Abstract

Objective

   The potential for brown adipose tissue (BAT) to be targeted as a
   therapeutic option to combat obesity has been heightened by the
   discovery of a brown–like form of inducible “beige” adipose tissue in
   white fat which has overlapping structural and functional properties to
   “classical” BAT. The likelihood that both beige and brown fat are
   recruited functionally by neural mechanisms, taken together with the
   lack of a detailed understanding of the nature of changes in the
   nervous system when white adipose tissue (WAT) is transformed to brown,
   provides the impetus for this study. Here, we aim to identify whether
   there is a shift in the gene expression profile in neurons directly
   innervating inguinal white adipose tissue (iWAT) that has undergone
   “beiging” to a signature that is more similar to neurons projecting to
   BAT.

Methods

   Two groups of rats, one housed at thermoneutrality (27 °C) and the
   other exposed to cold (8 °C) for 7 days, were killed, and their T13/L1
   ganglia, stellate ganglion (T1/T2), or superior cervical ganglion (SCG,
   C2/3) removed. This approach yielded ganglia containing neurons that
   innervate either beiged white fat (8 °C for 7 days), inguinal WAT
   (27 °C for 7 days), BAT (both 27 °C and 8 °C for 7 days) or non-WAT
   (8 °C for 7 days), the latter included to isolate changes in gene
   expression that were more aligned with a response to cold exposure than
   the transformation of white to beige adipocytes. Bioinformatics
   analyses of RNA sequencing data was performed followed by Ingenuity
   Pathway Analysis (IPA) to determine differential gene expression and
   recruitment of biosynthetic pathways.

Results

   When iWAT is “beiged” there is a significant shift in the gene
   expression profile of neurons in sympathetic ganglia (T13/L1)
   innervating this depot toward a gene neurochemical signature that is
   similar to the stellate ganglion projecting to BAT. Bioinformatics
   analyses of “beiging” related genes revealed upregulation of genes
   encoding neuropeptides proopiomelanocortin (POMC) and calcitonin-gene
   related peptide (CGRP) within ganglionic neurons. Treatment of
   differentiated 3T3L1 adipocytes with αMSH, one of the products cleaved
   from POMC, results in an elevation in lipolysis and the beiging of
   these cells as indicated by changes in gene expression markers of
   browning (Ucp1 and Ppargc1a).

Conclusion

   These data indicate that, coincident with beiging, there is a shift
   toward a “brown-like” neurochemical signature of postganglionic neurons
   projecting to inguinal white fat, an increased expression of POMC, and,
   consistent with a causative role for this prohormone in beiging, an
   αMSH-mediated increase in beige gene markers in isolated adipocytes.

   Keywords: Beige fat, RNA sequencing, Brown adipose tissue,
   Thermogenesis

   Abbreviations: αMSH, alpha melanocyte stimulating hormone; BAT, brown
   adipose tissue; BMI, body mass index; CGRP, Calcitonin gene-related
   peptide; CT, computed tomography; FDR, false discovery rate; IPA,
   Ingenuity Pathway Analysis; iBAT, interscapular brown adipose tissue;
   iWAT, inguinal white adipose tissue; PCA, principal component analysis;
   PET, positron emission tomography; POMC, Pro-opiomelanocortin; RNA-Seq,
   RNA Sequencing; SCG, Superior cervical ganglion; TH, tyrosine
   hydroxylase; UCP1, Uncoupling Protein 1; WAT, white adipose tissue

Graphical abstract

   graphic file with name fx1.jpg
   [33]Open in a new tab

Highlights

     * •
       RNA Seq showed shifts in neuronal gene expression following
       browning of white fat.
     * •
       Gene expression in ganglia projecting to white fat became
       brown-like with beiging.
     * •
       Bioinformatics analyses revealed neuronal gene candidates
       associated with beiging.
     * •
       Prominent gene candidates associated with beiging included POMC and
       CGRP.
     * •
       POMC cleavage product α-MSH caused beiging of cultured fat cells.

1. Introduction

   The rejuvenation of interest in BAT in the last decade has been driven
   by two major revelations. The first was the realization that brown fat
   exists in adult humans based on identification of supraclavicular
   uptake of 18F-FDG co-registered with PET/CT imaging [34][1], [35][2]
   and publication of the first retrospective analyses of 18F-FDG PET/CT
   scans [36][3], which preceded the three landmark publications in 2009
   that demonstrated that BAT is not only present in adult humans [37][4],
   [38][5], [39][6] but exists in amounts inversely proportional to BMI
   and fat mass [40][6]. The second was the discovery of an inducible form
   of brown-like fat, so–called “brite” or “beige” fat. While the
   nomenclature has implications that relate to its functional origins
   with the term “brite” referring to the derivation of brown from white
   fat [41][7] and “beige” being consistent with a unique progenitor
   [42][8], the term beige will be used here.

   It is striking that in its short history, beige fat has been at the
   center of several controversies. As alluded to above, its embryological
   origins have been described differently, and these views are supported
   by what appear to be equally compelling data; so, these disparate
   perspectives remain embedded in our understanding of BAT biology.
   Subcutaneous WAT, particularly in the inguinal depot is shown to
   undergo a process of “beiging” in which thermogenic, mitochondrial
   rich, UCP1-expressing multilocular cells develop within white
   adipocytes [43][9]. On one hand, these have been described as arising
   from the transformation of mature white adipocytes to brown–like/beige
   adipocytes in a reversible manner driven primarily, but not
   exclusively, by elevated levels of noradrenergic stimulation [44][10].
   The equally compelling alternative view is that beige adipocytes are
   derived under a range of conditions from dedicated beige precursors,
   identified by a characteristic complement of genes, that are already
   present in the tissue during development [45][11], [46][12]. More
   recently, another form of beige fat has been identified that is capable
   of thermogenic activity without what has been thought of as its
   essential thermogenic ingredient, UCP1 [47][13]. In addition to
   conflicting views about its origins, the question of the extent to
   which brown-like fat is represented in adult humans as beige or
   “classical” brown fat remains unresolved [48][14], [49][15], [50][16],
   [51][17], [52][18].

   Irrespective of the details of the origins of beige fat, whether it be
   by transformation from mature white adipocytes or derivation from
   dedicated precursors, the fact remains that there is evidence that it
   contributes in a functionally appropriate way to metabolic improvement
   under various conditions in both rodents and humans [53][19], [54][20],
   [55][21]. The question which forms the basis of this study is the
   following. How is it that WAT, which is supported in its primary
   functions of lipid storage and lipolysis by integrated neural circuits,
   becomes functionally more aligned with energy expenditure when mammals
   are exposed to cold and white fat is turned to beige? At face value,
   this transition between fat types would necessarily involve input from
   different central circuits recruited by very different sensory
   modalities controlling such disparate functions.

   A well-established tenet of the organization and function of the
   autonomic nervous system is that the sympathetic innervation of
   particular end organs is associated with a characteristic chemical
   profile which is most pronounced in neurons of the paravertebral
   ganglion innervating that structure [56][22], [57][23], [58][24]. As
   such subpopulations of neurons innervating different target tissues can
   express unique combinations of neurotransmitters, neuropeptides, ion
   channels and receptors and form a “neurochemical signature” which, if
   not unique, is characteristic of that neuron to target organ connection
   [59][23], [60][24], [61][25], [62][26], [63][27].

   In the case of the postganglionic innervation of the iBAT and iWAT,
   these populations of neurons are contained within the stellate ganglion
   and T13/L1 sympathetic ganglia, respectively. The neurochemical profile
   of the stellate ganglion neurons innervating either blood vessels or
   brown adipocytes has been previously established; neurons projecting to
   blood vessels contain TH and neuropeptide Y but not calbindin [64][28],
   whereas viral tracing studies have identified the neurochemical
   signature of neurons projecting to BAT as containing TH and calbindin
   but rarely neuropeptide Y [65][29]. While the chemical code of neurons
   in the T13/L1 ganglion projecting to iWAT is unknown, it is likely
   that, coincident with the transition from iWAT to beige fat after
   exposure to cold or otherwise elevated β-adrenoceptor activation, there
   is a corresponding shift in the neurochemical coding of neurons
   innervating that fat depot.

   In the present experiments, we have utilized cold exposure to generate
   a brown-like phenotype in the iWAT of rats. We hypothesize that when
   the function of iWAT changes from energy storing to energy burning with
   cold exposure, that the neurochemical phenotype of ganglionic neurons
   within the T13/L1 ganglia will shift towards the phenotype seen within
   the BAT-projecting neurons in the stellate ganglion. In order to
   identify the changes in the neurochemical profile of sympathetic
   ganglia, we have utilized next generation RNA sequencing (RNA-Seq) to
   identify differential gene expression. This method allows us to
   describe the nature of the innervation of beiged iWAT and compare it to
   that of neurons projecting to iWAT and iBAT at thermoneutrality.

2. Methods

2.1. Animals and housing

   Male Sprague–Dawley rats were obtained from Monash Animal Services
   (Clayton, VIC, Australia). Rats (n = 17) weighing 200–220 g were housed
   individually under standard housing conditions on a 12 h:12 h
   light/dark cycle, and were provided ad libitum access to water and
   standard laboratory chow (12% kcal from lipids). Rats were either
   housed in a thermoneutral environment (27 °C) or underwent exposure to
   cold (8 °C) for 7 days. All experiments were approved by the Monash
   University Animal Research Platform Ethics Committee (MARP/2013/099).

2.2. Tissue collection

   Rats were deeply anesthetized with an injection of pentobarbitone
   sodium (100 mg/kg, i.p) and then flushed with RNase-free 0.9%
   physiological saline. Stellate ganglia (T1/T2), T13/L1 ganglia, and
   superior cervical ganglia (SCG) were dissected under microscopic
   guidance from a ventral aspect following identification of the
   sympathetic chain. Individual ganglia were immediately stored in RNase
   Later (Thermo Scientific, Wilmington, DE, USA) before RNA extraction.

2.3. RNA extraction

   RNA was extracted using the RNeasy Mini Kit (Qiagen, VIC, Australia)
   and DNA fragments removed with the RNase-Free DNase Set (Qiagen, VIC,
   Australia) from the stellate ganglion, T13/L1 ganglia and SCG.

2.4. RNA-sequencing and bioinformatics analysis

   RNA was transcribed and amplified via Ovation RNA-Seq system V2 and
   Ovation® Ultralow Library Systems V2 (NuGEN Technologies). Sequence
   data was generated by the Medical Genomics Facility at Monash Health
   Translation Precinct (MHTP). Raw reads from the RNA-Seq libraries were
   quality checked using the Fastqc tool (v0.11.5). RNA-Seq libraries were
   mapped to the Rat genome (Ensembl version Rnor_6.0) using the STAR
   software v2.5.2 [66][30], with the number of reads mapped to each gene
   calculated using the featureCounts tool from the Subread package
   [67][31]. Mapping statistics were summarized using MultiQC tool
   [68][32]. Stellate and T13/L1 ganglia samples yielded 19–23 million
   reads that uniquely mapped to the genome, of which 14–20 million reads
   uniquely mapped to the known genes. SCG samples generated 40–64 million
   reads which uniquely mapped to the genome, of which 33–52 million
   uniquely mapped to the known genes. This difference in this sequencing
   depth was accounted for during the normalization of the datasets.

   Differential expression analysis was carried out using edgeR [69][33]
   along with the RUVseq [70][34] to remove unwanted noise in the data.
   The upper quartile normalization method was used and PCA plots showed
   expected clustering of samples into groups ([71]Supplementary
   Figure 1). Significant differentially expressed genes were selected
   using log-fold-change of ±1 and FDR cut-off of 0.05 and were used for
   performing pathway enrichment analysis using IPA (Qiagen
   Bioinformatics, Redwood City, CA).

2.5. Impact of neuropeptides (CGRP and αMSH) on markers of browning

   Cell culture studies: 3T3L1 preadipocytes were cultured in high glucose
   Dulbecco's modified Eagle's medium DMEM (DMEM + GlutaMAX) (Gibco, Life
   Technologies) supplemented with 10% (v/v) FBS (Gibco, Life
   Technologies) and 1% penicillin and streptomycin in a humidified 5%
   CO[2] atmosphere. The day after cells reached confluence, cells were
   differentiated using 3-isobutyl-1-methylxanthine (500 μM; Sigma),
   dexamethasone (5 μM; Sigma, St. Louis, MO, U.S.A.), biotin (0.1 μg/mL)
   and insulin (0.05U/mL) for 3 days, followed by culture with insulin
   (0.05U/mL) in DMEM containing 10% FBS and 1% antibiotics. Prior to drug
   treatment, insulin was removed from the media for 24 h. On the
   experimental day, increasing concentrations CGRP (0, 10^−12, 10^−9,
   10^−6 M; Bachem, Bubendorf, Swirzerland) or αMSH (0, 10^−13, 10^−10,
   10^−7 M; Tocris Bioscience, Bristol, United Kingdom) were applied to
   the mature adipocytes for 6 h. Cells were then washed with ice-cold PBS
   and collected for the assessment of gene expression.

   Gene expression analysis using quantitative real-time PCR: Total RNA
   was extracted from the harvested cells using Trizol reagent (Thermo
   Scientific, Wilmington, DE, USA) and quantity determined using a
   NanoDrop (Thermo Scientific, Wilmington, DE, USA). RNA was reverse
   transcribed into cDNA (iScript Reverse Transcription Supermix for
   RT-qPCR, Bio-rad Laboratories, Hercules, CA, USA), and gene expression
   was determined by quantitative real-time PCR using the TaqMan Universal
   PCR Master Mix and TaqMan Gene Expression Assays (Applied Biosystems,
   Foster City, CA) for Ucp1 (Mm01244861_m1) and 18S (Mm03928990_g1) and
   SYBR Green PCR Master Mix (QuantiNova SYBR Green RT-PCR Master Mix) for
   Ppargc1a (from M. Swarbrick) and 18S (from R. Stark). Amplifications
   were performed using a Real Plex4 Mastercycler (Eppendorf) for Taqman
   assays and CFX384 Touch Real-Time PCR Detection System (Bio-Rad,
   Gladesville, New South Wales, Australia) for SYBR green assays.
   Relative gene expression was calculated using the 2^−^ΔΔCT method.

3. Results

3.1. Gene expression profiles of neurons in the T13/L1 ganglia innervating
iWAT and beiged iWAT – comparison with stellate ganglion projections to iBAT

   In order to assess whether there is a characteristic gene expression
   profile in sympathetic ganglionic neurons that innervate iWAT that
   shifts as this fat depot becomes beige under conditions of cold,
   RNA-Seq was undertaken comparing levels of gene expression in T13/L1
   and stellate ganglia either at thermoneutrality (27 °C) or following 7
   days cold (8 °C) exposure. The T13/L1 ganglia and the stellate ganglion
   are the primary ganglia directing post-ganglionic fibers to iWAT and
   iBAT, respectively. The basic comparisons undertaken in this gene
   analysis were between neurons innervating iWAT versus iBAT, i.e. T13/L1
   versus stellate ganglia at 27 °C [[72]Table 1(A)]; iWAT versus beiged
   iWAT, i.e. T13/L1 ganglia at 27 °C and 8 °C [[73]Table 1(B)]; beiged
   iWAT versus iBAT, ie T13/L1 ganglia at 8 °C and stellate ganglion at
   8 °C [[74]Table 1(D)]. In the latter case, the analysis of stellate
   ganglion at 8 °C was included to compensate for any changes in the
   innervation of iBAT coincident with cooling. The final comparison of
   beiged iWAT versus ganglion deemed unlikely to have a significant
   projection to WAT, ie T13/L1 ganglia at 8 °C and superior cervical
   ganglia (SCG) at 8 °C [[75]Table 1(E)], was also included to control
   for effects on gene expression in sympathetic ganglia that were solely
   related to cooling. All comparisons and the rationale for their
   inclusion in the analyses are summarized in [76]Table 1.

Table 1.

   Differential gene expression comparisons used for RNA-Seq data and
   rationale for their inclusion.

   [77]3.1.
   [78]Open in a new tab

3.2. Changes in gene expression between neurons in ganglia projecting to iWAT
and iBAT under thermoneutral and cold conditions

   Fold changes, which represent the changes in gene expression between
   two variables, in this case ganglia and/or temperature, are shown in
   [79]Figure 1 both as “pie charts” and more conventionally as “smear
   plots”. The latter depicts gene fold change plotted against statistical
   significance value. When rats were housed at 27 °C, gene expression in
   T13/L1 ganglia and stellate ganglia were different with 3227 (1713
   upregulated [12.6%] and 1514 down-regulated [11.1%]) differentially
   expressed genes reflecting the functional differences in the target fat
   depots at thermoneutrality ([80]Figure 1A). Comparing T13/L1 ganglia
   from rats housed under the two different temperature conditions (27 °C
   and 8 °C), there were 3716 (2922 upregulated [21.4%] and 794
   down-regulated [5.8%]) differentially expressed genes, consistent with
   a significant shift (FDR<0.05) in gene expression in the neural input
   to iWAT coincident with beiging at cold temperatures ([81]Figure 1B).
   By comparison, there was a smaller shift of 2211 differentially
   expressed (1723 upregulated [12.6%] and 488 downregulated [3.6%]) genes
   in the stellate ganglion under the two temperature conditions
   ([82]Figure 1C).

Figure 1.

   [83]Figure 1
   [84]Open in a new tab

   Pie charts (left column) showing the percentage of differential gene
   expression with a fold change greater than log1 (FDR ≤ 0.05) and smear
   plots (right column) showing differentially expressed genes. Red, FDR
   ≤0.05; black, FDR >0.05. SCG, superior cervical ganglion.

   One of the most pertinent comparisons was that between T13/L1 and
   stellate ganglia at 8 °C. The results showed that after cold exposure,
   there was a reduction in the differentially expressed genes from
   3227 at 27 °C to 741 (494 upregulated [3.6%] and 247 downregulated
   [1.8%]) at 8 °C ([85]Figure 1D), indicating that with beiging, there
   was a corresponding shift in the gene expression of neurons in T13/L1
   to become more “brown–like” in their gene expression profile.

   In order to further isolate possible changes in gene expression as a
   result of a whole body physiological response to cold exposure, T13/L1
   and stellate ganglion were compared to the SCG, a ganglion with
   sympathetic neurons innervating structures in the head and neck and
   considered to have little involvement in the innervation of major
   adipose tissue depots. When rats were housed at 8 °C, the gene
   expression profile of SCG compared to that in either T13/L1 or stellate
   ganglia was significantly different, with 4284 (1142 upregulated [8.1%]
   and 3142 downregulated [22.4%]) genes differentially expressed in
   T13/L1 and 3729 (798 upregulated [5.7%] and 2931 downregulated [20.9%])
   genes differentially expressed in the stellate ([86]Figure 1E,F). These
   data indicate that under conditions of cold, there are significant
   differences in gene expression between these ganglia showing that the
   results above ([87]Figure 1A) are not a generalized response to cold.

3.3. Candidates involved in the innervation of beige adipose tissue based on
fold change in specific genes and gene pathways

3.3.1. Rationale for screening approach

   Under conditions of cold, comparison of shifts in gene expression
   profiles in the stellate and T13/L1 ganglia relative to those in the
   SCG, which has neuronal projections directed to the head and upper neck
   with a paucity of input to adipose tissue depots, allows an assessment
   of differentially expressed genes that are likely to be due to beiging
   rather than a generalized response to cooling. The rationale for this
   comparison is as follows: Genes that are specifically associated with
   “beiging” need to be both i) differentially expressed in T13/L1 when
   comparisons are made between 27 °C and 8 °C and ii) differentially
   expressed when comparisons are made between T13/L1 and SCG at 8 °C.
   This overlap represents the set of genes that are changed in T13/L1 but
   are not similarly expressed in the SCG. Those genes that are similarly
   expressed in the T13/L1 and SCG at 8 °C would be reasonably designated
   as responding to cold alone (see [88]Figure 2).

Figure 2.

   [89]Figure 2
   [90]Open in a new tab

   (A) Flow chart depicting RNA Sequencing analysis in T13/L1 ganglia at
   27 °C and 8 °C and (B) T13/L1 ganglia and SCG at 8 °C. (B) Venn diagram
   demonstrating overlapping beiging- and cold-related genes in T13/L1
   ganglia. Shaded area in (B) depicts the set of “beiging” related
   differentially expressed genes in T13/L1 ganglia.

3.3.2. Whole genome sequencing

   High-throughput RNA-Seq enabled the identification and measurement of
   transcript expression in response to cold exposure. Using the approach
   described above, we compared the gene expression profiles between the
   T13/L1 ganglia and SCG at 8 °C. Further analysis revealed 3502
   significantly differentially regulated genes (FDR≤0.05) in the T13/L1
   ganglia that were independent of the cold exposure and occurred
   concomitantly with the beiging of iWAT. The 40 most variable of these
   genes (20 upregulated and 20 downregulated) are listed in [91]Table 2.
   It was interesting to note the presence of Ucp1 (8.03 logFC) and Cidea
   (6.28 logFC) in this category; these genes were significantly
   up-regulated in T13/L1, and these changes are concomitant with the
   beiging of iWAT.

Table 2.

   Changes in gene expression in neuropeptides and neurotransmitters in
   T13/L1 ganglia after exposure to cold.
   Gene Gene Description LogFC FDR
   Upregulated genes (LogFC≥1)
   LOC500035 hypothetical protein LOC500035 10.07 4.74E-53
   [92]AC139642.2 5.23 1.32E-33
   [93]AABR07013776.1 8.75 3.80E-28
   [94]AC119603.1 6.33 8.37E-21
   LOC680579 similar to ribosomal protein L14 3.84 7.86E-20
   Gnal G protein subunit alpha L 2.83 9.87E-18
   RGD1564469 similar to Acidic ribosomal phosphoprotein P0 4.96 1.19E-17
   RGD1563581 similar to S100 calcium binding protein A11 (calizzarin)
   2.10 1.56E-17
   LOC100361920 dynein light chain 1-like 3.09 1.16E-15
   Ucp1 uncoupling protein 1 8.03 2.08E-13
   Dbp D-box binding PAR bZIP transcription factor 3.16 2.79E-13
   [95]AABR07030494.1 3.55 3.63E-13
   Fam111a family with sequence similarity 111, member A 4.84 6.30E-13
   Cidea cell death-inducing DFFA-like effector a 6.28 2.28E-12
   Tbca tubulin folding cofactor A 3.14 2.53E-12
   Cox8b cytochrome c oxidase, subunit VIIIb 6.11 3.53E-12
   Lag3 lymphocyte activating 3 4.28 5.17E-12
   Gpr146 G protein-coupled receptor 146 2.75 2.39E-11
   Cebpa CCAAT/enhancer binding protein alpha 4.02 4.13E-11
   Ybx3 Y box binding protein 2 4.26 4.64E-11
   Downregulated genes (LogFC ≤−1)
   RGD1562420 similar to hypothetical protein −5.80 1.29E-33
   Gm1141 predicted gene 1141 −6.19 2.06E-32
   Ppia peptidylprolyl isomerase A −3.08 3.70E-32
   LOC100359951 ribosomal protein S20-like −7.38 5.73E-32
   LOC690468 similar to 60S ribosomal protein L38 −8.29 8.47E-28
   Rpl13 ribosomal protein L13 −3.99 4.87E-27
   LOC689899 similar to 60S ribosomal protein L23a −7.84 6.21E-27
   LOC683961 similar to ribosomal protein S13 −6.00 1.67E-25
   [96]AABR07013410.1 −7.13 1.66E-21
   RGD1564606 similar to 60S ribosomal protein L23a −6.14 5.03E-19
   LOC100364191 hCG1994130-like −2.70 3.37E-18
   Actg1 actin, gamma 1 −3.52 5.40E-17
   Rpl37 ribosomal protein L37 −4.33 7.06E-17
   [97]AC136661.1 −3.88 8.32E-15
   M6pr cation-dependent mannose-6-phosphate receptor-like −3.30 1.20E-14
   [98]AABR07068955.1 −3.79 1.81E-14
   LOC108348118 −6.50 4.62E-14
   RGD1564839 similar to ribosomal protein L31 −4.63 2.36E-13
   Rfk riboflavin kinase −1.83 9.33E-13
   Ankrd13c ankyrin repeat domain 13C −1.75 1.10E-12
   [99]Open in a new tab

3.3.3. Screening known neuropeptides/neuromodulators in paravertebral ganglia

   Shifts in gene expressions for known neuropeptides and
   neurotransmitters present within sympathetic ganglia were established
   ([100]Table 3). The two genes that displayed the greatest significant
   (FDR≤0.05) shift in expression were those encoding calcitonin
   gene-related peptide (CGRP, Calca, up-regulated 2.03 logFC) and
   proenkephalin (Penk, up-regulated 1.90 logFC) ([101]Table 3). There was
   also a 2- and 1-fold increase in the expression of Tac1 and Nos1, the
   genes encoding Substance P and Nitric Oxide Synthase 1, respectively.
   The fold changes in these four peptides were further reflected in the
   total number of reads determined through RNA-Seq, showing significant
   elevation after cold exposure ([102]Figure 3).

Table 3.

   Changes in neuropeptides and neurotransmitters in T13/L1 ganglia after
   exposure to cold.
   Neuropeptide Gene Symbol Name of genes LogFC FDR
   Calcitonin Gene Related Peptide (CGRP) Calca Calcitonin-Related
   Polypeptide Alpha 2.03 6.93E-04
   Enkephalin (ENK) Pomc Proopiomelanocortin 1.48 3.91E-03
   Penk Proenkephalin 1.90 1.56E-03
   Noradrenaline Th Tyrosine Hydroxylase 0.89 5.79E-04
   Dbh Dopamine Beta-Hydroxylase −0.29 2.76E-01
   Ddc Dopa Decarboxylase (Aromatic l-Amino Acid Decarboxylase) −0.79
   3.15E-03
   [103]^aQdpr Quinoid Dihydropteridine Reductase −0.31 2.37E-01
   Substance P [104]^aTac1 Tachykinin, Precursor 1 1.93 5.16E-03
   Vasoactive Intestinal Peptide VIP Vasoactive Intestinal Peptide −0.22
   6.81E-01
   Neuropeptide Y Npy Neuropeptide Y not detected not detected
   Calretinin (CALRET) Calb1 Calbindin 1 −0.29 4.84E-01
   Glutamate Gad1 Glutamate Decarboxylase 1 not detected not detected
   Nitric Oxide [105]^aNos1 Nitric Oxide Synthase 1 (Neuronal) 1.08
   2.5E-02
   Acetylcholine Ache Acetylcholinesterase 0.69 1.02E-03
   Chat Choline Acetyltransferase 1.45 9.46E-03
   Pituitary Adenylate Cyclase-Activating Peptide (PACAP) Adcyap1
   Adenylate Cyclase Activating Polypeptide 1 not detected not detected
   [106]Open in a new tab
   ^a

   Designates cold-related gene i.e. similar expression between T13/L1 and
   SCG at 8 °C.

Figure 3.

   [107]Figure 3
   [108]Open in a new tab

   Number of reads (CPM) of CGRP (Calca), POMC (Pomc), Substance P (Tac1)
   and Nitric Oxide Synthase 1 (Nos1) following RNA Sequencing T13/L1
   ganglia at 27 °C and 8 °C.

3.3.4. Analysis of identified biosynthetic pathways by IPA

   In addition to the unbiased approaches to the analyses of shifts in
   gene expression, analyses of canonical pathways were carried out by
   subjecting the pre-filtered list of “beiging” related differentially
   expressed genes within the T13/L1 ganglia with a FDR≤0.05 to IPA. The
   significance score was expressed as the negative logarithm of the P
   value, indicating the likelihood that the focus genes were assembled
   into a pathway. IPA identified 476 altered pathways in the T13/L1
   ganglia when assessing genes that are associated with the beiging of
   iWAT. Interestingly, there was an overrepresentation of genes
   associated with mitochondrial dysfunction, oxidative phosphorylation,
   fatty acid oxidation, and insulin receptor signaling following
   cold-induced beiging of iWAT ([109]Figure 4A). Analysis of
   differentially expressed genes (FDR≤0.05) with respect to their
   relationship to specific diseases and biological functions revealed a
   substantial enrichment of processes related to the synthesis, oxidation
   and metabolism of lipids, carbohydrates, and amino acids
   ([110]Figure 4B).

Figure 4.

   [111]Figure 4
   [112]Open in a new tab

   Classification of differentially expressed genes in the T13/L1 ganglia
   that are associated with the beiging of iWAT according to the top
   twenty (A) canonical pathways, (B) diseases and functions, and (C)
   predicted upstream regulators. Bars in (A) indicate the likelihood
   [−log(P-value)] that the specific pathway is implicated in regulating
   the beiging of iWAT and the p-value of overlap of molecules detected in
   dataset with those associated with specific (B) diseases and functions
   and (C) upstream regulators.

   IPA also provides an exploratory analysis platform to predict upstream
   regulators where the activation or inhibition may explain the observed
   gene expression patterns. The basis of this analysis is that genes are
   assigned to upstream regulators based on previous experimental evidence
   from the literature. To investigate the possible molecular bases for
   the observed transcriptomic changes, we investigated the predicted
   upstream regulators of the list of differentially expressed genes
   (P ≤ 0.05) within the T13/L1 ganglia that were related to the beiging
   of iWAT. The most significant predicted regulator was leptin (P < 0.05)
   with similarly important representation of the leptin receptor
   ([113]Figure 4C). It is notable that in this leptin and leptin receptor
   network, there were a significant number of molecules from the dataset
   that have been defined as downstream targets. Of these overlapping
   molecules, the majority were upregulated (leptin: 17/18 and leptin
   receptor: 10/11) in response to the beiging of iWAT, suggesting an
   activation of these signaling pathway.

3.3.5. Analysis of impact of candidate neuropeptides on browning in cultured
3T3L1 adipocytes

   In order to assess the impact of CGRP and αMSH on browning, we
   differentiated 3T3L1 preadipocytes into mature adipocytes, followed by
   treatment with the two candidate neuropeptides. Increasing
   concentrations of CGRP did not affect lipolysis (glycerol release) or
   the gene expression levels of Ucp1; however, treatment with αMSH
   resulted in a dose dependent elevation in gene expression, most
   pronounced at the highest concentration (10^−7 M) (see [114]Figure 5).
   Furthermore, assessment of another marker of browning, Ppargc1, also
   revealed a significant elevation following incubation with αMSH at the
   dose (10^−7 M) that achieved maximal expression of Ucp1. In order to
   determine whether there was an interaction between stimulation of the
   beta-adrenergic receptor and treatment with the candidate peptides,
   cells were incubated with isoproterenol. Isoproterenol alone caused an
   increase in lipolysis and browning (Ucp1 expression) following
   submaximal (10 nm) and maximal (10 μM) stimulation. Co-incubation of
   CGRP or αMSH with a submaximal (10 nM) dose of isoproterenol did not
   modulate the impact of the two peptides on browning beyond what would
   be an indicative of an addictive effect, as determined by Ucp1
   expression (data not shown).

Figure 5.

   [115]Figure 5
   [116]Open in a new tab

   (A) Glycerol release (change from baseline), (B) Ucp1 and (C) Ppargc1a
   gene expression in 3T3L1 adipocytes following treatment (6 h) with
   candidate neuropeptides, CGRP (0, 10^−12, 10^−9, 10^−6 M) and/or αMSH
   (0, 10^−13, 10^−10, 10^−7 M). *P < .05, compared to αMSH (0);
   ^#P < .05, compared αMSH (10^−13 M), ***P < .0001, compared to αMSH
   (0); ^###P < .0001, compared αMSH (10^−13 M).

4. Discussion

   These data indicate that with beiging of iWAT, there is a coincident
   shift in the gene expression profile of neurons in paravertebral
   ganglia projecting to this adipose tissue pad. Significantly, this
   pattern of gene expression becomes more aligned with that of neurons
   projecting to BAT rather than to WAT. Of the biosynthetic pathways and
   individual genes coding for known neurotransmitters and neuropeptides
   that are enhanced in their expression in ganglionic neurons following
   cold-induced beiging, POMC and CGRP emerge as prime candidates. Of
   these, POMC is shown to induce the expression of brown-like or beige
   gene markers in cultured adipocytes.

   The extended autonomic neuraxis providing the innervation of peripheral
   tissues via sympathetic preganglionic neurons projecting to the SCG
   (head and neck), stellate ganglion (predominantly brown fat, heart and
   trachea), and T13/L1 ganglia (inguinal fat) is illustrated in
   [117]Supplementary Figure 2. Essentially, this is comprised of
   “premotor” neurons in the brain stem and hypothalamus, sympathetic
   preganglionic neurons in the thoracolumbar spinal cord, and ganglionic
   neurons housed in the ganglia noted above.

   In addition to these major topographical divisions, the present studies
   are based on the premise that there is a basic neurochemical code that
   exists in neurons of the paravertebral ganglia and other parts of
   sympathetic outflow to particular endpoints that is characteristic of
   that end organ. A corollary of this fact is that with the changing
   structure and function of iWAT that occurs with browning in response to
   cold or other stimuli, there is (necessarily) a reorganization of the
   nervous system, including sympathetic postganglionic neurons to
   accommodate that change and facilitate the coordinated physiological
   recruitment of the new “brown-like” phenotype of iWAT.

   The notion that a “chemical code” [118][24] or “neurochemical
   signature” [119][25] exists in postganglionic and even preganglionic
   sympathetic neurons is not new (for review see [120][22]). In fact, the
   idea that a combination of transmitters, modulators, receptors, or
   intracellular signaling entities form a code that might be unique to a
   functional class of neurons has driven, and continues to drive, a
   number of studies of regions deeper in the autonomic neuraxis,
   particularly in the hypothalamus. It seems likely, however, that the
   association between a chemical signature and a functional endpoint is
   most robust in those parts of the nervous system closest the endpoint,
   namely the postganglionic neuron and as such “upstream populations”
   including preganglionic neurons were not assessed for gene changes.
   Postganglionic neurons, for example, projecting directly to salivary
   glands are noradrenergic and enkephalinergic [121][36], [122][37],
   [123][38], whereas those sudomotor inputs to sweat glands are uniquely
   cholinergic and vasoactive intestinal peptide and CGRP-containing
   [124][39], [125][40]. While these few examples and those noted in the
   Introduction are part of a compendium of codes, the broader question of
   whether the chemical phenotype of postganglionic neurons is
   predetermined and directs axons during development to seek out a
   specific end tissue (target independent), or conversely, whether target
   tissues induce a ganglionic neuron to adopt an “appropriate” chemical
   phenotype (target dependent differentiation), remains unresolved.
   Certainly, the last attempt to summarize the evidence supporting either
   of these propositions was inconclusive [126][22]. Importantly, the
   present RNA-seq data, which show a dramatic shift in gene expression
   profiles from 24% differentially expressed genes in the T13/L1 ganglia
   at thermoneutrality between iWAT and iBAT to just 5% after cold
   exposure and browning of iWAT, are consistent with a primary role of
   the target tissue in the determination of neurochemical phenotype. More
   relevant to the question in this study, these data begin to define a
   phenotype that is related to the browning of WAT. In this respect, it
   has been assumed throughout these analyses that the neurons best
   representing the paravertebral ganglionic input to iBAT are those in
   the stellate ganglion. While this is true, it should also be recognized
   that there is input to iBAT from adjacent ganglia in the upper thoracic
   sympathetic chain, namely from T3-T5 [127][35]. It should also be
   recognized that while the gene changes associated with cold exposure in
   the stellate ganglion are likely a reflection of the input of stellate
   ganglionic neurons to the iBAT, neurons in the ganglion also project to
   the heart and trachea. The implication of this is that the magnitude of
   the gene changes is likely to be underestimated given the presence of
   neurons that are less likely to respond to the stimulus.

   The premise that there must be some reorganization of neural pathways
   after beiging of iWAT is supported indirectly by studies showing that
   it can be functionally recruited to promote better metabolic outcomes
   in rodent models and humans (for review, see [128][41]). In other
   words, despite the transition from an adipose tissue pad that is
   primarily involved in lipid storage to one that contributes
   significantly to energy expenditure [129][42] and, after
   transformation, is driven by the sensory modalities of temperature and
   diet rather than stimuli that promote lipolysis, beiged iWAT is
   activated in a physiologically meaningful way. The weight of evidence
   suggests that this must involve central integrated neural circuits
   [130][43], [131][44]. In fact, a recent viral tracing study indicates
   that, coincident with the beiging of iWAT, there are changes in
   discrete supraspinal brain regions [132][45], which, together with the
   shifts in gene expression in post ganglionic neurons described here,
   support the thesis that the beiging of WAT involves a coordinated
   response in the nervous system and targeted adipose tissue depot.

   The strategy employed in the present study involved a comparison of
   RNA-Seq generated data from T13/L1 (to iWAT) and stellate (to iBAT)
   ganglia after cold exposure with gene expression profiles in the SCG,
   which has no substantial projection to WAT, that, taken together,
   increases the likelihood of defining changes that are related to
   beiging rather than to cold exposure itself. Analysis of a set of
   “beiging” related genes provided a list of up- and down-regulated genes
   that predominantly have structural or ribosomal functions, or, in many
   cases, do not have a previously described function. Further assessment
   using IPA enabled a more refined analysis, integration, and
   understanding of the gene expression data following RNA-Seq in the
   context of canonical pathways and specific diseases and functions. In
   this respect, the top ranked canonical pathways were related to
   mitochondrial function (mitochondrial function and sirtuin signalling)
   and substrate metabolism (oxidative phosphorylation and fatty acid
   oxidation). In the same vein, assessment of beiging related genes with
   respect to diseases or functions indicated an enrichment of genes
   involved in synthesis, metabolism, and/or oxidation of carbohydrates,
   fatty acids, and amino acids. Given that the maintenance of neuronal
   activity is critically dependent on an adequate energy supply, which
   functions to regulate pre- and post-synaptic action potentials,
   transmitter release, and post-synaptic currents [133][46], it is not
   surprising that there in an upregulation of processes that will
   ultimately lead to the generation of ATP from oxidative
   phosphorylation.

   Assessment of predicted upstream regulators demonstrated leptin as the
   most likely candidate molecule to explain the observed gene expression
   profiles. Leptin has an undeniable role in the regulation of energy
   homeostasis; however, this is primarily based on evidence derived from
   its function in the hypothalamus, including the arcuate nucleus [for
   review, see [134][47]]. There is accumulating evidence that, besides
   its involvement in appetite regulation, the arcuate nucleus also
   regulates thermogenic activity. For example, viral tracing studies have
   established that leptin receptor expressing neurons in the arcuate
   nucleus are polysynaptically connected to BAT [135][29], and leptin
   administration into the arcuate nucleus results in an elevation in
   sympathetic drive to BAT [136][48], whereas deletion of leptin
   signaling in the arcuate nucleus abolishes sympathetic activation of
   BAT [137][49]. The interaction between leptin and the autonomic nervous
   system was further corroborated in a recent study which demonstrated
   the leptin-induced lipolysis in WAT is directly mediated by sympathetic
   neurons that innervate white adipocytes [138][43]. Importantly, in the
   context of the current findings that beige adipose tissue is associated
   with a coincident shift in neuropeptide gene expression, this recent
   study suggests that the leptin-induced production of noradrenaline from
   sympathetic neurons could act through additional receptors (i.e. non
   β-adrenergic receptors) and highlights the possibility that sympathetic
   post-ganglionic neurons may co-release other neurotransmitters or
   neuropeptides that signal through non-adrenergic receptors.

   The upregulation of Pomc in the targeted gene analysis is consistent
   with the well-established function of POMC, or more specifically
   peptides cleaved from this protein, in the regulation of energy balance
   [139][50]. In this respect, we and others have shown that manipulation
   of melanocortin receptor signaling, either via central administration
   of αMSH, a POMC-derived peptide, or the melanocortin receptor
   antagonist SHU9119, results in a shift in BAT activity [140][51],
   [141][52], [142][53]. In addition, the involvement of POMC neurons in
   the regulation of thermogenesis was also recently shown to be dependent
   on the function of phosphatases within these neurons (PTP1B and TCPTP)
   and co-administration of leptin and insulin to mice exerts a
   synergistic effect by the promotion of browning in iWAT [143][54].
   Consistent with a role of POMC products in the beiging process, albeit
   by a peripheral mechanism, is the recent observation that
   methionine-enkephalin peptides released from group 2 innate lymphoid
   cells can act directly on adipocytes to upregulate Ucp1 expression
   in vitro and promote beiging in iWAT with a corresponding shift in
   oxygen consumption in vivo [144][55]. To our knowledge, however, ours
   is the first report of POMC processing in paravertebral ganglia. This
   combined with the present result of αMSH induced expression of UCP1 and
   PCG1α in cultured 3T3L1 adipocytes, highlights the potential for a
   direct action of POMC products on inguinal WAT to promote browning.

   The targeted gene analysis also demonstrated a significant upregulation
   of CGRP and Substance P, the latter of which occurred as a generalized
   response to cold. In addition, TH was also elevated after cold
   exposure, consistent with the sprouting of (presumably) noradrenergic
   fibers amongst newly formed beige adipocytes in the iWAT, an
   observation made in a previous study [145][45] and in a number of
   different visceral and subcutaneous adipose tissue depots [146][56].
   CGRP and SP-containing nerve fibers are present within peri-ovarian
   WAT, and their density is increased in the vicinity of brown-like
   transformed adipocytes after cold exposure [147][57]. Much of this
   increase in fiber density was reversed by capsaicin treatment,
   consistent with a proportion of these being sensory in nature
   [148][57], a result which is not surprising given the widespread
   representation of both peptide transmitters in sensory projections.
   Consistent with capsaicin treatment in peri-ovarian adipose tissue
   depots, epididymal WAT shows a reduction in CGRP-containing nerve
   fibers, but no significant decrease in TH-containing nerve fibers after
   capsaicin treatment [149][58]. However, after surgical denervation of
   epididymal WAT, which necessarily involves separation of both
   sympathetic and sensory fibres from the adipose tissue pad, there is a
   greater reduction in the number of CGRP-containing nerve fibers
   compared to only the elimination of sensory fibers, and a similar
   decrease in TH-containing fibers [150][58]. The reduction in
   TH-containing nerve fibers and the further reduction in CGRP-positive
   fibers suggests a role for CGRP in sympathetic input. The simplest
   explanation of these data from others and those from the present
   experiments is that the population of CGRP fibers in iWAT includes
   those with parent cell bodies in both the dorsal root ganglia (sensory)
   and the T13/L1 ganglia (sympathetic). In fact, CGRP mRNA is present in
   T13/L1 ganglia as demonstrated with hybridization histochemistry (data
   not shown).

   Intriguingly, UCP1, the enzyme responsible for uncoupling oxidative
   phosphorylation in BAT and, CIDEA, a lipid droplet protein enriched in
   brown adipocyte, are both elevated in T13/L1 ganglia. While the
   upregulation of neuronal Ucp1 and Cidea is interesting and more
   specifically, their association with the beiging process is
   “eye–catching”, their function is more likely to reflect the cold
   stimulus used to induce the beiging process. Further evidence for the
   involvement of UCP1 in response to cold exposure is derived from
   studies in torpid mammals (ground squirrels), in which, coincident with
   reduced body temperatures during hibernation, there is an increase in
   functional UCP1 protein within both the cortex and dorsal root ganglia
   [151][59]. Similarly, carp, a non-mammalian species, have also been
   shown to have increased UCP1 within brain regions after cold
   acclimation [152][60]. These observations are consistent with the idea
   that mammalian hibernators and other non-hibernating species are able
   to use neuronal UCP1 to supply heat, aiding in the maintenance of the
   basal activity of both the central and peripheral nervous systems
   during exposure to cold or low metabolic activity.

   Taken together, these data demonstrate that there is a significant
   shift in the gene expression profile in neurons directly innervating
   iWAT, concomitant with the beiging process, to a signature which is
   more aligned with that of neurons innervating iBAT. Among the genes
   that are changed that may modulate the beiging process are those coding
   for POMC and CGRP, and, of these, based on in vitro experiments, POMC,
   or specifically, its cleavage product αMSH, appears to be the most
   viable candidate contributing to the process. This represents the first
   direct insight into the nature of the innervation of beige adipocytes
   and supports the long-held and general notion that ganglionic neurons
   contain a neurochemical signature that relates them directly to the
   structures that they innervate. The results are also consistent with a
   recently identified central reorganization that occurs coincident with
   the browning of iWAT. Both the changes in central integrative neural
   pathways and gene expression in peripheral ganglia provide a basis for
   the functional recruitment of beiged white adipocytes.

Declaration of interest

   This work is supported by a grant from the NHMRC Australia (APP1082599)
   and a NHMRC Fellowship to BJO (APP1022451) and MJW (APP1077703).

Footnotes

   ^Appendix A

   Supplementary data related to this article can be found at
   [153]https://doi.org/10.1016/j.molmet.2018.01.024.

Conflict of interest

   None declared.

Appendix A. Supplementary data

   The following is the supplementary data related to this article:
   [154]mmc1.docx^ (4.9MB, docx)

References