Recent advances in the understanding of how neuropeptide Y and α-melanocyte stimulating hormone function in adipose physiology

ABSTRACT

Communication between the brain and the adipose tissue has been the focus of many studies in recent years, with the “brain-fat axis” identified as a system that orchestrates the assimilation and usage of energy to maintain body mass and adequate fat stores. It is now well-known that appetite-regulating peptides that were studied as neurotransmitters in the central nervous system can act both on the hypothalamus to regulate feeding behavior and also on the adipose tissue to modulate the storage of energy. Energy balance is thus partly controlled by factors that can alter both energy intake and storage/expenditure. Two such factors involved in these processes are neuropeptide Y (NPY) and α-melanocyte stimulating hormone (α-MSH). NPY, an orexigenic factor, is associated with promoting adipogenesis in both mammals and chickens, while α-MSH, an anorexigenic factor, stimulates lipolysis in rodents. There is also evidence of interaction between the 2 peptides. This review aims to summarize recent advances in the study of NPY and α-MSH regarding their role in adipose tissue physiology, with an emphasis on the cellular and molecular mechanisms. A greater understanding of the brain-fat axis and regulation of adiposity by bioactive peptides may provide insights on strategies to prevent or treat obesity and also enhance nutrient utilization efficiency in agriculturally-important species.

KEYWORDS:

• adipocyte
• alpha-melanocyte simulating hormone
• hypothalamus
• neuropeptide Y

Introduction

Many different factors are involved in regulating adipose tissue physiology. Because white adipose tissue is comprised of many different cell types, including preadipocytes, adipocytes, blood cells, immune cells, and mesenchymal stem cells among others, and is innervated by the sympathetic nervous system and is highly vascularized, there are multiple routes through which factors can act on cells in the adipose tissue (neural, endocrine, paracrine, autocrine, etc.) and numerous cells that play a role in the acquisition and storage of energy, including the adipocyte that stores triacylglycerols and cholesterol esters in lipid droplets that occupy most of the volume of the cell. The majority of studies in adipose physiology focus on mammalian models, with fewer studies in birds, however, with the ease of availability of chickens and the bulk of avian research being conducted in the domestic chicken, it has emerged as an attractive model for understanding the relationship between appetite regulation and adipocyte lipid metabolism.

Two peptides that play a major role in regulating food intake are neuropeptide Y (NPY) and α-melanocyte stimulating hormone (α-MSH). These factors are regarded as neurotransmitters, however because most studies do not distinguish between actions in vivo that originate centrally or peripherally (both factors and their receptors are produced in adipose tissue and both are found in the circulation), they will be referred to as “peptides” for the remainder of this review. NPY is highly orexigenic in many species, and its receptor sub-types are well characterized in mammals,¹ while α-MSH is anorexigenic and is part of the melanocortin system. The two peptides are also associated with each other, with the NPY system acting to regulate the activity of α-MSH in the hypothalamus by decreasing its phosphorylation capacity and amount present in the paraventricular nucleus (PVN).² Both peptides also affect adipose physiology; NPY mainly affects adipogenesis, while α-MSH primarily affects lipolysis.

Neuropeptide Y, a 36 amino acid peptide, was first isolated from the pig brain,³ and its orexigenic properties have been studied in a multitude of species, including rodents,⁴ rabbits,⁵ teleost fish,⁶ white-crowned sparrows,⁷ and chickens.^8,9 It is involved in promoting adipogenesis in mouse 3T3-L1 cells,¹⁰ and chicken preadipocytes.¹¹

Adrenocorticotropic hormone (ACTH) is a peptide derived from pro-opiomelanocortin (POMC), and α-MSH is then further derived from ACTH, as the first 13 amino acids of ACTH are what make up α-MSH.¹² It is involved in energy balance and is an agonist to 4 out of 5 melanocortin receptors (MCR).¹³ It is also anorexigenic in multiple species, including rats,¹⁴ goldfish,¹⁵ and chicks.¹⁶ A lipolytic effect mediated through MC5R was reported in mouse 3T3-L1 cells,¹⁷ but is yet to be determined in chicken or any other avian species.

Although our group recently published a review on hypothalamus-adipose tissue crosstalk with a focus on NPY,¹⁸ many developments have since been reported on the study of appetite-regulating factors and their role in adipose tissue metabolism. The purpose of this review is to incorporate recent information about the actions of NPY and α-MSH in adipose tissue, with an emphasis on the molecular mechanisms. Additionally, because the information reviewed is in the subject of adipose physiology; adipogenesis, adipose tissue expansion, and models used for studying adipogenesis are also discussed.

Adipogenesis and adipose tissue expansion

Adipose cells originate from mesenchymal stem cells, which are found in the adipose tissue.¹⁹ Although accepted as being of mesenchymal origin, adipose cells are also derived from areas outside of the mesoderm such as the neural crest (NC).^20,21 Mesenchymal stem cells can differentiate into a multitude of different cell types, including adipocytes, chondrocytes, osteoblasts, and myoblasts^22-28 as well as neuron-like cells,^29-31 with considerable transcriptional regulation by a diverse array of factors to determine the route of differentiation. Adipogenesis encompasses the many steps of differentiation; from a stem cell being determined to become a preadipocyte to the preadipocyte during the early (clonal expansion) and late (terminal) stages of differentiating into the fully mature adipocyte.³² For the remainder of this review, differentiation will refer to terminal differentiation of the adipocyte, unless stated otherwise.

Transcription factors and adipocyte differentiation

There are several major transcription factors that have been widely studied for their role in adipogenesis, with mechanisms underlying their actions well-understood in mammals. The transcription factors discussed herein and the times during differentiation that they are expressed are described in Table 1.

Table 1. Transcription factors involved in adipocyte differentiation.

Transcription Factor^1	Time when expression is induced during differentiation	Citation
PPARγ	Early and throughout	^34-36
C/EBPβ	Early	^49,50,52
C/EBPδ	Early	^49,50
C/EBPα	Following expression of C/EBPβ and C/EBPδ	^49-51
SREBP	Early	^44,48

Note.

¹ PPARγ: Peroxisome proliferator-activated receptor γ; C/EBPβ, C/EBPδ, and C/EBPα: CCAAT/Enhancer Binding Protein β, δ, and α, respectively; SREBP: sterol regulatory element-binding protein

Peroxisome proliferator activated receptor gamma (PPARγ) is part of the nuclear receptor superfamily, and is known as the “master regulator” of preadipocyte differentiation,³³ and is necessary for development of adipose tissue.³⁴ Expression is induced early in differentiation,³⁵ and induced expression in fibroblasts can cause differentiation into adipocytes,³⁶ as well as trans-differentiation of myoblasts into adipocytes.³⁷ Expression after adipocyte maturity is also important, as 3T3-L1 cells containing a dominant-negative PPARγ, a mutant form that is able to bind DNA but not ligands, are induced to de-differentiate.³⁸ Knockout (KO) of PPARγ in adipocytes in vivo leads to adipocyte death due to necrosis, which then stimulates the generation of new adipocytes,³⁹ cells that the authors hypothesized were derived from fibroblast-like cells; adipose progenitor cells are widely distributed in connective tissue and possess the ability to proliferate and differentiate into adipocytes.^39,40

Another transcription factor, sterol regulatory element binding protein (SREBP), plays a role in adipogenesis. Its main function is to control expression of enzymes involved in cholesterol, fatty acid, triacylglycerol, and lipid synthesis, as reviewed.⁴¹ SREBP 1 and 2 activate transcription of more than 30 genes that are related to lipid synthesis and transport, particularly in the liver.^42,43 Diet-derived polyunsaturated fatty acids suppress hepatic expression of genes contributing to fatty acid synthesis, with SREBP 1 and 2 purported to be involved.⁴³ SREBP increases the activity of PPARγ by producing endogenous ligands that bind to the ligand-binding domain of PPARγ, as conditioned media from NIH 3T3 cells expressing SREBP activated PPARγ;⁴⁴ although it was not specified which specific endogenous ligands were produced, authors noted that secreted lipid(s) in the fatty acid family serve as endogenous ligands of PPARγ.^44-47 SREBP is also involved in insulin-mediated signaling to induce lipid accumulation by increasing expression of the lipogenic genes fatty acid synthase (FAS) and lipoprotein lipase (LPL).⁴⁸

The CCAAT enhancer binding protein (C/EBP) family of transcription factors is also important during adipocyte differentiation, with early expression of C/EBPβ and C/EBPδ during the first 2 days of differentiation leading to induction of C/EBPα.^32,49,50 C/EBPα KO mice die shortly after birth, but when expression is limited to the liver they survive with greatly reduced amounts of white adipose tissue.⁵¹ When C/EBPα null mice have the C/EBPα gene replaced with C/EBPβ, leading to expression of C/EBPβ from the C/EBPα gene locus in addition to its own endogenous expression, mice are able to survive, but amounts of white adipose tissue are still reduced,⁵² demonstrating that C/EBPβ is not sufficient to fully replace the function of C/EBPα in regulating adipose mass, although it is sufficient for survival.

While the C/EBP's are important for differentiation, they cannot induce differentiation without PPARγ. While C/EBPα deficient mice produce less adipose, they also do not induce endogenous PPARγ gene transcription, as fibroblast cell lines isolated from C/EBPα KO mice lack expression of PPARγ that is rescued by partially restoring expression of C/EBPα with a retroviral vector expressing C/EBPα;⁵³ thus showing the importance of communication between these 2 transcription factors. Additionally, treating mouse embryo fibroblasts (MEF) lacking a functional PPARγ gene with C/EBPβ does not induce expression of C/EBPα,⁵⁴ further supporting the role of communication between the C/EBP's and PPARγ in orchestrating adipocyte differentiation.

Adipose depots

Adipose tissue is divided into 2 major types, white adipose tissue (WAT) and brown adipose tissue (BAT), with WAT being an energy storage and endocrine tissue,⁵⁵ and BAT being involved in the production of heat, present in greater amounts in neonates and hibernators.^56,57 Existence of BAT is still questionable in avian species,^58-61 and is not reviewed here. The two main classifications of WAT depots in humans are subcutaneous adipose tissue and visceral adipose tissue, the visceral adipose tissue being the adipose surrounding the inner organs.⁶²

The different depots have many distinctions including differences in cellularity, adipokine production, and gene expression, as recently reviewed.⁶³ In the developing chicken embryo, fat is first deposited subcutaneously,⁶⁴ and develops through a combination of hyperplasia and hypertrophy.⁶⁵ Subcutaneous and clavicular fat depots are heavier than abdominal fat at 4 days post-hatch,⁶⁶ but as a percentage of body weight, the amount of abdominal fat increases with age.⁶⁶ It is unclear if birds are similar to humans in regards to metabolic differences between adipose tissue depots, although we have reported that there are differences in gene expression between adipose depots in chickens.^66-68

In addition to the differences we have reported in meat-type broiler chickens, we have also found adipose depot differences in a line of chickens selected for high and low body weight, the Virginia lines of chickens, which have undergone divergent selection for either low (LWS) or high (HWS) juvenile body weight at 56 days of age for 58 generations. This selection has resulted in a 10-fold difference in body weight at age of selection, with associated physiological and behavioral differences in feeding, adiposity, development, and body composition, with the LWS being lean and hypophagic, while the HWS are obese and hyperphagic as juveniles.^69-72 The LWS has greater PPARγ mRNA than HWS in the subcutaneous and clavicular adipose tissue, and pyruvate dehydrogenase kinase 4 (PDK4), and forkhead box 01A (FOX01), 2 enzymes important in metabolic flexibility,^73-77 also differ in expression between the lines, with greater expression of PDK4 in the clavicular adipose of the LWS and subcutaneous adipose of the HWS, and greater expression of FOX01 in the subcutaneous adipose of the HWS.⁶⁸ These differences in adipose depot expression of metabolic factors may contribute to the differences in adiposity of these chicken lines.

Models for studying adipogenesis

With the adipose being such a diverse tissue with numerous cell types present, and cross-talk with other systems in the body, many different models have emerged for studying the adipose tissue. A popular practice for in vitro studies is the use of cell lines. A common preadipocyte model, the mouse preadipocyte 3T3-L1 cell line, is very widely used to study adipogenesis. Their ability to differentiate into adipocytes and the ease through which different experiments can be conducted make them an attractive model. However, information on how these cells communicate with other cell types normally found in close proximity in vivo is impossible to determine through studies with single cell types. Cell lines are not available for every species, thus many studies in alternative animal models make use of a primary cell culture model to study adipogenesis.

Using 3T3-L1 cells allows for the study of a single cell type free of contamination by other cells that are present in the tissue. On the other hand, when generating a primary cell culture model, isolating one cell type proves more difficult especially in the absence of cell sorting technology, and thus there are usually multiple cell types present. There are numerous cell lines available, or types of cells and cell groups that can be isolated from a primary cell model, as noted in an excellent review on cell models used to study differentiation.⁷⁸ Additionally, while some protocols enrich for a specific cell type through isolation, other primary cell models enrich for a certain cell type through the media containing known factors that either push cells toward a certain goal, like differentiation, or enrich for a particular cell type. For example, a study by Billon et al., 2007, used a primary and secondary culture method of developing quail neural crest (NC) cells. Although these cells are not preadipocytes, they found that by including specific factors in the media cocktail, these cells were able to differentiate into adipocytes.²⁰ Another example would be isolating cells from the stromal vascular fraction (SVF), the cells isolated after completion of enzymatic digestion, centrifugation, cellular filtration, and red blood cell lysis of the adipose tissue,⁷⁹ a mixture of cells that include mesenchymal stem cells, preadipocytes, endothelial cells, and immune cells.^80-82 This technique has been applied in mice,⁸³ humans,⁸⁴ and chickens.¹¹

In addition to generating single cell suspensions for culture, whole tissue can also be cultured as explants. By isolating the tissue, but preserving the cross-talk between cells in the tissue, explants offer the benefit of more closely mimicking the in vivo environment of the cells.^85,86 As in vitro studies provide further understanding in fields of physiology, culturing whole tissue explants will become increasingly important to study conditions in vivo.

While there are numerous cell lines available for mammalian species, for avian species in vitro studies on adipogenesis often use a primary cell culture model. Some differences in methodology are adapted when studying differentiation, including using chicken serum (CS) in the differentiation cocktail, and using a combination of insulin, dexamethasone, and heparin.⁸⁷ The concentration of insulin is also greater than what is included in mammalian studies, because birds are naturally insulin resistant.^88,89 These studies are also conducted with tissue collected from the abdominal fat of young birds, usually 2–4 weeks old, because there is sufficient fat accumulation to yield ample cells for culture from a single chicken, and cell viability and proliferative capacity of adipose-derived mesenchymal stem cells decreases with age.⁹⁰ We also recently showed that cells isolated from birds at this age had robust proliferative capacity and ability to differentiate.¹¹

Factors that regulate both energy intake and storage in the animal

Identifying factors that regulate how adipose tissue is remodeled, as well as how energy is stored in different animals becomes increasingly important as more advances are made in understanding diseases related to the adipose tissue. The adipose is indeed an endocrine organ associated with the release of multiple hormones.⁹¹ While these hormones are important in energy balance and function, crosstalk between the brain and hypothalamus, as well as the sympathetic nervous system is also important in providing reciprocal signals that serve as feedback loops in times of energy restriction or excess. Multiple studies from the Bartness group used the pseudorabies virus (PRV) to elucidate the brain network controlling the sympathetic innervation of WAT⁹² and BAT.⁹³ The PRV is a neurotropic virus that is able to infect neurons trans-synaptically, thus providing the ability to trace a neural network in a retrograde fashion when injected into a specific tissue.^94,95 In Siberian hamsters (a species that displays seasonal changes in appetite and adipose mass in response to alterations in photoperiod) and rats, they found that areas of the brain stem, midbrain, and hypothalamus were connected to the WAT and BAT, with the paraventricular nucleus (PVN) and medial preoptic area (MPA) in the hypothalamus showing greatest concentration of the virus in connection to the WAT and BAT.^92,93 They also showed that the WAT is not innervated by any parasympathetic pathways in Siberian hamsters, rats, or mice, an idea that has been contested.⁹⁶

Many neurotransmitters are expressed in areas other than neurons, and have effects on the fat. Serotonin is involved in regulating energy homeostasis, as KO and inhibition of tryptophan hydroxylase 1, one of the enzymes responsible for conversion of tryptophan into serotonin, causes a reduction in body weight gain, improved glucose tolerance, increased thermogenic activity in BAT and decreased lipogenesis in WAT.⁹⁷ Other amino acid neurotransmitters are also found in the adipose tissue; with immunohistochemical labeling used to detect gamma amino butyric acid (GABA), glutamate, the GABA producing enzyme glutamate decarboxylase, and transporters and receptors for glutamate and GABA.⁹⁸ There is also regulation by POMC peptides in adipose tissue to promote browning of WAT and lipolysis,^99,100 and sympathetic innervation of the adipose tissue.¹⁰¹ The neurotransmitter NPY has received much attention in recent years, highlighted in our recent review,¹⁸ but is still an active area of research, which is reviewed herein. The POMC-derived peptide α-MSH has also been identified as a regulator of fat mass, and while developments in relation to adipose are relatively new, they are also reviewed herein.

Adipose physiology: NPY

While well regarded as an orexigenic peptide in the brain, NPY functions in many other aspects of physiology, such as learning and memory,¹⁰² tumor growth and progression,¹⁰³ and other processes related to adipose tissue. The effects of NPY that are related to adipose tissue and energy balance are summarized in Table 2 and Figure 1.

Figure 1. Adipogenic effects of NPY during adipocyte differentiation. When neuropeptide Y (NPY) is included in the differentiation cocktail of preadipocytes and cells from the stromal vascular fraction (mesenchymal stem cells (MSC) and preadipocytes (PreAd)) there is increased cellular proliferation of the precursor cells, a process thought to be mediated primarily via NPY receptor 2 (NPY2R). Throughout differentiation, there is increased expression of peroxisome proliferator activated receptor (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα), and decreased expression of uncoupling protein 1 (UCP1), thereby leading to increased lipid accumulation in the terminally differentiated adipocyte. Yellow represents the cell nucleus and red represents the lipid droplet.

Table 2. Physiological actions of Neuropeptide Y (NPY) related to energy balance and the receptors (NPYR) involved.

Action^1	Impact (+/−)	Receptor	Citation
Lipid Accumulation	+	NPYR 2 and 5	^11,112,126,184
Lipolysis	−	NPY1R	^134,185
Cellular Proliferation	+	NPY2R	^10,11,113,138
Food Intake	+	NPYR 1 and 2	^121,122,186
Body Weight Gain	+	NPYR 1, 2 and 5	^109,187,188
WAT Formation	+	NPYR 1 and 2	^109,137,189
BAT Formation	−	NPYR 1 and 5	^114,136,190
Insulin Resistance	+	NPY5R	^141
Glucose Consumption	−	NPY5R	^141
Bone Formation	−	NPYR 1 and 2	^115-119

Note.

¹ WAT: white adipose tissue; BAT: brown adipose tissue

NPY co-exists with norepinephrine (NE) in sympathetic nerve terminals.¹⁰⁴ The specific action of co-release of NPY and NE is often studied in the cardiovascular system,^105,106 but is not well annotated in the adipose tissue. Obese (fa/fa) Zucker rats reduce food intake and display enhanced metabolic rates when infused with both NPY and NE, suggestive of a synergistic effect.¹⁰⁷ In 3T3-L1 cells, NPY is able to potentiate lipolysis that is stimulated by isoproterenol, a β-adrenergic agonist,¹⁰⁸ further showing synergy between NPY and NE. In another study, mice overexpressing NPY in noradrenergic neurons show increased body, WAT, and BAT weight.¹⁰⁹ The effect is possibly determined by how much of each is present, distribution and abundance of receptor sub-types, with increases in adipose tissue development observed when NPY is more abundant, but the synergetic lipolytic effect being observed when amounts are similar.

Stem cells to preadipocytes to adipocytes

Prenatal stress can increase the chances of progeny developing obesity,¹¹⁰ and most adipocytes originate from adipose progenitor cells that were committed prenatally or early postnatally.¹¹¹ To understand the effect of prenatal stress on development of adipose tissue, epinephrine (EPI) was included in the adipocyte differentiation cocktail of murine embryonic stem cells to stimulate stress, after which NPY and NPY1R and NPY2R mRNAs were up-regulated.¹¹² Treatment with EPI was also associated with increased adipogenesis, through increased proliferation, mRNA expression of FABP4 and PPARγ, and enhanced neutral lipid accumulation. This effect was blocked by combined antagonism of NPYR's 1, 2, and 5,¹¹² indicating that the NPY system is important in the generation of more adipose cells from mesenchymal stem cells in response to stress.

Preadipocytes of the 3T3-L1 cell line were more viable and proliferated more when treated with a low dose of NPY (10⁻¹⁵M and 10⁻¹³M), but less viable and proliferative when treated with a much higher dose (10⁻⁷M).¹¹³ It was speculated that high concentrations of NPY may be contributing to differentiation of the preadipocyte rather than proliferation, explaining why the effect on proliferation is not observed at a higher concentration of NPY.¹¹³ When included in the differentiation cocktail, NPY up-regulates PPARγ and C/EBPα protein expression, and increases lipid accumulation in cells.¹¹³ These data indicate that NPY stimulates preadipocytes to multiply and then undergo hypertrophy during differentiation.

NPY also has effects on brown fat during cellular differentiation and after isolation of cells from the SVF of brown fat. Results from a study that used a mesenchymal stem cell line, C3H10T1/2, that is able to be induced to behave in a brown-like manner, demonstrated that including NPY in the differentiation cocktail at either 0, 1, 10, or 100 nM concentration had no effect on brown fat cell adipogenesis (it did not cause browning of the cells induced to differentiate), but was associated with inhibition of brown fat cell-associated marker uncoupling protein-1 (UCP-1),¹¹⁴ providing more evidence for NPY's role in suppressing brown fat activation, as reviewed earlier.¹⁸

Bone-adipose crosstalk

The bone and adipose tissue have considerable cross-talk, and both contribute to energy balance, with NPY and its receptors also being involved in the regulation of these 2 systems to control energy balance. A 40% reduction of plasma NPY was observed in mice that have an osteoblast-specific deletion of p38α mitogen activated protein kinase (p38 MAPK). A 20% increase in energy expenditure, measured by locomotor activity, O₂ consumption, CO₂ expenditure and energy expenditure monitored through an indirect calorimetric analysis, and up-regulation of UCP-1 without a change in energy intake was also observed in WAT and BAT, as well as a decrease in body weight and total adiposity. The mRNA distribution of NPY was evaluated in bone, primary osteoblasts, whole brain, hypothalamus, sympathetic superior cervical ganglia, adrenal gland, muscle, adipose, and jejunum, and was reduced in bone and primary osteoblasts, and increased in the hypothalamus and adipose of p38 MAPK-osteoblast-null mice. Although NPY was increased in the hypothalamus, the distribution of brain NPY immunoreactivity did not differ between the wild and KO mice. An increase in feeding for the first few hours was observed, but the effect normalized over time. Intraperitoneal injection of NPY partially restored adipose tissue mass in the mice lacking p38 MAPK in their osteoblasts.¹¹⁵

A review by Shi and Baldock, 2012, highlighted the relationship between NPY, bone, and adipose tissue.¹¹⁶ In bone, NPY represses bone formation, mainly through NPYR 1 and 2, and a lack of NPY expression increases osteoblast activity (bone formation), and bone mass. The two systems may be coordinately regulated although effects of NPY on bone and adipose homeostasis are complicated by how the brain perceives the negative energy balance state. Bone formation is similarly affected by elevated NPY in various energy states, whereas the adipose reacts differently. When there is elevated NPY, for instance during a negative energy balance state, bone metabolism is optimized for energy conservation and inhibition of bone formation, while in the adipose tissue, elevated NPY leads to increases in body weight and WAT mass.^117,118

Although the interaction between the adipose and bone remains to be further elucidated, they seem to be an attractive target for pharmacological intervention. NPY KO mice show increased bone formation,¹¹⁹ thus while targeting NPY to reduce obesity, an intervention currently underway,¹²⁰ bone formation could possibly be positively affected.

NPY knockout, knockdown, and NPYR antagonism

There are reports of NPY KO mice, knockdown in specific tissues, as well as pharmacological inhibition of receptor sub-types (Table 3); all models to better understand the role of NPY in adipose physiology and to identify the specific receptor(s) through which it mediates various functions in adipose tissue.

Table 3. Effects of NPY receptor (NPYR) antagonism in preadipocytes and adipocytes.

Receptor Antagonized	Effect of Antagonism	Citation
NPY1R	Blocks NPY-induced inhibition of lipolysis	^134
NPY2R	Decreased cell number	^138
NPY5R	Blocks decrease in glucose uptake	^141
NPYR 1, 2, and 5	Blocks increase in adipogenesis by epinephrine	^112

As reviewed earlier,¹⁸ NPY and NPYR 1, 2, and 5 are expressed in the adipose tissue and downstream effects related to energy balance are summarized in Table 2. The NPY1R has the widest body distribution, with expression being greatest in the paraventricular nucleus (PVN) of the hypothalamus.¹²¹ It is regarded as the NPYR most involved in regulating food intake, although NPY2R is also expressed in the arcuate nucleus (ARC) of the hypothalamus and contributes to appetite regulation.¹²² The NPY5R is most similar to NPY1R in amino acid sequence identity,¹²³ and is co-localized with NPY1R in many areas of the nervous system where NPY5R is expressed.¹²⁴ Adipose-specific production of NPY5R remains debated, as it has been detected in the adipose of rodents^125,126 and chickens,⁶⁷ but at other times is undetectable.^127,128 Additionally, NPY2R was expressed in human,¹⁰ mouse¹⁰ and chicken preadipocytes,¹¹ while in another study NPY2R was not detected in mouse preadipocytes.¹²⁸ While in vivo studies may show the importance of each receptor in whole body adipose biology, in vitro studies allow more specific quantification and study of regulation in a particular cell type.

Sirtuin 1 (SIRT1) has been reported to play a role in fat metabolism and may mediate some NPY-induced changes in adipose physiology. It is a deacetylase involved in lipid mobilization from fat¹²⁹ that also promotes brown fat cellular remodeling,¹³⁰ thereby contributing to an increase in energy release.¹³¹ A transcription factor, cAMP response element binding protein (CREB), is also important in this role as it induces expression of UCP-1,¹³² which as described earlier is a factor important for the thermogenic function of BAT.¹³³

In NPY-KO mice, adipogenesis-associated factor mRNAs are down-regulated, while expression of genes involved in lipolysis increase in gonadal white adipose tissue (gWAT).¹³⁴ Age related decreases in gWAT lipogenesis, and thermogenesis in inguinal WAT are also reduced in NPY KO mice.¹³⁴ SIRT1 has been reported to promote fat mobilization due to repressed PPARγ activity,¹²⁹ and the changes due to NPY KO are thought to be associated with activation of SIRT1, and subsequent inactivation of PPARγ- mediated pathways. This is supported by the finding that NPY treatment inhibits lipolysis in 3T3-L1 cells via NPY1R-mediated signaling and reduced CREB and SIRT1 protein expression.¹³⁴ Deletion of NPY in mice clearly affects pathways involving SIRT1, as well as regulation of transcription factors PPARγ and CREB. Because the NPY system is widely distributed, targeting factors that are affected by NPY, such as SIRT1, may thereby target the fat, especially since this factor is associated with the loss of fat, a major goal of modulating the NPY system to treat obesity.¹²⁰

Dorsomedial hypothalamic NPY has effects independent of ARC NPY, and is a major source of NPY that has been well studied in relation to energy balance.¹³⁵ Additionally, this nucleus was shown to be connected to the innervation of the adipose through the afore-mentioned PRV studies.^92,93 Knockdown of NPY expression in the dorsomedial hypothalamus (DMH) via adeno-associated virus-mediated RNA interference reduced fat depot weights in rats and ameliorated high-fat diet-induced hyperphagia and obesity. Knockdown also promoted brown adipose tissue (BAT) development and increased UCP-1 expression.¹³⁶

Daily IP injection of the NPY antagonist S.A.0204 for 30 days caused a decrease in body weight, and an increase in WAT lipolysis and apoptosis in diet-induced obese rats.¹³⁷ Intraperitoneal injection of the NPY2R antagonist BIIE 0246 into male mice for 14 days decreased adipocyte cell number but did not change cell size, indicating that NPY2R is important in producing new cells, but may not necessarily be involved in hypertrophy of adipocytes.¹³⁸ Although this study did not measure gene expression, future studies could evaluate whether the genes involved in adipogenesis/lipolysis are affected by NPYR antagonism. Serum deprivation of 3T3-L1 cells can induce lipolysis, an effect that is inhibited by NPY treatment, with NPY's effects in turn blocked by the NPY1R antagonist BIBO3304, demonstrating that the ability of NPY to rescue cells from the lipolytic effect of serum starvation is specific to NPY-mediated signaling.¹³⁴

Insulin resistance and sensitivity

Obesity is associated with insulin resistance, a pathology where cells are unable to normally respond to insulin,¹³⁹ thus it is worth noting the relationship of NPY function to insulin resistance in the adipose tissue. In biopsies from human subjects, mRNA abundance of adipose-derived hormones involved in insulin sensitivity, including adiponectin, visfatin, and omentin, was correlated with mRNA expression of NPY and its receptors. In subcutaneous fat all 3 were positively correlated with expression of NPY, and adiponectin, visfatin, and omentin were positively correlated with expression of NPYR 1 and 5, NPYR 1, 2, and 5, and NPY5R, respectively. All three were also positively correlated with NPY expression in the visceral fat, and visfatin was positively correlated with NPYR 1, 2, and 5 in visceral fat. There was no correlation of expression between depots, implying that the depots function differently.¹⁴⁰

Long-term over-expression of NPY in the PVN leads to increased adipose weight and insulin resistance in mice, as determined by a euglycemic-hyperinsulinemic clamp test and intravenous glucose tolerance test. Glucose uptake was decreased in adipose tissue but was not affected in skeletal muscle, suggesting that effects of NPY overexpression on insulin resistance were tissue specific.¹⁴¹ In the same study, 3T3-L1 cells were differentiated and treated with NPY, which decreased basal glucose consumption and decreased glucose uptake. This effect was not blocked by antagonism of NPY1R, but was blunted by antagonism of NPY5R.¹⁴¹ It is difficult to directly attribute the increased PVN NPY to the insulin resistance in the adipose tissue, but that the in vitro effect of NPY can be blocked by antagonism of NPY5R suggests that this may be the case; nonetheless, more studies are needed to confirm this hypothesis. These studies provide evidence that NPY5R may be the key NPYR through which NPY signaling leads to insulin resistance, as overexpression of NPY is associated with insulin resistance that can be blunted when NPYR5 signaling is blocked. That adiponectin,^142,143 visfatin,¹⁴⁴ and omentin¹⁴⁵ are involved in insulin sensitivity and are all correlated with NPY and NPY5R expression in subcutaneous adipose tissue¹⁴⁰ also implies that NPY and NPY5R play a role in insulin sensitivity.

Although NPY is already a target for drug development to combat obesity,¹²⁰ these correlations must be considered when targeting the NPY system pharmacologically. Antagonizing the NPY system could decrease fat accretion while increasing bone formation, a potentially positive side effect of treating obesity; but because of their correlated expression, decreased expression of adiponectin, visfatin, or omentin could exacerbate insulin resistance. Additionally, in addition to obesity and bone physiology, NPY also effects many other systems and diseases, including anxiety, depression, epilepsy, alcoholism, pain, cancer, cardiovascular regulation, gastrointestinal tract, and circadian regulation, as reviewed.¹ Drug development has proved difficult when targeting NPY, as targeting central NPY is not very specific and concerns the blood brain barrier, thus targeting the periphery warrants further exploration.¹⁴⁶

NPY and adipose development in chickens

Adipose tissue develops as a consequence of both hyperplasia and hypertrophy, with hyperplasia leading to hypertrophy. We reported changes in the adipose tissue of broiler chicks during the first 2 weeks post-hatch.⁶⁶ Specifically, for the subcutaneous, abdominal, and clavicular fat depots at days 4 and 14 of age, there were depot-specific differences with larger adipocytes and the greatest glycerol–3–phosphate dehydrogenase (G3PDH) activity in the subcutaneous fat at day 4. The abdominal fat grew more rapidly than the other 2 depots from day 4 to 14, and clavicular fat was intermediate for most traits that were measured. These changes were most likely due to a combination of hypertrophy and hyperplasia.⁶⁶ Neuropeptide Y and its receptors were also measured in the study; from day 4 to 14 there was an increase in mRNA abundance of NPY and NPY receptor 5 (NPY5R) and a decrease in NPY2R in the clavicular adipose tissue. The orexigenic properties of NPY are mostly associated with NPY1R,¹²² mRNA of which was not affected by age in clavicular adipose, but did increase in the hypothalamus of broiler chicks from day 4 to 14.⁶⁶ NPY receptors 1, 2, and 5 were all increased in the liver at 14 days post-hatch, implying that the NPY system may function in the liver during development.⁶⁶ Thus, NPY and several of its receptors are transcribed in chick adipose tissue and liver and appear to be differentially regulated during early post-hatch development, although functional implications are still unclear.

We also conducted an in vitro study showing that 4 hours of NPY treatment increased mRNA abundance of NPY and NPY2R in abdominal fat preadipocytes isolated from 14 day-old broilers.¹¹ With NPY being included in the differentiation cocktail, abundance of NPY was also increased on days 2 and 6 post induction of differentiation, whereas NPYR2 expression was not affected during this time.¹¹ That NPY2R mRNA expression was upregulated in preadipocytes in response to NPY may imply that NPY2R is more important to NPY function in the preadipocyte than in the differentiating adipocyte. On day 5 and 6 post-induction of differentiation, there was increased cell proliferation and G3PDH activity, respectively. This indicates that NPY treatment was associated with increased hyperplasia, as well as fat synthesis, as G3PDH activity is an indirect marker of triacylglycerol synthesis.^147,148 Thus in chicken adipose cells, NPY may contribute to both hyperplasia and hypertrophy. While NPYR 1 and 5 seem to play a role based on data from our in vivo studies, expression of NPYR 1 and 5 could not be detected in adipose cells in our in vitro studies.¹¹ This may be due to the difference in cell types included in each study. The in vivo studies included the whole adipose tissue, whereas in the in vitro studies isolated the stromal vascular fraction of cells from adipose and then enriched for mesenchymal stem cells and preadipocytes.

Interestingly, we reported heterosis for NPYR 1 and 5 mRNA in the adipose tissue of chickens from the Virginia body weight lines, suggesting that the NPY system is important for energy balance in chickens.⁶⁷ HWS chickens expressed more NPY and NPYR 1 and 5 mRNA in the abdominal fat,⁶⁷ while LWS chicks expressed more NPY and NPYR 2 and 5 in the hypothalamus.¹⁴⁹ Additionally, expression of NPY5R in the hypothalamus is regulated differently between the fed vs fasted state, where in the fed state, NPY5R was more highly expressed in the LWS, and the magnitude of difference between the LWS and HWS was lessened by fasting.¹⁵⁰

Adipose phyiology: α-MSH

The peptide α-MSH is a part of the melanocortin system, which is made up of α, β, and γ – MSH, ACTH, melanocortin receptors (MCR) 1–5, and 2 endogenous MCR antagonists, agouti and agouti-related peptide (AgRP), as reviewed.^151,152 All of the melanocortin peptides are derived from POMC,¹² and have some function in regulating food intake. Our group recently showed that ACTH, and the C-terminal fragment of ACTH, β-cell-tropin (βCT) have effects on food intake in chicks, with ACTH being anorexigenic,¹⁵³ and βCT being orexigenic;¹⁵⁴ the same effects on food intake were reported in rats.¹⁴ All of the MSH peptides decrease food intake in mice, with α-MSH being the most potent,¹⁵⁵ but in chicks β and γ-MSH had no effect on food intake.¹⁶

MC1R is mainly associated with pigmentation,¹⁵⁶ but is also involved in the control of inflammation,^157,158 and has the highest affinity for α-MSH.¹⁵⁹ MC2R is the ACTH receptor, and is mainly associated with the action of ACTH on the periphery,¹⁶⁰ namely the release of glucocorticoids.¹⁶¹ It is also expressed in the adipocyte, and may function in regulating lipolysis.¹⁶² MCR 3 and 4 are highly expressed in the brain,^163,164 and are involved in food intake and energy balance,¹⁶⁵ and MC3R-deficient mice have increased fat mass.¹⁶⁶ MC4R is the receptor for both ACTH and α-MSH in the brain of rats,¹⁴ and α-MSH in the brain of chicks.¹⁶ MC5R is expressed in many sebaceous and exocrine glands,¹⁶⁷ regulates fatty acid oxidation in skeletal muscle,¹⁶⁸ and is associated with lipolytic effects in the adipose.^17,162

In addition to its anorexigenic function, α-MSH is associated with decreasing fat mass and body weight (Fig. 2).^13,14 α-MSH promoted lipolysis in vitro¹⁷ and in mice reduced their fat mass.^169,170 Signaling via MC5R increased lipolysis and impaired fatty acid re-esterification in 3T3-L1 adipocytes,¹⁷ providing a plausible route of action of α-MSH in adipose tissue. Because POMC is a precursor to multiple peptides, KO of POMC does not correlate well with determining the function of a specific POMC cleavage product, which has led to other techniques to overexpress the specific peptides in order to ascertain their function. In two month-old male C57BL/6N mice, overexpression of α-MSH by a lentiviral vector (LVi-α-MSH-EGFP) in the nucleus tractus solitarius decreased fat mass, food intake, and body weight.¹⁶⁹ The phosphatidylinositol 3-kinase (PI3K) pathway is important in the regulation of food intake and energy homeostasis in the hypothalamus,^171,172 and inhibition of 2 PI3K's, PI3Kβ and PI3Kγ, led to increased NE concentrations in the adipose tissue, which then led to decreased fat mass and was associated with increased lipolysis and browning of white adipose tissue in adult mice.¹⁷⁰ It was suggested that the increase in NE was due to increased α-MSH-dependent sympathetic drive as cAMP production was also increased in the intermediolateral nucleus, an area where energy expenditure is regulated by α-MSH.¹⁷³ Moreover, intracerebroventricular injection of α-MSH into wild-type mice caused increased phosphorylation of hormone sensitive lipase (HSL), a lipase known to be activated by NE,^174,175 to the same extent as in PI3Kβ- and PI3Kγ-inhibited mice.¹⁷⁰ In humans, α-MSH stimulated lipolysis in intact white adipose tissue but not in isolated preadipocytes that were differentiated into adipocytes, suggesting that the effect may be dependent on neuronal innervation.¹⁷⁶ Clearly, α-MSH has effects on the adipose tissue, some of which may relate to its role in the sympathetic nervous system.

Figure 2. Actions of α-MSH on the adipocyte. When adipocytes are treated with α-melanocyte stimulating hormone (α-MSH), there is increased expression of nuclear hormone receptor subfamily 4 group A member 1 (NR4A1), and increased lipolysis and inhibition of re-esterification. In animals, this leads to a decrease in fat mass and body weight. These effects are thought to be mediated by melanocortin receptor 5 (MC5R). Yellow represents the cell nucleus and red represents the lipid droplet.

The nuclear hormone receptor (NR) 4A subgroup is involved in adipose metabolism, with effects on differentiation and proliferation.^177,178 Treatment of differentiated 3T3-L1 cells with an α-MSH agonist (NDP-MSH) for 2 hours was associated with increased mRNA expression of members of the NR4A subgroup, but not other subgroups in the NR superfamily.¹⁷⁹ Furthermore, siRNA-mediated knockdown of nuclear hormone receptor subfamily 4 group A member 1 (NR4A1) attenuated the effects of NDP-MSH treatment on gene expression in 3T3-L1 adipocytes. Intraperitoneal (IP) injection of NDP-MSH into 7-week-old C57 BL/6J male mice was associated with increased expression of NR4A1, suggesting that expression of NR4A1 is necessary for the action of α-MSH.

These studies collectively indicate that α-MSH has effects on the adipose tissue that are lipolytic, and signaling initiated from the brain and sympathetic nervous system can act to decrease fat mass. Involvement of the NR superfamily has also been implicated in the action of α-MSH, and MC5R may be an important receptor for mediating effects of the melanocortin system in the adipose tissue. More in vivo studies are needed to confirm that MC5R is important for adipose function, and whether or not there are different effects through the various receptors. To date, effects of the melanocortin system in adipose tissue has not been reported in non-mammalian species.

Communication between α-MSH and NPY

Both NPY and α-MSH have strong, opposing effects on food intake, with effects conserved across a range of vertebrate species. In chicks, combined intracerebroventricular (ICV) injection of both α-MSH and NPY results in a decrease in food intake,¹⁸⁰ the orexigenic effect of NPY likely overpowered by the anorexigenic effect of α-MSH. Because α-MSH is a peptide derived from POMC, post-translational processing becomes very important in regulating the quantity of the active peptide, and serves as a point of entry for intervention, either through pharmaceutical means, feedback loops, or action of other neurotransmitters. NPY has been implicated in inhibiting post-translational processing of POMC to yield α-MSH by decreasing prohormone convertase 2 (PC2) protein; this effect is associated with downregulation of early growth response protein 1 (EGR-1) and is likely occurring via NPY1R, as blocking NPY1R is able to restore PC2 activity and α-MSH release.² After ICV injection, NPY decreased the phosphorylation of CREB, an effect normally caused by α-MSH, and NPY also decreased the amount of α-MSH in the PVN.² When co-injected, α-MSH overpowers the orexigenic effect of NPY,¹⁸⁰ but when promoting positive energy balance NPY signals to decrease the amount of α-MSH in the hypothalamus.² This may imply that the effect of combined injection is more of a pharmacological response than physiological.

Potential physiological routes through which NPY and α-MSH affect adipose tissue

Although most studies concerning the interaction of NPY and α-MSH are in relation to the brain and control of food intake, there may also be interactions between the 2 in adipose tissue. Melanotan-II is a non-selective MCR agonist, and a 7 day-long central infusion into rats reduced feeding and adiposity, and when infused simultaneously with NPY reduced feeding and attenuated NPY-mediated increases in fat pad weight. Although combined injection is thus able to inhibit some of the effects of NPY, it does not block NPY-mediated inhibition of the gonadotropic and somatotropic axes,¹⁸¹ suggesting that crosstalk between MSH and NPY is tissue and/or axis specific rather than global.

Circulating plasma levels of NPY are increased in obese and overweight humans, but circulating α-MSH levels are similar between normal, overweight and obese individuals.¹⁸² This indicates that increased weight from adipose tissue has impacts on circulating NPY, but may not impact plasma α-MSH. Moreover, in obese subjects after weight loss, cerebrospinal fluid (CSF) concentrations of NPY decrease whereas α-MSH levels remained unchanged.¹⁸³

The interaction of NPY and α-MSH is complex, and much is still unknown as there is little reported on the role of α-MSH and its interaction with NPY and other factors in the adipose tissue. The fat-loss effect of α-MSH may not be a direct effect on the adipose, but rather an indirect effect of decreasing the amount of energy available from reduced energy intake, thus forcing the animal to tap into its energy reserves in the adipose tissue to maintain energy balance. Likewise, the fat-synthesizing effect of NPY in the adipose tissue is likely a response to increased energy intake (and thus availability) mediated via the hypothalamus.

Conclusions

Factors involved in regulating energy balance and adipose physiology are crucial to understand in light of the current obesity epidemic and increasing importance of enhancing nutrient utilization efficiency and meat production in agricultural species to meet the demand for dietary protein as the global population continues to increase. The adipose tissue is a diverse endocrine organ, with many cell types and models used to study its function. Because of its complicated physiology, there are multiple cellular pathways to explore for pharmacological targeting, but also many functions that may not directly relate to whole-body energy balance. NPY exerts multiple functions in the adipose tissue mediated by distinct receptor sub-types, but also regulates metabolism in other tissues such as bone. Knockout and antagonism of NPY are associated with reduced adiposity and body weight, but because of its diverse physiological functions, affecting solely the lipid metabolism in adipose tissue is a challenge in targeting this system to treat obesity. Regulation of α-MSH could also be a potential route of intervention, as it is associated with not only a decrease in feeding, but also decreased body weight and increased lipolysis. The interaction of these 2 peptides is also important and multi-faceted; the two have opposing effects on appetite and adipocyte metabolism and at times one may inhibit the expression and activity of the other. Further study of NPY and α-MSH functions in the brain-fat axis, as well as identification of other appetite-regulatory factors that affect adipose tissue physiology will provide useful information in the development of pharmacological strategies to battle obesity and also approaches to modulate energy utilization in animals raised for food consumption.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.