In order to better understand the genetics of adiposity in a bird that has huge seasonal variation in fat deposition and metabolism, we have selected 7 adipokine genes for examination that have been established to be involved with fat deposition and metabolism. Of the 7 genes (eFABP4, eSCD1, eAdipoQ, eAdipoR1, eAdipoR2, eLept and eLepR), we were not able to detect any eLept mRNA expression in the emu adipose tissue. Based on the lack of Leptin expression in the adipose tissue of zebra finch , jungle fowl , several lines of commercial chickens , rock dove , and quail , Friedman and Seroussi  concluded that Leptin is not expressed in avian adipose tissue. Our result from emu, a ratite that is phylogenetically distant from the birds examined so far, provided support to their observations. In birds, leptin is expressed in brain tissue, adrenal glands and gonads, but is not expressed in the liver and is generally not detectable in the blood. Leptin receptors are predominantly expressed in the pituitary. Seroussi et al  reported that in chicken, ducks, and quail adipose tissue, Lept and LepR were scarcely transcribed, and the expression level was not correlated to adiposity. They proposed that leptin in birds may act as an autocrine or paracrine instead of being a circulating hormone as in mammals. These observations, mostly from chicken studies, allowed Friedman and Seroussi  to speculate that avian adipose tissue does not control appetite, insulin resistance, or inflammation.
We have detected low expression levels of eLepR in emu fat tissue. Expression level was highest in April and stepwise decreased to the lowest level in November, which was opposite to the fat weight gain trend. The amount of back and retroperitoneal fat gain between June and August regressed significantly but negatively on eLepR June expression level. Since there was no eLept expression in emu fat tissue, it seems likely that leptin in emu is still a circulating hormone that affects fat deposition and metabolism. Our phylogenetic analysis found that emu LepR is closer to those of migrating waterfowl than other bird species examined. In mammals, leptin specially repressed the expression of SCD-1 and reduced the accumulation of hepatic triglycerides, cholesterol esters and VLDL synthesis [26, 45, 46]. It is suspected that the role of leptin in governing adipose tissue regulation of appetite and energy expenditure has been altered in birds . Never the less, the relationship between leptin and the loss of appetite over the winter breeding period in emus remains to be studied.
eSCD1 is expressed in emu adipose tissue. There was no seasonal variation in expression except that fat samples collected from females in August had a significant 35-fold increase in expression. Fat weight gained between June and August regressed significantly on June eSCD1 expression level. Individuals that had high fat weight gain would have high eSCD1 expression but low eLepR expression and vice versa for individuals that had little fat weight gain. This would indicate that emu leptin suppresses the expression of eSCD1 as seen in mammals. SCD-1 is also transcriptionally regulated by a number of factors in mammals, including sterol regulatory element-binding protein-1 (SREBP-1) and polyunsaturated fatty acids [47, 48].
SCD-1 is predominately located in the endoplasmic reticulum and catalyzes the rate-limiting step in the cellular synthesis of mono-unsaturated fatty acids from saturated fatty acids [15, 49]. SCD-1 converts the saturated fatty acids, palmitic acid (16:0) and stearic acid (18:0), to generate the mono-unsaturated fatty acids, palmitoleic (16:1 n7) and oleic acid (18:1 n9), which are accumulated as triglycerides in adipose tissues [49, 50, 51, 52]. Oleic acid is the predominant fatty acid in emu adipose tissue. A proper ratio of saturated fatty acids to mono-unsaturated fatty acids contributes to membrane fluidity. In mice, SCD-1, known as a lipid synthesis enzyme, also plays a role in upregulating lipid mobilization through its desaturation product, oleic acid . Specific unsaturated fatty acids are preferentially used during metabolism over saturated fatty acids [54, 55, 56].
Fat storage and usage in birds are mainly for survival, migration and reproductive performance [57, 58, 59, 60]. Catbirds increased adipose storage during spring and autumn migration, showing increased rates of basal lipolysis during migration and tropical overwintering . In our study, emus started gaining fat in April and the rate of gain was maximized between June and August. Fat weight gain between June and August significantly regressed on June eSCD-1 expression. From August to November fat gain was minimal. It was during this period when female emus were getting ready to lay eggs. There was a 35-fold increase in eSCD-1 expression in females in August. They may be optimizing the fatty acid composition of the adipose tissue to get ready for the mobilization of lipids into the ovary for formation of the egg yolk.
FABPs are a family of proteins known as intracellular lipid chaperones that regulate lipid trafficking and responses in cells . FABP gene has been shown to be associated with lipid metabolism (lipogenesis and lipolysis), homeostasis in adipocytes, marbling and back fat deposition [63, 64, 65]. FABP4 is highly expressed in adipocytes and its expression can be highly induced during adipocyte differentiation which is transcriptionally controlled by peroxisome proliferator-activated receptor (PPAR) ℽ agonists, fatty acids, dexamethasone and insulin [61, 66]. It has also been postulated that FABP4 can activate hormone sensitive lipase (HSL) in adipocytes to regulate lipolysis [67, 68]. In chickens, earlier studies that examined the relationship of FABP4 with growth and fat accumulation reported results ranging from no association with fat accumulation in hybrid chickens , significant positive correlation with abdominal fat in Luyuan chickens , to correlation with growth depression in Arbor Acre genotype but strong positive association with growth performance Cobb genotype . In our study, eFABP-4 expression in emu adipose tissue was high both in April and November and relatively low in June and August. Fat gain from April to August regressed positively on April and June eFABP-4 expression, respectively. However, fat gain from August to November regressed negatively on August eFABP-4 expression. From August to November, fat gain was minimal and a couple birds even had negative fat gain. By this time of the year, emus started to draw on the energy from the accumulated fat and the role of FABP4 switched from lipogenesis to lipolysis . eFABP-4 expression was highest in November and this may be an indication that the birds were more and more dependent on fat for energy because they have very little feed intake during breeding. In geese, FABP4 was found to be involved in lipid transportation and metabolic process, follicle development and final egg production. eFABP-4 was upregulated in the laying group compared with the pre-laying group .
Adiponectin has been originally identified as a protein secreted and expressed exclusively in adipose tissue [72, 73]. In chicken, the coding region of chicken adiponectin shares 67% and 65% identity with human and mouse, respectively . In addition, the chicken AdipoR1 cDNA was found to be 80–83% homologous to human, mouse, rat, or pig AdipoR1 cDNA, while the deduced protein sequence was 91% similar to mammalian AdipoR1. Similarly, the chicken AdipoR2 cDNA was 76–78% homologous to human, mouse, or pig AdipoR2 cDNA, while the deduced protein sequence was 82% similar to mammalian AdipoR2 . In comparison, we found that eAdipoQ nucleotide sequence was 82% similar to chicken and 71% similar to human. eAdipoR1 nucleotide sequence was 93% similar to chicken and 83% similar to human AdipoR1. Whereas eAdipoR1 amino acids sequence was 98% similar to chicken and 84% similar to human AdipoR1 amino acids sequence. eAdipoR2 nucleotide sequence was 94% similar to chicken and 81% similar to human AdipoR2 nucleotide sequence. Amino acids sequence of eAdipoR2 was 98% similar to chicken and 82% similar human AdipoR2.
Adiponectin showed many functions like expanding fatty acids oxidation, controlling glucose level and managing receptor activity. In humans, Adiponectin is known to stimulate the expression of FABP . In chickens, adiponectin plays important roles in energy homeostasis, body weight, lipid metabolism, and insulin sensitivity [75, 76, 77]. In emus, eAdipoQ expression was low in April, with a slight increase in June, peaking in August, and back to April level in November. In broiler chickens, Tahmoorespur et al.  showed that AdipoQ expression in adipose tissue was inversely related to chicken abdominal fat deposition levels. Adiponectin has an effect on the impairment of adipocyte differentiation, which contributes to the negative regulation of fat deposition in chicken . From April to November, female emu eAdipoQ expression was significantly higher than male. In adipose tissue of adult chickens, AdipoQ expression is higher in females than males, but AdipoR1 expression was higher in males than females . In female birds, Adiponectin is secreted into the blood from adipocytes with a higher serum level . Emu fat gain from June to August regressed positively on June eAdipoQ expression but fat gain from August to November regressed negatively on August eAdipoQ expression. Similarly, fat gain from June to August also regressed positively on June eAdipoR1 expression while fat gain from August to November regressed negatively on August eAdipoR2 expression. White-throated sparrows increase fat deposits during pre-migratory periods and rely on these fat stores to fuel migration. In the adipose tissue, there was a significant change in the biological control of adipokine expression from pre-migratory conditions to migratory conditions. It was proposed that Adiponectin may play a role in the switch from fat deposition to lipid metabolism as the main source of energy to fuel migratory flight in birds . In emus, eAdipoR1/R2 expression was highest in April, before the birds started gaining fat. eAdipoR1 expression took a dip in June and came back up in August and November. eAdipoR2 expression gradually declined until the lowest level in November. Interestingly, in the oil extracted from emu fat in November, the % of Palmitic Acid (FAC16:0) regressed significantly but negatively on November eAdipoR1 expression. In emus, back fat showed a higher level of protein, cholesterol, C16:1 and the elements K, P, Si, Na, Ca, Mg, Fe, Zn, Se and Cu. Abdominal fat was characterized by higher content of fat and ash, as well as Mn and Ba. Regardless of back or abdominal fat, there was generally high content of MUFA and PUFA. Males have higher content of Si, Ca, Cu, Sr in the adipose tissue than female . In chickens, the most promising candidate genes affecting polyunsaturated fatty acids percentage were FADS2, DCN, FRZB, OGN, PRKAG3, LHFP, CHCHD10, CYTL1, FBLN5, and ADGRD1 .