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\begin{document}

\title{Bringing equine adipose tissue into focus}



\author[1]{Sophie McCullagh}%
\author[2]{Miranda Dosi}%
\author[3]{John Keen}%
\author[4]{Ruth Morgan}%
\affil[1]{The University of Edinburgh}%
\affil[2]{The University of Edinburgh Royal Dick School of Veterinary Studies}%
\affil[3]{University of Edinburgh}%
\affil[4]{SRUC}%


\vspace{-1em}



  
  \date{June 07, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Adipose tissue is not only required for energy storage but is an
essential endocrine organ with a central role in the pathology of
obesity. The understanding of its role, both in human and equine
medicine, is continually evolving. With obesity being an ever-growing
problem in the equine population, gaining owner compliance is critical
when implementing management plans. The aim of this review is to
encourage the inclusion of the concept of adiposity in discussions with
horse owners on obesity and metabolic syndrome. In doing this, we hope
to improve clients understanding and therefore maximise the impact of
diagnostic tests, monitoring tools and management.%
\end{abstract}%



\sloppy


\emph{Bringing equine adipose tissue into focus}

\textbf{Sophie Elizabeth McCullagh\textsuperscript{1}, John A
Keen\textsuperscript{1}, Miranda Carlotta Maria
Dosi\textsuperscript{1}and Ruth Anna Morgan\textsuperscript{1,2}}

\textsuperscript{\textbf{1}}\textbf{Equine Hospital, The University of
Edinburgh, Royal Dick School of Veterinary Science, Easter Bush EH25
9RG}

\textsuperscript{\textbf{2}} \textbf{Scotland's Rural College, Roslin
Institute, Easter Bush, EH25 9RG}

\textbf{Contact details for primary author (Sophie McCullagh)}

Telephone number: 07521 955541

Email address: smcculla@ed.ac.uk

\textbf{Keywords;} Horse, Adipose, Obesity, Metabolic Syndrome, Insulin
Resistance

\textbf{Summary}

Adipose tissue is not only required for energy storage but is an
essential endocrine organ with a central role in the pathology of
obesity. The understanding of its role, both in human and equine
medicine, is continually evolving. With obesity being an ever-growing
problem in the equine population, gaining owner compliance is critical
when implementing management plans. The aim of this review is to
encourage the inclusion of the concept of adiposity in discussions with
horse owners on obesity and metabolic syndrome. In doing this, we hope
to improve clients understanding and therefore maximise the impact of
diagnostic tests, monitoring tools and management.

\textbf{Introduction}

Adipose tissue is a multifaceted organ, essential for health. While its
role in lipid storage is well established, understanding the importance
of adipose tissue as an endocrine organ, with a central role in the
pathology of obesity, is evolving. Helping clients to understand the
systemic impacts of excess adipose tissue can maximise the impact of
diagnostic tests, monitoring tools, management and owner compliance.

\textbf{Adipose as a storage organ}

The amount of adipose tissue varies hugely between individual horses and
is affected by factors such as age, sex and breed (Wallis and Raffan,
2020). Estimation of body fat content by deuterium oxide dilution found
adipose to account for 2.7-35.6\% of total bodyweight in horses (Dugdale
et al., 2012). There are two distinct types of adipose: white adipose
tissue and brown adipose tissue. The white adipose tissue predominates
and is specialised for energy storage. The brown adipose, named due to
its colouration from the huge concentration of mitochondria, has a role
in thermoregulation and, as a consequence, is abundant in new-borns and
animals in hibernation (Kiranmayi and Bhargav, 2019). Most adult mammals
have very low quantities of brown adipose and indeed, this form has not
been described at all in the adult horse. There is great interest in
brown adipose activation as a novel therapeutic target in human obesity
(Liu et al., 2022); further research into brown adipose in the horse may
therefore be of interest.

White adipose tissue is primarily an energy storage organ and is
composed of adipocytes, in which lipid accumulates, as well as
connective tissue, immune cells and blood vessels. Adipose can store
almost 100 times more megajoules of energy than muscle and this resource
allows mammals to cope with changes in energy availability over time.
Humans with congenital lipoatrophy (Berardinelli-Seip syndrome), who
have a functional failure of their adipose, rapidly develop severe
insulin resistance and hepatic lipidosis, leading to liver failure and
cardiovascular disease (Garg, 2004). This demonstrates that adipose an
essential component of energy homeostasis and should not be viewed as
always detrimental to health.

Adipocytes are formed from mesenchymal stem cells in a process called
adipogenesis; a complex multistep process which includes the formation
of pre-adipocytes from undifferentiated stem cells and the formation of
mature adipocytes from these preadipocytes by accumulation of lipid.
Equine adipose mesenchymal stem cells (MSCs) are well understood due to
their regular use in the developments of treatments of musculoskeletal
disorders (stem cell therapy) which has evolved due to their relative
ease of acquisition and robust response to \emph{in vitro} manipulation
(Marycz et al., 2016). The \emph{in vitro} characteristics of equine
MSCs are very similar to those of humans, including the pre-adipocyte
response to insulin and glucocorticoid stimulation which induces lipid
accumulation and differentiation to mature adipocytes (Bukowska et al.,
2021).

Adipocytes store fat as triglyceride (triacylglycerol) in one large
lipid droplet per cell. Triglycerides from dietary fat are absorbed from
the gut and transported to the adipose in the form of chylomicrons.
Adipose, as well as liver, can also synthesise triglycerides from excess
carbohydrates, a process known as de novo lipogenesis (DNL) (Ameer et
al., 2014). The starting compound for DNL varies greatly between
species, with horses using acetate, unlike humans who primarily use
glucose or pigs who can use either (Suagee et al., 2010). Adipose
tissue, as opposed to the liver, is the primary site of DNL in horses in
contrast to humans (Suagee et al., 2010; Adolph et al., 2019), which may
go some way to explain why fatty liver is less common in horses than
other species. Aberrant DNL is associated with insulin resistance and
cardiovascular risk in humans and is a potential link between excess
carbohydrate intake and these conditions (Ameer et al., 2014), however
very little is known about changes in DNL in equine disease.

In the face of increased calorie intake, adipose can expand by
hyperplasia (increased adipocyte numbers derived from preadipocytes) or
by hypertrophy (increase in individual cell size by lipid accumulation).
The capacity for hyperplasia varies between adipose depots, as discussed
later. When required, the stored triglycerides are broken down into
glycerol and free fatty acids by lipolysis. These fatty acids are then
available to mitochondria for respiration. The ability and speed at
which adipose tissue breaks down stored triglycerides is one of the main
distinguishing features between adipose depots.

\textbf{Adipose as an endocrine organ}

In addition to its primary role in storage, adipose tissue is the
largest endocrine organ in the body, producing and responding to
hormonal signals and critical in the cross-talk between metabolic organs
which govern energy homeostasis. Adipocytes secrete bioactive peptides
(adipokines and adipocytokines) which can act locally
(autocrine/paracrine) or systemically. Adipose also possesses a complex
receptor profile which allows it to respond to endocrine and nervous
input. Finally, adipocytes are able to exert fine control over endocrine
signalling through their enzyme machinery, important in the metabolism
of hormones, particularly steroid hormones. Our understanding of the
equine adipocyte endocrine profile is more limited than that of humans
or rodents but some data exist.

\emph{Adipokines}

\textbf{Leptin} is a critical regulator of energy storage through
appetite/satiety control: when energy stores are adequate, leptin
signals to the hypothalamus to reduce appetite drive, thus reducing food
intake. As in other species, leptin is secreted from equine adipocytes
in proportion to body fat mass. Indeed, animals with increased body fat
(without disease) have higher plasma and adipose tissue expression of
leptin (Staub et al., 2019; Buff et al., 2002).

\textbf{Adiponectin} is produced almost exclusively by adipocytes (Fang
\& Judd, 2011) and it acts primarily on muscle and liver to increase
insulin sensitivity and reduce inflammation. In most species, including
horses, there is an inverse relationship between fat mass and plasma
adiponectin (Kearns et al., 2006).

\textbf{Resistin} is also an adipose-specific protein, whose
transcription is induced during differentiation of adipocytes. In
rodents, it has been shown to decrease gluconeogenesis in the liver
(Banerjee et al., 2004). Numerous human studies have failed to
demonstrate a reliable association between resistin levels, obesity
and/or insulin dysregulation. In contrast, resistin may be a marker of
inflammation (Banerjee and Lazar, 2003), consistent with findings in
horses (Fuentes-Romero et al., 2021).

\emph{Adipocytokines}

Adipocytes, stromovascular and inflammatory cells all contribute to
adipocytokines secretion. Cytokines released include IL-6, TNF\selectlanguage{greek}α, \selectlanguage{english}MCP1,
CCL2 and IL1\selectlanguage{greek}β. \selectlanguage{english}The actions of the adipocytokines are diverse and are not
limited to just inflammatory effects such as chemoattraction and immune
cell activation. For example, TNF\selectlanguage{greek}α \selectlanguage{english}suppresses free fatty acid and
glucose uptake into adipocytes in humans (Ruan et al., 2002). Little is
known about the actions of these cytokines in equine adipose but
expression of several, including \selectlanguage{greek}\emph{TNFα} ,\selectlanguage{english}\selectlanguage{greek}\emph{~IL1β} , \selectlanguage{english}IL-6
and~\emph{CCL2} , has been shown (Reynolds et al., 2019; Basinska et
al., 2015). The analysis of these cytokines may also be a potential
diagnostic target in equine metabolic syndrome.

\emph{Steroid hormones}

Adipose is crucial for the action and metabolism of steroid hormones,
particularly glucocorticoids and sex steroids. Furthermore,
glucocorticoids are essential in differentiation of mature adipocytes.
Adipose tissue in humans and horses contains high levels of
glucocorticoid receptor (GR) and also the metabolizing enzyme
11\selectlanguage{greek}β-\selectlanguage{english}hydroxysteroid dehydrogenase type 1 (HSD1,) which metabolises
inactive cortisone into active cortisol and thus potentiates activation
of GR (Morgan et al., 2018). Horses predominantly metabolise their
cortisol through the carbonyl reductase 1 pathway which is very active
in adipose tissue and produces a metabolite which impacts glucose
homeostasis (Morgan et al., 2017). Less is known about sex steroid
metabolism and receptors in equine adipose tissue, though sexually
dimorphic patterns of adipose distribution are noted (such as the large
nuchal crest adipose depots in stallions) suggesting androgens may
impact adipose deposition.

\emph{Insulin}

Adipocytes are exquisitely sensitive to the effects of insulin. Binding
of insulin to its receptor initiates the movement of glucose transporter
GLUT4 to the plasma membrane to allow uptake of glucose by the
adipocyte. Adipocyte GLUT4 trafficking not only keeps substrate
available for DNL but also seems to mediate the cross-talk of adipose
with liver and muscle to maintain systemic glucose homeostasis (Abel et
al., 2001). GLUT4 trafficking is evident in equine adipocytes but there
is a suggestion that more complex and additional insulin signalling
pathways may also contribute (Warnken et al., 2017). Insulin also
supresses lipolysis in equine and human adipocytes (Warnken et al.,
2017; Duncan et al., 2007) and this acts to regulate the availability of
free fatty acids and glycerol to the liver for gluconeogenesis.

Figure 1 Diagram showing some of the key functions of adipose tissue.
Created with BioRender.com.

\textbf{Not all adipose is created equal}

The anatomical location of adipose tissue appears to impart several
important characteristics, most likely due to the slightly different
role adipose will play in each different site. The simplest distinction
is that of subcutaneous adipose tissue (SAT) which lies immediately
underneath the skin and visceral (VAT) which surrounds organs. In
addition to these two major categories, adipose tissue within the bone
marrow, around the heart (epicardial) and blood vessels and in/around
skeletal muscle have all been found to have distinct phenotypes in
humans and rodents. Very little is known about these tissue specific
depots in the horse and so this review will focus on what is known about
SAT and VAT.

\emph{Subcutaneous versus visceral adipose tissue}

The distinction between SAT and VAT is important because of the
independent association of VAT with increased metabolic risk. People who
carry their weight around their femorogluteal region (SAT) which is the
predominant pattern seen in women, are relatively protected from
metabolic risk in obesity (Booth et al., 2014). In contrast, those who
predominantly lay down visceral adipose are at increased risk (Kwon et
al., 2017). The reason for this different impact on metabolic health is
not clear, but several features of SAT and VAT have been implicated.

\begin{itemize}
\tightlist
\item
  SAT has a greater capacity for expansion by hyperplasia, which results
  in more abundant but smaller and ``healthier'' adipocytes. VAT however
  favours hypertrophy for expansion which results in large and less
  healthy adipocytes, which can more readily induce lipolysis and
  release fatty acids into the circulation or directly to the liver via
  the portal circulation (Bergman et al., 2006). A similar pattern of
  adipocyte size is found in horses, with peri-renal and retroperitoneal
  adipocytes having a significantly larger cross-sectional area than
  nuchal adipose (Bruynsteen et al., 2013).
\item
  In humans, VAT has a more inflammatory phenotype, compared with SAT.
  This is the case both in `normal' individuals and those in the face of
  persistent calorie excess (Ibrahim, 2010). In horses there was
  increased mRNA levels of inflammatory cytokines in visceral depots
  compared with the nuchal depot (Bruynsteen et al., 2013).
\item
  Leptin secretion is greater from SAT relative to VAT in humans and
  horses (Bruynsteen et al., 2013). In humans, adiponectin expression is
  higher in SAT than VAT (Fain et al., 2004) but the opposite is found
  in horses (Bruynsteen et al., 2013). There is also some evidence in
  humans and horses (Warnken et al., 2017) that SAT is more insulin
  sensitive than VAT, potentially contributing to ``safer'' storage of
  lipids.
\end{itemize}

This distinction between SAT and VAT is relevant in horses because
clinical measures of adipose tissues (body condition scoring, weigh
tapes) almost exclusively measure SAT. It is important to recognise this
as a limitation and remember that visceral adipose may be more important
in predicting disease risk. Body condition score is only strongly
correlated with total body fat as determined by deuterium oxide dilution
(eTBF\%) (Dugdale et al., 2011) in lean or non-obese horses. As BCS
increases, the predictive ability of BCS reduces significantly (Dugdale
et al., 2012). In addition, reliance on BCS may lead us to miss horses
with body fat carried almost exclusively around the viscera, a state
referred to as TOFIs in human medicine: Thin on the Outside Fat on the
Inside (Thomas et al., 2012). Whilst we cannot use the MRI techniques
employed in human medicine, it is worth considering abdominal ultrasound
in horses if a TOFI phenotype is suspected.

\textbf{Adipose tissue in obesity}

Obesity is defined by the World Health Organisation (WHO) as `abnormal
or excessive fat accumulation that may impair health.' In horses we have
yet to define this parameter, so our definition of obesity is based
solely on measures of subcutaneous fat. We do know that in obese horses,
as defined by BCS, adipose can account for up to 35\% of body mass
(Dugdale et al, 2012).

Obese adipose tissue, particularly VAT, is markedly dysfunctional in
humans and directly related to the development of obesity associated
morbidities including insulin resistance and cardiovascular risk
(Santillana et al., 2023). In horses, dysfunction of obese adipose
tissue is also clear. Reynolds \emph{et al} (2019) found marked
hypertrophy of VAT adipocytes, a hallmark of dysfunctional adipose in
humans. Fibrosis, a key feature of human dysregulated adipose, was not
found in the adipose of these horses but has been reported by others
(Basinska et al., 2015), this discrepancy perhaps due to the chronicity
of disease in the animals studied. Dysfunctional adipose has also been
shown to have increased expression of leptin (Reynolds et al., 2019).
There is conflicting data regarding adipocytokine expression in obese
adipose of horses, as there is in the human literature. Whilst
Burns\emph{et al} (2010) showed no change in TNF\selectlanguage{greek}α \selectlanguage{english}and IL1\selectlanguage{greek}β \selectlanguage{english}in obese
adipose (Burns et al., 2010), several groups have since shown increases
associated with obesity and insulin dysregulation in VAT (Reynolds et
al., 2019; Jayathilake et al., 2022) and SAT (Basinska et al., 2015).
Basinska \emph{et al} also reported macrophage infiltration. It should
be noted that the populations studied are invariably diverse in terms of
disease state. In humans, obese adipose expansion by hypertrophy fails
to stimulate angiogenesis which is driven by increased cell number
(hyperplasia), therefore the blood supply eventually becomes limited and
hypoxia occurs which contributes to inflammation (Hammarstedt et al.,
2018). Although this has not yet been investigated, the same process may
occur in the horse.

A key question in understanding how obesity results in insulin
resistance, is whether obese adipose tissue is itself insulin resistant.
There is currently not a clear answer to this question in humans as it
appears to be the case in some individuals but not in others. This
alludes to the increasing acceptance that many sub-types of insulin
resistance syndromes exist (Imi et al., 2023). Very little is known
about insulin sensitivity of obese adipose in horses. A crude measure of
insulin signalling components in adipose showed no differences between
lean horses and those with equine metabolic syndrome (Reynolds et al.,
2019); however, insulin signalling is dynamic and relies on an altered
phosphorylation state, so much more work is required in this area.

Figure Diagram showing some of the known and proposed effects of
unhealthy adipose tissue seen in overweight/obese patients. Created with
BioRender.com

The next question is whether dysfunctional obese adipose tissue
contributes to whole body insulin dysregulation. The answer is
invariably yes in human and rodent models, although there is still
debate over the temporal nature of this relationship (Bl\selectlanguage{ngerman}üher, 2016; Kahn
and Flier, 2000; Kahn et al., 2006). In general, it is thought that
factors released in greater or lesser quantities from obese adipose
perturb insulin signalling, whether that is a function of the insulin
receptor itself or the downstream signalling cascade, particularly in
liver and muscle but also in adipose. These downstream factors include
adipocytokines (Blüher, 2016), leptin and adiponectin (Yadav et al.,
2013). Overloaded adipocytes, especially in VAT, can release free fatty
acids into the portal circulation and overwhelm hepatic gluconeogenesis
thus impacting insulin sensitivity (Longo et al., 2019). If adipocytes
have reached capacity for safe storage then lipid will be deposited
ectopically such as in muscle and in liver which is detrimental to
insulin sensitivity in those tissues (Longo et al., 2019).

\textbf{Conclusion}

The aim of this review was to demonstrate our growing understanding of
the complexities of adipose tissue and its essential role in the
morbidity of equine obesity. It is vital that knowledge gleaned from
this work should be translated into practical benefits in terms of
equine welfare; helping owners and veterinary surgeons to mitigate the
impact of obesity on the domestic equine population. The authors have
found it is useful to include discussion of ``unhealthy'' adipose when
communicating with owners about obesity. In doing this, the owner should
become aware that the horse is not just fat, but this fat is producing
factors which negatively impact most other organs in the body.

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\begin{figure}[H]
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\includegraphics[width=0.70\columnwidth]{figures/Healthy-adipose/Healthy-adipose}

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\end{figure}\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Unhealthy-adipose/Unhealthy-adipose}

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