 iWAT. Lipogenesis and
   lipolysis are both tightly regulated processes, and they work
   oppositely to accumulate lipid. Among the genes that were up-regulated
   in the F0 males, PPARα is a key regulator of fatty acid oxidation,
   which targets many genes in this pathway, including Cpt1, Cpt2, and Aco
   ([113]Figure 3C). PPARα also can inhibit lipogenesis by suppressing
   Srebp, Acc, and Scd expression or by reducing the expression of
   glycolytic genes ([114]Finck and Kelly 2002). The up-regulation of
   fatty acid oxidation is usually associated with greater energy
   production and thermogenesis. Consistent with this, genes involved in
   the TCA cycle and uncoupling protein 1 (Ucp1) were up-regulated by LPD
   feeding, according to the RNA-seq data (Figure S1B, File S2)
   ([115]Figure 3C). Thus, in contrast to the reduction in lipogenesis in
   offspring iWAT, mediated through altered ChREBP expression, lipogenesis
   may be down-regulated in F0 iWAT secondary to enhanced lipolysis,
   mediated through PPARα.

Comparison of the pLPD-regulated genes in offspring iWAT and liver

   To understand how pLPD-induced differences in gene expression in the
   iWAT and liver of the offspring contribute to the glucose intolerance
   that was previously reported ([116]Watkins and Sinclair 2014), we also
   determined the DEGs in F1 liver, which was previously analyzed
   following a similar feeding study ([117]Carone et al. 2010). We again
   performed pathway analysis of the up- and down- regulated genes. In
   liver, of the 1,559 DEGs (FDR < 0.001), 759 genes were up-regulated and
   836 genes were down-regulated. KEGG pathway analysis identified the
   pathways that were enriched among the up- and down-regulated genes,
   respectively ([118]Figure 4A and [119]4B). Of the top 21 pathways
   represented among the up-regulated genes, 10 are involved in
   cholesterol biosynthesis and lipid metabolism, whereas none of the
   pathways represented among the down-regulated genes are related to
   lipid metabolism. Comparison of the DEGs in F1 iWAT and liver showed
   almost no overlap, with only one and five genes overlapping among the
   up- and down-regulated lists, respectively ([120]Figure 4C), and none
   of these are involved in metabolism. To confirm that the result of
   [121]Carone et al. (2010) are reproducibly obtained in the present
   study, we performed qRT-PCR to examine the expression levels of typical
   genes involved in lipid and cholesterol biosynthesis, including sterol
   regulatory element binding transcription factor 2 (Srebf2), squalene
   spoxidase (Sqle), isopentenyl-diphosphate delta-isomerase 1 (Idi1),
   stearoyl-CoA desaturase (Scd1) and fatty acid synthase (Fasn). As shown
   in [122]Figure 4D, our results were consistent with previous study
   ([123]Carone et al. 2010), and indicated that the expression levels of
   these lipogenic genes were significantly increased by pLPD feeding. The
   up-regulation of many genes involved in lipid and cholesterol
   biosynthesis suggests that lipogenesis may be up-regulated by pLPD
   feeding in F1 liver. To assess whether the lipogenic genes and
   regulators which were differentially expressed in iWAT are oppositely
   expressed in liver, we measured the expression of these genes in the
   livers of the offspring as in [124]Figure 4E. Except for Tbxas1,
   expression levels of Acacb, Acly, Elovl5, Elovl6 and Tecr that were
   reduced in iWAT were strongly increased in pLPD offspring liver
   compared to CD mice, which was also consistent with previous study,
   indicating the opposite expression of lipogenic genes in liver and
   iWAT. The expressions of ChREBP-α, ChREBP-β, Pfkl, Ppp2r5b and Igfbp2
   were unchanged, suggesting that ChREBP did not contribute to the
   increase of lipogenesis in the liver. Instead, we found the
   up-regulation of lipogenic transcriptional factor Srebf2 ([125]Figure
   4D). These results support the previous study showing that
   up-regulation of hepatic lipogenesis is mediated by Srebf2 ([126]Carone
   et al. 2010). Also, high expression of genes related to lipogenesis in
   pLPD offspring liver can be due to higher expression of
   SREBP-activating gene Scap ([127]Carone et al. 2010). The findings here
   are in agreement with the fact that SREBP is the key regulator of
   lipogenesis in liver, whereas ChREPB plays dominant roles in regulation
   of lipogenesis in adipose tissue ([128]Horton et al. 2002, [129]Herman
   et al. 2012). Threfore, our results demonstrate that lipogenic genes
   are reciprocally expressed in iWAT and liver in the pLPD offspring, and
   their expressions are regulated by different transcriptional factors in
   a tissue-specific manner. It has previously been shown that lipogenesis
   in WAT and liver oppositely correlate with insulin resistance: a lower
   lipogenic rate in WAT and a higher lipogenic rate in liver cause
   insulin resistance ([130]Cao et al. 2008; [131]Yang et al. 2011;
   [132]Huo et al. 2012; [133]Chavez and Summers 2012; [134]Beaven et al.
   2013; [135]Abdul-Wahed et al. 2017; [136]Vijayakumar et al. 2017).
   Taken together, our results therefore suggest that disorders of lipid
   metabolism in liver and white adipose tissue in juvenile mice of LPD
   fathers may play pivotal roles in the development of metabolic
   disorders ([137]Figure 4F).

Figure 4.

   [138]Figure 4
   [139]Open in a new tab

   Comparison of pLPD-regulated genes in iWAT and liver of F1 offspring
   mice. (A and B) The up- and down-regulated genes previously reported by
   Carone et al. in the liver of F1 offspring ([140]Carone et al. 2010)
   were used for this analysis. Gene ontology analysis for biological
   processes (GO-BP) for up- (A) and down-regulated (B) genes,
   respectively, was performed as described in [141]Figure 1A. Only top 21
   terms for upregulated genes and 20 terms for downregulated genes are
   shown. Genes involved in cholesterol biosynthesis and fatty acid
   metabolism are enriched among the up-regulated genes, and shown in
   orange. (C) Comparison of the pLPD-regulated genes in the liver and
   iWAT of F1 offspring. The Venn diagram shows the numbers of up- and
   down-regulated genes in the liver, as reported by Carone et al.
   ([142]Carone et al. 2010), and in the iWAT (shown in [143]Figure 1) of
   F1 offspring. (D) Expression levels of genes related to lipid and
   cholesterol biosynthesis in the offspring liver measured by qRT-PCR
   were consistent with the previous study ([144]Carone et al., 2010) (n =
   3). (E) Validation of expression levels of genes of interest in iWAT in
   the offspring liver (n = 3). (For D and E) Expression level was
   normalized to the reference gene 36B4, and actin was used as a negative
   control. Bars represent the mean ± SD *, P < 0.05; **, P < 0.01; ns,
   not significant; CD, control diet; LPD, low-protein diet. (F) Model
   depicting how the gene expression changes induced by pLPD feeding in
   iWAT and liver could cause metabolic disorders in offspring.

Discussion

   The present study indicates that pLPD down-regulates multiple lipogenic
   genes in offspring iWAT, which may reduce lipogenesis and increase the
   risk of metabolic disorders. Down-regulation of ChREBP-β, a key
   transcription factor for lipogenic genes, which might be the result of
   an increase in Igfbp2 expression, is likely to play an important role
   in this down-regulation. We also identified pLPD-induced up-regulation
   of genes involved in the immune response, including pellino-1 (Peli1)
   and high mobility group box 2 (Hmgb2), in offspring iWAT. It was
   previously shown that inflammation prevents adipocyte differentiation
   by inhibiting PPARα and PPARγ ([145]Rosen & MacDougald 2006). However,
   there is no clear evidence to show that some of these up-regulated
   genes enhance inflammation, although some reports suggest the
   possibility that PELI1 and HMGB2 may regulate inflammation ([146]Aird
   et al. 2016; [147]Kim et al. 2017). Thus, up-regulation of immune
   response-related genes may not directly contribute to the
   down-regulation of lipogenic genes by inhibiting adipocyte
   differentiation.

   Many studies have shown an effect of pLPD on offspring gene expression
   in rodents, but comparison of the data in the reports is not always
   possible because of the use of different diets and feeding regimens. To
   enable comparison of our data with those in the previous report by
   Carone et al. ([148]Carone et al. 2010), we used the same diet and
   feeding regimen. However, there still exist differences between two
   studies. First, 3-week-old mice were used in the study by [149]Carone
   et al. (2010). In this study, because iWAT of 3-week-old-mice were
   quite small and contains high amount of lipid which affects the
   efficiency of RNA extraction, we used the 5-week-old offspring in which
   the offspring are still young and their adipose tissues are also
   sufficient for experimental manipulation. Second, C57BL/6 mice in
   [150]Carone et al. (2010) were obtained from Jackson Labs and from
   Charles River Laboratories, whereas our C57BL/6 mice were obtained from
   Shizuoka, Japan. These mice are substrains of B6 mice. Despite these
   differences, in the offspring liver, we also found the up-regulation of
   genes relating to lipid and cholesterol biosynthesis, which was
   consistent with [151]Carone et al. (2010). This data suggested that
   lipid metabolism is sensitive to pLPD in spite of some of genetic
   background differences among B6 substrains ([152]Mekada et al. 2009).
   The Technology and Development Team for Mouse Phenotype Analysis of
   RIKEN BRC also confirmed that there is no significant difference in the
   glucose tolerance test between C57BL/6 mice from SLC and Charles River
   Laboratories (data not shown). In contrast to liver, we found that
   lipogenic genes were reduced in iWAT, which was partly due to the
   reduction of ChREBP-β. It was demonstrated that loss of adipose-ChREBP
   results in insulin resistance and increased WAT inflammation
   ([153]Vijayakumar et al. 2017). In our RNA-seq data, we noted that mRNA
   level of TNF-α gene in the iWAT had a tendency to increase, albeit at
   the border of significance (log[2]FC = 2.18, FDR = 0.07, data not
   shown), suggesting a low-grade inflammation in adipose tissue of pLPD
   offspring. Previously, [154]Watkins and Sinclair (2014) reported that
   the adult offspring from LPD-fed fathers developed impaired glucose
   tolerance and increased serum TNF-α level ([155]Watkins and Sinclair
   2014). Our results could imply that lipid metabolic disorders in liver
   and white adipose tissue during early life contribute to the
   development of such diseases in these mice. However, between our study
   and the study by [156]Watkins and Sinclair (2014), there are many other
   differences in experimental components, including protein
   concentration, feeding scheme, and age at mating, in addition to the
   difference of C57/BL6 genetic background. Nevertheless, the important
   factors that are likely to contribute to the effect of pLPD may include
   protein concentration and exposure time. In both studies, protein
   amount was reduced by about half (11% and 9% in this study and Watkins
   & Sinclair study, respectively); more importantly, the time of LPD
   exposure of at least 7 weeks in two studies was assured for the
   exposure of LPD during a complete cycle of male germ cell formation.
   Therefore, we speculate that although the adult offspring in our study
   may also develop metabolic syndrome, but the types of disease and/or
   the degree of progress of disease may differ from study of [157]Watkins
   and Sinclair (2014). More studies are needed to investigate the traits
   during the development of the pLPD offspring. Overall, our findings
   based on enriched genes and pathways suggest that lipid metabolism was
   disturbed in liver and white adipose tissue in the young offspring from
   LPD fathers, which may coordinately increase risk of metabolic
   disorders.

   Almost no overlap was found between the DEGs in iWAT and liver, and it
   is as yet unclear how a pLPD might differentially affect the expression
   of genes in these two offspring tissues. LPD may induce an epigenetic
   change, such as histone modification or RNA induction, in the
   testicular germ cells (TGCs), which may be transmitted to the mature
   sperm. Although it has been shown that LPD does not affect DNA
   methylation pattern in mature sperm, it is unknown whether LPD can
   cause histone modification or alter RNA content in mature sperm
   ([158]Shea et al. 2015). Specific histone modifications and/or RNAs in
   sperm may be transmitted to the zygote, which could induce an
   epigenetic change and/or alter gene expression in the early embryo.
   Because differentiation into tissues such as liver and WAT occurs in
   the post-blastocyst embryo, such changes in the early embryo would be
   expected to affect gene expression in the embryo at a later stage.
   Thus, further analysis is required to dissect the mechanism underlying
   the differential effects of a pLPD on the expression of genes in iWAT
   and liver.

   The molecular mechanism underlying the transmission of pLPD effects is
   not fully understood. It has also been recently reported that sperm
   from mice fed a LPD and those fed a high-fat diet contain higher
   concentrations of tRNA fragments ([159]Chen et al. 2016; [160]Sharma et
   al. 2016), and that injection of sperm small RNAs from HFD-fed mice
   into zygotes causes metabolic disorders and alters the expression of
   genes in metabolic pathways in the offspring ([161]Chen et al. 2016).
   However, the type of epigenetic change induced by LPD in TGCs and
   mature sperm remain to be established. Our previous studies showed
   that, in response to various environmental factors, including heat
   shock, psychological stress, inflammatory cytokines, and pathogen
   infection, the stress-responsive epigenetic regulator Drosophila ATF2
   (dATF2) and the vertebrate ortholog ATF7 are phosphorylated by p38 and
   released from the chromatin, resulting in a reduction in histone H3K9
   tri- and di-methylation (H3K9me3/2), which can be transmitted to the
   subsequent generation in some instances ([162]Maekawa et al. 2010;
   [163]Seong et al. 2011; [164]Seong et al. 2012; [165]Yoshida et al.
   2015). We hypothesize that ATF7 may play role in this transmission
   because it is reported that LPD feeding reduces the concentration of
   glutathione (GST) ([166]Leaf and Neyberger 1947), an abundant
   cysteine-containing tripeptide that removes ROS ([167]Hayes and
   McLellan 1999), which activate p38 ([168]Ito et al. 2006). Hence, it is
   possible that LPD induces phosphorylation of ATF7 via p38, finally
   leading to a change of histone H3K9me3/2, as previously reported
   ([169]Maekawa et al. 2010; [170]Seong et al. 2011; [171]Seong et al.
   2012; [172]Yoshida et al. 2015). In addition, epigenetic changes may be
   induced by the inhibition of α-ketoglutarate-dependent dioxygenases
   that are involved in DNA and histone demethylation, secondary to
   altered levels of fumarate and succinate, key metabolites in the TCA
   cycle ([173]Xiao et al. 2012; [174]Sciacovelli et al. 2016). Future
   experiments are required to dissect the mechanism underlying the
   inheritance of pLPD effects.

   A LPD up-regulates the expression of lipid oxidation genes in the iWAT
   of F0 mice, but down-regulates lipogenic gene expression in offspring
   iWAT. Differences in gene expression in offspring WAT may be the result
   of epigenetic changes in the TGCs of their male progenitors. Although
   epigenetic changes may be similar across all types of cells, including
   iWAT and TGCs, epigenetic changes appear to be induced on different
   genes by LPD in iWAT and TGCs. This may be due to differences in
   chromatin structure, such as differences in heterochromatin in each
   cell type. In addition to pericentromeric and subtelomeric
   heterochromatin, many genes in the euchromatin region also have
   heterochromatin-like structures enriched in H3K9me3/2, and this differs
   among cell types. Therefore, LPD may induce epigenetic changes on
   different genes depending on cell type, despite similar mechanisms of
   epigenetic modification.

Conclusions

   Many studies have shown that parental diet affects the metabolism and
   health of their offspring ([175]Barker et al. 1993; [176]Anderson et
   al. 2006; [177]Ng et al. 2010; [178]Carone et al. 2010; [179]Radford et
   al. 2014; [180]Watkins and Sinclair 2014). To understand the
   relationship between parental diet and offspring health,
   tissue-specific analysis of gene expression changes induced by paternal
   dietary manipulation is essential. The present study of gene expression
   in iWAT and liver may provide useful insight into the molecular
   mechanisms underlying the effect of a pLPD on gene expression patterns
   in the offspring.

Acknowledgments