Adipose tissue-derived neurotrophic factor 3 regulates sympathetic innervation and thermogenesis in adipose tissue

Activation of brown fat thermogenesis increases energy expenditure and alleviates obesity. Sympathetic nervous system (SNS) is important in brown/beige adipocyte thermogenesis. Here we

discover a fat-derived “adipokine” neurotrophic factor neurotrophin 3 (NT-3) and its receptor Tropomyosin receptor kinase C (TRKC) as key regulators of SNS growth and innervation in adipose

tissue. NT-3 is highly expressed in brown/beige adipocytes, and potently stimulates sympathetic neuron neurite growth. NT-3/TRKC regulates a plethora of pathways in neuronal axonal growth

and elongation. Adipose tissue sympathetic innervation is significantly increased in mice with adipocyte-specific NT-3 overexpression, but profoundly reduced in mice with TRKC

haploinsufficiency (TRKC +/−). Increasing NT-3 via pharmacological or genetic approach promotes beige adipocyte development, enhances cold-induced thermogenesis and protects against

diet-induced obesity (DIO); whereas TRKC + /− or SNS TRKC deficient mice are cold intolerant and prone to DIO. Thus, NT-3 is a fat-derived neurotrophic factor that regulates SNS innervation,

energy metabolism and obesity.

Obesity has become a serious health problem that poses as a major risk factor for the development of a panel of metabolic diseases such as insulin resistance/type 2 diabetes, dyslipidemia,

hypertension, and cardiovascular diseases1. Obesity is caused by a chronic energy imbalance that results from energy intake over energy expenditure1. Adipose tissue is one of the most

important organs that regulate energy homeostasis in the body2. There exist two distinct types of adipose tissues: white adipose tissue (WAT) that is specialized in energy storage and brown

adipose tissue (BAT) that is unique for energy dissipation2,3. BAT has the capacity to dissipate energy through adaptive thermogenesis, a biological process in which energy is burned as heat

instead of being trapped in ATP2,4. The thermogenic activity of BAT largely depends on the unique action of uncoupling protein 1 (UCP1) in the inner mitochondrial membrane, which actively

uncouples oxidative phosphorylation from ATP synthesis, thereby profoundly increasing energy expenditure2,5. However, recent studies also discovered UCP1-independent thermogenesis mediated

by SERCA2b-induced calcium cycling and creatine-driven substrate cycling6,7. Brown fat thermogenesis was traditionally viewed as a defense mechanism against cold in rodents. A recent

discovery of metabolically active brown fat in adult humans has further implicated BAT thermogenesis as a promising therapeutic target for the treatment of obesity8,9,10.

There are two kinds of UCP1-positive brown adipocytes identified in rodents: traditional brown adipocytes residing in an anatomically defined area (e.g., interscapular (iBAT)) and beige

adipocytes dispersed in white fat depots2,5. Unlike brown adipocytes that develop prenatally and persist throughout lifetime, beige adipocytes are mostly induced by cold and β-adrenergic

agonists2,5. Xue et al. previously reported the existence of a unique subset of beige adipocytes, so-called developmentally induced beige adipocytes, which are transiently induced in

postnatal mice11.

Adipose tissue is innervated by SNS that plays a key role in adipose lipolysis and BAT/beige thermogenesis12,13,14,15,16. Catecholamines released by sympathetic nerve terminals in response

to cold stimulate lipolysis and activate BAT/beige thermogenesis via β-adrenergic receptors17,18. Although extensive studies have been devoted to the role of neuronal network and diverse

neural-endocrine pathways in the regulation of SNS activation19,20, much less is known about the role of SNS-targeted tissues (e.g., BAT and WAT) in regulating the development and activation

of SNS. Recent data demonstrated that S100, a BAT-derived secretory protein, promotes SNS innervation into adipose tissue21. However, the mechanism underlying the neurotrophic effect of

S100 on SNS is not entirely clear.

It is noteworthy that the developmentally induced beige adipocytes appear transiently, peaking at postnatal day 20 and then disappearing thereafter toward adulthood11. However, the

mechanisms mediating the induction and disappearance of the developmental beige adipocytes are not known. Here, we found that the disappearance of the developmental beige cells in adult mice

is associated with the diminished sympathetic innervation in white adipose tissue. Our data indicate that the physiological pathway(s) required to maintain the vigorous innervation of

sympathetic nerves could decline in adult mice, leading to the disappearance of the developmental beige adipocytes. We discovered that the expression of the fat-derived neurotrophic factor

NT-3 is higher in mouse BAT than WAT; NT-3 expression coincides with the appearance of developmentally induced beige adipocytes in postnatal mice and is induced in WAT by cold exposure in

adult mice. Using pharmacological and genetic approaches, we further determined the role of NT-3 and its receptor neurotrophic receptor 3/Tropomyosin receptor kinase C (TRKC) in the

regulation of SNS growth and innervation in adipose tissue and energy metabolism during the cold- and diet-induced thermogenesis.

Xue et al. (a senior author in this paper) previously found that beige adipocytes can be induced in WAT not only during cold exposure in adult animals but also in newborn pups, which peaked

at 20 days of age11. These beige adipocytes then gradually disappeared and replaced by mature white adipocytes by the time when mice were 2 months of age11. Indeed, here we found that Ucp1

mRNA in inguinal WAT (iWAT) was profoundly reduced in 3-month- and 6-month-old mice compared to that of postnatal day 20 (P20) pups (Fig. 1a). A similar reduction of UCP1 protein levels in

iWAT was observed in 3-month-old adult mice compared to P20 pups (Fig. 1b). The reduction of UCP1 expression in adult iWAT was associated with reduced expression of the SNS marker tyrosine

hydroxylase (TH) measured by immunoblotting (Fig. 1b). A similar reduction of UCP1 and TH protein levels was also observed in epidydimal WAT (eWAT) of 3-month-old adult mice compared to that

of P20 pups (Supplementary Fig. 1a). We also established a whole-mount adipose tissue clearing approach (Adipo-Clear) to permit more comprehensive and accurate three-dimensional

visualization of sympathetic innervation with immunostaining of TH22. Compared to iWAT from P20 pups, iWAT from 3-month-old mice had significantly diminished SNS nerve innervation (Fig. 1c,

left panel). Quantitation of SNS innervation in iWAT using Imaris Image Analysis Software revealed that mean nerve fiber density, mean nerve fiber length, and mean nerve branching points

were significantly reduced in iWAT of 3-month-old mice compared to that of P20 pups (Fig. 1c, right panel). A similarly diminished sympathetic innervation was also observed in eWAT of

3-month-old adult mice compared to that of P20 pups (Supplementary Fig. 1b). These data suggest that the reduced SNS innervation may lead to the disappearance of the developmental beige

adipocytes in adult mice.

a Ucp1 mRNA expression in iWAT of 20-day-old postnatal pups, and 3- and 6-month-old mice (n = 5/group, one-way ANOVA, F (2,12) = 18.04, P = 0.0002, *indicates statistical significance vs.

20D with Turkey’s multiple comparisons test). b UCP1 and TH protein levels in iWAT of 20-day-old postnatal pups and 3-month-old adult mice (n = 5/group, * indicates statistical significance

vs. 20D using unpaired two-tailed t test). c Representative images of TH-positive sympathetic nerve innervation in iWAT (left panel, from three replicates/group, scale bar = 2000 µm) and

quantitation of mean nerve fiber density, mean nerve fiber length, and mean branching points normalized to total adipose tissue area (right panel) in iWAT of 20-days-old postnatal pups and

3-month-old adult mice (n = 3/group, * indicates statistical significance vs. 20D using unpaired two-tailed t test). d Tissue distribution of Nt-3 expression (iBAT n = 7; iWAT n = 5; others

n = 4) (*P