Coordinated changes in rate of lipid synthesis in mammary, liver and adipose during the transition from pregnancy to lactation
The in-vitro rate of incorporation of 14C-labeled glucose into lipids was used as an indicator of the in-vivo metabolic capacity of mammary, liver and adipose tissue on pregnancy day 20 (P20) and lactation day 1 (L1). Rate of lipid synthesis on P20 was low in mammary tissue, when the mammary gland was not synthesizing milk. With the onset of lactation there was approximately a 10-fold increase in the rate of lipid synthesis in the mammary gland (). This increase in lipid synthesis is needed to supply milk fats to the neonate.
In-vitro rate of incorporation of glucose into lipids.
Although the rate of lipid synthesis was relatively low in liver, there was a significant increase during the transition from late pregnancy to lactation (; P<0.005). We verified these results using acetate as a substrate, and found that the response to the onset of lactation was similar but the rate of lipid synthesis using acetate as a substrate was greater in liver (data not shown). The four-fold increase in lipid synthesis likely reflects the liver's role in providing lipids for synthesis of milk in the mammary gland. The decrease in rate of fat synthesis from P20 to L1 in adipose tissue () indicates a decrease in its ability to store nutrients, thus indicating metabolic changes during this transition insure that nutrients are available for milk synthesis in the mammary gland.
Coordinated changes in gene expression among multiple tissues during the transition from pregnancy to lactation
The orchestrated switch in lipogenesis from adipose tissue to the mammary gland is controlled by the hormonal environment which results in tissue-specific changes in the transcription and activity of enzymes that regulate lipogenesis 
. However, there is limited information on the coordinated transcriptional regulation among the mammary, liver and adipose tissues during the transition from pregnancy to lactation.
In order to characterize the global gene expression patterns in liver, mammary and adipose tissues, total RNA was isolated from mammary, liver and adipose tissue from rat dams on P20 and L1 and gene expression was measured using Rat 230 2.0 Affymetrix GeneChips. Two types of gene expression analysis were performed. Linear regression was used to identify genes that were uniquely up and down-regulated in each tissue following the transition from P20 to L1 (i.e. mammary tissue on L1 versus mammary tissue on 20), and univariate regression was performed to identify individual genes that were commonly up-regulated and down-regulated across all the L1 tissues versus all the P20 tissues 
When nominal P-values were adjusted with false discovery rate, mammary tissue had by far the greatest number of statistically significant changes in gene expression during the transition from pregnancy to lactation (). Only 18 genes were significantly changed at the P<0.001 level in liver, and no genes were significant at this level in adipose tissue. The lack of large transcriptional changes in liver and adipose during this transition was not surprising. We expected moderate changes in gene expression relative to mammary, as the dam is already in a catabolic state in late pregnancy, which is enhanced in these two tissues at the onset of lactation to accommodate the increased energetic demands of milk synthesis 
. Changes in metabolism in liver and adipose during this transition are thus likely to be subtle and may include regulation at the post-transcriptional level.
No. genes differentially expressed between pregnancy and lactation in each tissue and common to all tissues.
There were 68 genes (P<0.001) commonly up- and down-regulated across all three tissues (Supplemental Table S1
). Several of the genes commonly up regulated encode proteins involved in chaperone and stress response, actin cytoskeleton assembly, transcellular/intracellular trafficking as well as neural related signaling. The fact that a greater number of genes were significantly changed when examined for common regulation, was in part, due to the greater number of arrays compared (n
15 across tissues versus n
5 within tissues).
Changes in the molecular signature of the mammary gland during the transition from pregnancy to lactation
Gene sets enriched with genes up regulated during the pregnancy to lactation transition in the mammary gland reflect the turning on of secretory processes in this tissue. These gene sets included the GO: endomembrane system, endoplasmic reticulum, transport, establishment of protein localization
as well as the KEGG pathway SNARE interactions in vesicular transport
(Supplemental Figure S1A
). Up regulated genes were also enriched in the ubquitin related proteolysis
KEGG pathways; enrichment of genes in these sets reflect the important role of ubiquitination pathways in the hormonal regulation of secretory activation in the mammary gland 
and suggest that post-translation regulation is also needed for initiation of lactation. The mTor signaling pathway
was enriched with genes up regulated during the transition from pregnancy to lactation (Supplemental Figure S1B
). mTOR plays a central role in signaling caused by nutrients and mitogens. mTOR positively regulates translation and ribosome biogenesis while negatively controlling autophagy, and is believed to set protein synthetic rates as a function of the availability of translational precursors 
Gene sets enriched with genes down regulated in the mammary gland during the pregnancy to lactation transition included GO: autophagy
and aminopeptidase activity
, as well as the KEGG pathways: N-glycan degradation, ABC transporter activity
and PPAR signaling
(Supplemental Figure S1
). Genes enriched in the PPAR signaling pathway encode proteins involved in lipid transport, lipid metabolism, particularly beta-oxidation and 2 nuclear receptors: RXR and the orphan receptor NR1H3 (aka LXR-alpha) that activates RXR (see Supplemental Table S2
for genes in this pathway). This molecular signature is consistent with the function and activity of the mammary gland during lactation, i.e. a down regulation of catabolic activity and sequestering of substrates to be used for milk synthesis through the down regulation of membrane transporters. The enrichment of down regulated genes in GO–regulation of cell shape/cell morphogenesis, Rab/Ras GTPase binding
, and activation of JNK activity
–is indicative of completion of mammary differentiation at the onset of lactation.
Changes in the molecular signature of the liver during the transition from pregnancy to lactation
Gene sets enriched with genes up regulated in liver were related to P450 pathways which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids (Supplemental Figure S2
). Genes within these genes sets were found to encode proteins involved in synthesis of estrogen and retinoic acid, conversion of cholesterol to bile acids, or function within the arachidonic acid pathway. Enrichment of up regulated genes in glutathione transferase activity
may be indicative of the increase in metabolic activity of liver during the pregnancy to lactation transition. Enzymes with this activity participate in the detoxification of reactive electrophilic compounds that are often by-products of metabolism.
The adipocytokine signaling pathway
was enriched with 8 genes (P<0.05, nominal p-value; Supplemental Table S3
) down regulated in liver at the onset of lactation. Genes within this set included: LEPR (the leptin receptor), PPARGC1A, CPT2, CPT1A, and PRKAB1. PPARGC1A encodes a transcriptional coactivator that regulates genes involved in energy metabolism. CPT2 stimulates beta oxidation of fatty acids, and CPT1A encodes a key enzyme involved in carnitine-dependent transport of long-chain fatty acids across the mitochondrial inner membrane and its deficiency results in a decreased rate of fatty acid beta-oxidation. PRKAB1 encodes a protein that positively regulates AMP-activated protein kinase (AMPK), an important energy-sensing enzyme that monitors cellular energy status.
Leptin plays a role in regulating food intake and adiposity centrally 
but also acts peripherally to exert an antilipogenic, pro-oxidative action on its peripheral nonadipose target tissues, by lowering expression of lipogenic transcription factors, such as sterol regulatory element-binding protein (SREBP)-1c in liver and peroxisome proliferator-activated receptor (PPAR)-γ2 as well as lipogenic enzymes, including acetyl CoA carboxylase and fatty acid synthase (for review 
). This transcriptional signature is likely a homeorhetic adaptation that reduces breakdown of fatty acids in the liver so that fats can be spared for milk synthesis in the mammary gland, and may be partly responsible for the 4-fold increase in the rate of lipogenesis we report for the liver ().
The gene set transmembrane receptor activity
, was enriched with 33 genes (P<0.05, nominal p-value; Supplemental Table S4
; Supplemental Figure S2
) down regulated in liver during the transition from pregnancy to lactation. Many of the genes within this set encoded proteins involved in feeding behavior, satiety and homeostasis, and included: LEPR, PRLR (prolactin receptor), INSRR (insulin-receptor related receptor), PPRY1 (receptor for neuropeptide Y and peptide YY), GNAT2 (a G-protein involved in transmission of visual impulses), GRPR (gastrin releasing peptide receptor), GPR50 (an orphan receptor that heterodimerizes with melatonin receptor), HTR7 (5-hydroxytryptamine (serotonin) receptor 7) CHRNA2 (a cholinergic receptor), HRH1 (a histamine receptor), and GFRA3 (receptor for neurotroph ARTN, artemin). Expression of many of these genes are classically associated with the central and enteric nervous system and regulate energy balance and feeding behavior 
, thus providing clues to the endocrine and neuroendocrine responses that need to be investigated to fully understand the homeorhetic response to lactation.
Changes in the molecular signature of the adipose tissue during the transition from pregnancy to lactation
Adipose tissue has traditionally been viewed as an inert energy storage tissue containing a fixed number of adipocytes, but now it is designated as a very dynamic endocrine organ with pleiotropic functions 
. Adipocytes secrete factors that play a central role in the regulation of energy balance, immunological responses and inflammation 
. The enrichment of the complement and coagulation cascade
KEGG pathway and the GO complement activation
and activation of plasma proteins
(Supplemental Figure S3
) during the transition from pregnancy to lactation indicate that both innate immunity and the complement system are up regulated, and suggests that an inflammatory response may be activated at the onset of lactation 
. The enrichment of up regulated genes within sets related to intracellular transport, membrane trafficking, and secretion endoplasmic reticulum, Golgi apparatus, melanosome
and vesicle mediated transport
are likely due to the increased rate of lipolysis and transport of stored fats out of adipose tissue into circulation to supply energy and fats needed for milk synthesis.
GO enriched with genes down regulated during the pregnancy to lactation transition were overwhelmingly related to muscle contraction sacromere, myofibrils, and cytoskeleton. Twelve genes enriched the contractile fiber
gene set (P<0.05, nominal P-value; Supplemental Table S5
) and included: ACATA1 (actin alpha 1), MYH3 (myosin), and TNNT2 (troponin). Interestingly, insulin-induced translocation of glucose through GLUT4 protein is dependent on microtubules, and without microtubules glucose transport is highly diminished 
, suggesting that the decrease in expression of cytoskeletal genes in adipose tissue helps to spare glucose use by peripheral tissues. Interestingly, the transcriptome pattern revealed in adipose tissue during the transition from pregnancy to lactation showed a striking similarity to that observed with long-term caloric restriction. The molecular signature of adipose tissue between rats exposed to long-term caloric restriction and control rats revealed that 120 out of 345 differentially expressed genes were associated with metabolism (carbohydrate, lipid, amino acid and central aspects of energy metabolism) 
, and the other 108 differentially expressed genes were classified as within ontology related to the cytoskeleton, ECM, inflammation and angiogenic activities 
Gene sets of commonly up and down regulated in mammary, liver and adipose tissue during the pregnancy to lactation transition
Not surprising, the majority of the gene sets enriched with genes commonly up regulated among all three tissues were related to metabolic processes (primary metabolic process, macromolecular complex assembly, cellular protein metabolic process) (). Interestingly the most highly enriched KEGG pathways with commonly up regulated genes were Pathogenic Escherichia coli infection – EHEC and Pathogenic Escherichia coli infection- EPEC. Genes within these pathways were involved in the toll-like receptor pathway and adherens junctions. Gene sets enriched with commonly up regulated genes, also indicated apoptosis/programmed cell death was being inhibited during the transition from pregnancy to lactation in all three tissues.
It is interesting to point out that ATRN, attractin, was one of the most significantly commonly up regulated genes (Supplemental Table S1
). Attractin is a low affinity receptor for agouti, and both of these molecules regulate pigmentation. Agouti is an antagonist for the melanocortin receptors, MC1R and MC4R 
,  
. Chronic antagonism of the cutaneous MC1-R by Agouti results in yellow fur and Agouti competition at the hypothalamic MC4-R results in obesity. Attractin (mahogony) knock out mice have increased basal metabolic rate and activity 
. These data suggest that inhibition of melanocortin signaling through ATRN may be a major homeorhetic adaptation for lactation 
In order to gain insight into what is stimulating these changes, we took a closer look at genes that were clustered into the gene ontology (GO: 0003700) transcription factor activity
(Supplemental Table S6
). There were 112 genes commonly up regulated among mammary, liver and adipose in this category. Many of these genes encoded proteins that functioned: to regulate metabolism (NR1I3, PTRF, MLX), as coactivators for nuclear receptors (NCOA1, NCOA3, NCOA4, MED13L, POU2F), or to transcriptionally regulate progression through the cell cycle (ARID4A, HELLS, MCM6). However, most interesting to us, was the common up regulation of 2 core molecular clock genes, ARNTL (aka BMAL1) and CLOCK as well as the up regulation of SREBF2. ARNTL and CLOCK gene products make up core clock elements that generate circadian rhythms. Heterodimers of ARNTL/CLOCK gene products activate transcription of numerous target genes that in turn show circadian patterns of expession either directly via E-box regulatory element in their promoter regions, or indirectly by other transcription factors whose expression is under clock control 
. SREBF2 encodes a sterol receptor binding protein transcription factor that activates enzymes important to de novo lipid synthesis.
There were 97 genes commonly down regulated among mammary, liver and adipose tissues that enriched the transcription factor activity
GO gene set (Supplemental Table S7
). Many of the products of these genes regulate developmental processes and included several classes of homeobox genes, the homeobox genes (HOXC9, HOXA5), sry (sex determining region) homeobox genes (SOX4, SOX10, SOX 15, SOX 21), and the Iroquois homeobox genes (IRX1, IRX3, IRX4, IRX5), a signature that indicates completion of differentiation at the onset lactation. There were also three genes within the transcription factor activity ontology cluster that were associated with the setting of the intracellular molecular clock and included: NR1D1, DBP, and BHLHB2. These three genes also enriched the GO Rhythmic process
(GO:0048511; ; Supplemental Table S8
) gene set that additionally included HTR7 and OPN4. HTR7 encodes a serotonin receptor (5-hydroxytryptamine receptor 7). Serotonin regulates tissue metabolism as well as entrains circadian rhythm phases 
. OPN4 encodes a photoreceptor, melanopsin, required for regulation of circadian rhythm. It is intriguing that the expression of a retinal associated gene is regulated in non-ocular tissues, and suggests that another role may be attributed to melanopsin: regulator of peripheral tissue rhythms.
Other gene sets enriched with genes commonly down regulated among the three tissues were related to perception and transduction of external stimuli, light and taste, and included the GO sets, Sensory perception of taste, Phototransduction
, and Detection of stimulus
(). There were 24 commonly down regulated genes (P<0.05 adjusted, within common genes; Supplemental Table S9
) that enriched the gene set Sensory perception of light stimulus
. Genes within this set encoded proteins classically known to receive, integrate and transmit light stimuli. Interestingly, a mutation of one of the genes in this set, BBS7, is associated with Bardet-Biedl syndrome. This syndrome is a genetically heterogeneous disorder characterized by severe pigmentary retinopathy and early onset obesity. Secondary features include diabetes mellitus, hypertension and congenital heart disease 
. Mice with knockout of this gene are not responsive to leptin signaling and have decreased expression of, the α-MSH precursor, pro-opiomelanocortin 
Molecular signatures in peripheral tissues suggest that metabolic changes may be regulated by changes in molecular clocks
Our data show that multiple pathways and gene sets related to energy homeostasis are changed in peripheral tissues at the onset of lactation. Molecular signatures common to all the three tissues showed enrichment of gene sets associated with reception, integration and response to environmental and internal stimuli that are normally associated with the central nervous system. Transcriptomes of all three tissues also showed changes in molecular clock genes during the transition from pregnancy to lactation (; Supplemental Table S10
Changes in molecular signatures of circadian clocks genes and genes that regulate fatty acid synthesis during the transition from pregnancy to lactation.
Circadian rhythms coordinate endogenous processes and circadian clocks are synchronized (entrained) to the external world, principally via light-dark cycles. Synchronization of circadian clocks to the external world enables organisms to anticipate and prepare for periodic and seasonal changes in their environment 
Daily and seasonal rhythms are coordinated in mammals by the master clock that lies in the suprachiasmatic nuclei (SCN) of the hypothalamus. Internal and external synchronizing factors affect the autoregulatory transcription–translation feedback loop of core clock genes that generate circadian rhythms 
. Molecular clocks are also distributed in every organ and perhaps in every cell of the organism 
. These tissue clocks are synchronized by endocrine, autonomic and behavioral cues that are dependent on the SCN, and in turn they drive the circadian expression of local transcriptomes, thereby coordinating metabolism and physiology of the entire organism.
Intracellular circadian rhythm generation occurs through an auto regulatory transcription–translation feedback loop 
. The positive loop consists of ARNTL (aka BMAL1) and CLOCK gene products (and NPAS2 outside of the SCN), and the negative loop consists of the PER and CRY gene products 
. ARNTL forms heterodimers with CLOCK and NPAS2; these complexes function as transcription factors that drive rhythmic expression of numerous output genes including their own repressors, PERS and CRYS 
. ARNTL expression is also regulated by Rev-erbα (NR1D1) and Rorα (RORA) that respectively repress or activate ARNTL transcription 
. The genes that RORA and NR1D1 regulate are often coordinately regulated by these two molecules, and crosstalk between RORA and NR1D1 likely acts to fine-tune their target physiologic networks, such as circadian rhythms, metabolic homeostasis, and inflammation 
. Additionally, the basic helix loop helix transcription factors BHLH2 and BHLH3, aka Dec1 and Dec2, repress Clock-Arntl promoter activation 
. CSNK1E, casein kinase 1, epsilon also acts as a negative regulator of circadian rhythmicity by phosphorylating PER1 and PER2 
During the transition from pregnancy to lactation there was a significant (P<0.05) induction of ARNTL (4 fold), CLOCK (1.4 fold), NPAS2 (5 fold) and RORA (3 fold) in the mammary gland (). Although an important clock gene, NPAS2, was not well measured on the gene expression arrays 
, we determined the expression levels of this gene by qPCR (). A significant decrease in expression of genes that generate the negative limb of circadian rhythms occurred in PER1, CRY1, NR1D1, BHLHB2 and CSNK1E, respectively, by 40%, 60%, 60%, 70% and 80% during the transition from pregnancy to lactation in the mammary gland. Significant expression changes in ARNTL, RORA, NR1D1, BHLHB2, CSNK1E and DBP during the pregnancy to lactation transition were confirmed and validated for mammary using qPCR (). It is important to note that since we collected the tissues at the same time of day on P20 and L1, these differences in gene expression are not due to sampling times; rather, differences are indicative of changes in amplitudes and/or patterns of genes that show circadian rhythms.
When expression statistics of core clock genes were examined for common up or down regulation during the transition from pregnancy to lactation, these data suggested that ARNTL, CLOCK and RORA genes were significantly induced, and expression of BHLHB2 and NR1D1 were significantly reduced in all three tissues (adjusted P<0.05; across the 15 arrays on P20 and L1; Supplemental Table S10
). However when changes in expression of genes were examined within liver and adipose, only ARNTL was found to be significantly induced in the liver (1.3 fold). The fact that subtle changes in expression in core clock genes can only be picked up when arrays are examined across all three tissues may be due to the fact that the majority of transcriptome changes in these tissues occurs in an earlier phase of reproduction. The intimate interaction of metabolism and circadian clocks in peripheral tissues, suggests that the subtle changes evident in transcriptomes picked up when examined across the three tissues have a real biological significance. Further, the fact that the dam switches to a “catabolic condition” in late pregnancy to support rapid fetal growth 
, which is geared up with the onset of lactation, suggests that in order to capture a window of large transcriptional changes in circadian clock and metabolic genes in liver and adipose we would need to compare non-pregnant and/or early pregnant animals with late pregnant and/or lactating animals.
Thus in general our data showed an induction of expression of the positive limb core clock genes and a suppression of expression of the negative limb of core clock genes. The transcriptional signature of the molecular clock suggests that the basal level of output genes that show a circadian rhythm of expression may be up-regulated at the onset of lactation, particularly in the mammary gland. Global temporal expression profiles of tissues, including liver, adipose, heart and SCN showed that a significant portion of the genome is under circadian control (in mammals, approximately 3–10% of all detectably expressed transcripts) 
. Tissue-specific clock-controlled genes were found to be involved in rate-limiting steps of processes critical to the function of the organ. For example, in the liver coordinated circadian expression of genes encoding components of sugar, lipid, cholesterol and xenobiotic metabolic pathways were reported 
. Transcription factors and enzymes involved in fatty acid synthesis including SREBF1, acetyl-CoA carboxylase (ACACA), fatty acid synthase (FASN) have also been reported to show circadian patterns of expression 
. Thus it is plausible that a circadian clock in mammary gland controls a unique set of genes important for its major function, lactation.
We examined the expression changes in genes involved in fatty acid synthesis during the transition from pregnancy to lactation in all three tissues in relation to changes in core clock genes (). The fatty acid synthesis genes (SREBF1, ACYL, ACACA, FASN) were selected based on their importance in milk fat synthesis and the fact that these genes respond to circadian entrainment 
. The induction of core clock genes ARNTL, CLOCK, NPAS2, and RORA corresponded to the up regulation of genes that regulate fatty acid synthesis in mammary tissue during the transition from pregnancy to lactation. Significant changes in fatty acid synthesis genes were confirmed and validated in mammary tissue using qPCR (data not shown). It is interesting to speculate that the up regulation in expression of genes that regulate fatty acid synthesis and that have been shown to have diurnal patterns of expression are due to changes in molecular clocks at the onset of lactation in the mammary gland.
Although we only examined one time point across 2 days, others have shown that there are amplitude changes in core circadian clock genes in the mammary gland during the transition from pregnancy to lactation. Specifically, in mouse dams there is an increase in the amplitude of expression of Bmal1 (Arntl) and decrease in amplitude of expression of Per2 during the transition from pregnancy to lactation 
. Preliminary work in our lab supports that mammary tissue in fact possesses a functional clock that can be reset by external signals. We tested the ability of a mammary epithelial cell line, MAC-T, to be synchronized in culture by serum treatment. Our studies showed that treating mammary epithelial cell cultures with serum for 2 hrs initiated a circadian pattern of expression of Bmal1
(ARNTL) as measured with qPCR every 4 hrs for 48 hrs.
Interestingly, homozygous Clock
mutant mice, which have a genetic mutation that disrupts circadian rhythms, exhibit severe alterations in energy balance, with a phenotype associated with metabolic syndrome, including obesity, hyperlipidemia, hepatic steatosis, high circulating glucose, and low circulating insulin 
. Offspring of these Clock
mutant mice fail to thrive, suggesting that their milk production may not be adequate enough to nourish their young 
. The effect of circadian clocks on milk production is evident in both the diurnal variation in milk composition 
as well as the photoperiod effect on milk quality and quantity in cattle and other ruminants 
. These studies have shown that altering the photoperiod in cows influences milk production and composition and results in changes in circulating levels of hormones known to be important for milk production.
We hypothesize that the master clock modifies peripheral clocks and hormonal levels at the onset of lactation in order to coordinate the changes needed to stimulate lactogenesis and accommodate the increased metabolic demands of milk synthesis. Following modification of the clocks there is a change in the mammal's metabolome that results in the partitioning of nutrients to the mammary which in turn are used to synthesize milk (). Based on this hypothesis we believe that environmental inputs and physiological inputs received through the master clock in the suprachiasmatic nucleus (day light, food availability, metabolic stores, social cues, stress, etc.) can profoundly influence milk production and composition.
Figure 5 Schematic of how molecular clocks affect metabolic output, as modified from .
Multiple pathways and gene sets related to energy homeostasis are changed in mammary, liver and adipose tissues during the transition from pregnancy to lactation. Gene sets enriched with genes up regulated during the pregnancy to lactation transition in the mammary gland reflect the turning on of secretory processes in this tissue and the down regulation of catabolic processes. Gene sets enriched with genes up regulated in liver were related to P450 pathways which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids, and the transcriptional signature of genes down regulated in liver at the onset of lactation suggest that there is a reduction in breakdown fatty acids, so that fats can be spared for milk synthesis in the mammary gland. There was a similarity between the molecular signature of adipose tissue at the onset of lactation and adipose tissue from rats exposed to long-term caloric restriction, in particular the enrichment of up regulated genes in inflammation related genes sets and the enrichment of down regulated genes in cytoskeletal and ECM gene sets. The majority of the gene sets enriched with genes commonly up regulated among all three tissues were related to metabolic processes. Genes commonly down regulated among the three tissues were related to perception and transduction of external stimuli, light and taste as well as rhythmic processes.
Molecular signatures of mammary, liver and adipose were also enriched with gene sets classically associated with central nervous systems reception, integration and response to environmental and internal stimuli. In particular we found that core clock genes were commonly changed among the three tissues at the onset of lactation. These signatures illustrate the complexity of homeorhetic adaptations as well as the role of the nervous system in orchestrating the response, and suggested that changes in multiple tissues may be coordinated by changes in molecular clocks. We envision that environmental gene interactions leading to taxonomic variation in milk composition are mediated through changes in molecular clocks, which in turn mediate changes in the animal's transcriptome, proteome and metabolome, and thus metabolic output, milk.