A brown fat-enriched adipokine, ASRA, is a leptin receptor antagonist that stimulates appetite

Abstract

The endocrine control of food intake remains incompletely understood, and whether the leptin receptor (LepR)-mediated anorexigenic pathway in the hypothalamus is negatively regulated by a humoral factor is unknown. Here, we identify an appetite-stimulating factor - ASRA - that represents a peripheral signal of energy deficit and orthosterically antagonizes LepR signaling. Asra encodes an 8 kD protein that is abundantly and selectively expressed in adipose tissue and to a lesser extent, in liver. ASRA associates with autophagy vesicles and its secretion is enhanced by energy deficiency. In vivo, fasting and cold stimulate Asra expression and increase its protein concentration in cerebrospinal fluid. Asra overexpression attenuates LepR signaling, leading to elevated blood glucose and development of severe hyperphagic obesity. Conversely, either adipose- or liver-specific Asra knockout mice display increased leptin sensitivity, improved glucose homeostasis, reduced food intake, resistance to high-fat diet-induced obesity, and blunted cold-evoked feeding response. Mechanistically, ASRA acts as a high affinity antagonist of LepR. AlphaFold2-multimer prediction and mutational studies suggest that a core segment of ASRA binds to the immunoglobin-like domain of LepR, similar to the 'site 3' recognition of the A-B loop of leptin. While administration of recombinant wild-type ASRA protein promotes food intake and increases blood glucose in a LepR signaling-dependent manner, point mutation within ASRA that disrupts LepR-binding results in a loss of these effects. Our studies reveal a previously unknown endocrine mechanism in appetite regulation and have important implications for our understanding of leptin resistance.

Figure 1.. Identification of ASRA as a BAT-enriched adipokine induced by fasting and cold and associated with autophagy vesicles.

a, Asra mRNA expression in mouse tissues (n=4). b, Asra mRNA expression in BAT, iWAT, and liver of mice subjected to non-fasting (N), 12-hr fasting (F), and 3-hr re-feeding (R). n=7/group. c, Asra mRNA expression in BAT, iWAT, and liver of mice subjected to 8-hr cold exposure. n=5/group. d, Asra mRNA expression in liver of mice fed with a regular diet (RD) (n=11) or a high-fat diet (HFD) for three weeks (n=10). The RNA-seq data were downloaded from GSE88818 dataset. e, ASRA secretion from BAT ex vivo. f, g, Detection of ASRA in mouse serum (f) or human serum (g) after immunoprecipitation with ASRA antibodies. h, Co-localization of endogenous ASRA and LC3 in mature adipocytes cultured in FBS-free DMEM medium containing high glucose (4.5 g/L) or low glucose (1 g/L) for 6 hr. Bar=50 μm. i, ASRA-GFP localization in mature adipocytes. Bar=50 μm. j, ASRA secretion from mature adipocytes cultured in FBS-free DMEM medium containing high glucose or low glucose for 6 hr. k, ASRA-GFP secretion from HEK293 cells cultured in FBS-free DMEM medium containing high glucose or low glucose for 6 hr (n=6).

Figure 2.. Exogenous expression of ASRA attenuates leptin receptor signaling that leads to hyperphagic obesity.

a, Body weight of male aP2-Asra transgenic mice (TG) (n=12) and littermate controls (n=13) on a chow diet. b, Fat mass and lean mass of aP2-Asra TG mice (n=5) and littermate controls (n=5) at seven-week-old. c, Body length of male (n=9–12) and female (n=8–9) aP2-Asra TG mice and littermate controls. d, Body weight of male aP2-Asra TG mice (n=5) and littermate controls (n=7) on a high fat diet. e, Cumulative food intake of aP2-Asra TG mice (n=10) and littermate controls (n=11). f, g, Circulating leptin (f) and glucose (g) levels in aP2-Asra TG mice (n=5) and littermate controls (n=10) at nine-weeks-old. h, i, Circulating leptin (h) and glucose (i) levels in pair-fed aP2-Asra TG mice (n=8) and ad libitum-fed littermate controls (n=6). j, Phosphorylation of STAT3 at Tyr705 in hypothalamus of aP2-Asra TG mice and littermate controls that were pre-fasted for 3 hr. k, Phospho-STAT3 immunostaining in the ARC, VMH and DMH of hypothalamus in aP2-Asra TG mice and littermate controls that were fasted for 5 hr followed by leptin injection. Bar=200 μm. l, m, Circulating leptin (l) (n=4) and glucose levels (m) (n=6) of mice injected with either GFP or ASRA adenoviruses. n, Phosphorylation of STAT3 in adenovirus-infected mice that were fasted for 3 hr. o, Phospho-STAT3 immunostaining in the ARC, VMH and DMH of hypothalamus in adenovirus-infected mice that were fasted for 5 hr followed by leptin injection. Bar=200 μm.

Figure 3.. Adipose-specific and liver-specific Asra knockout mice have lower food intake and are resistant to DIO.

a, Cumulative food intake of male Asra ADKO mice (n=37) and littermate controls (n=19) on a regular diet. b, Body weights of mice in (a) on a regular diet. n=19–37/group. c, Cumulative food intake of mice in (a) on a high fat diet. n=19–37/group. d, Body weights of a second cohort of male Asra ADKO mice (n=17) and littermate controls (n=20) on a high fat diet. e, Tissue weights of mice in (d). n=17–20/group. f, Cumulative food intake of Asra LKO mice (n=16) and littermate controls (n=16) on a regular diet. g, Body weights of mice in (f) on a regular diet. n=16/group. h, Cumulative food intake of mice in (f) on a high fat diet. i, Body weights of a second cohort of male Asra LKO mice (n=15) and littermate controls (n=18) on a high fat diet. j, Tissue weights of mice in (i). n=15–18/group.

Figure 4.. Peripheral ASRA-deficiency sensitizes leptin action and suppresses acute cold-evoked feeding.

a, Circulating leptin level in five-month-old Asra ADKO (n=8) and littermate controls (n=8). b, Circulating leptin level in five-month-old Asra LKO (n=10) and littermate controls (n=10). c, Blood glucose of Asra ADKO (n=37) and littermate controls (n=19) fed ad libitum or after 12-hr fasting. d, Blood glucose of Asra LKO (n=16) and littermate controls (n=16) fed ad libitum or after 12-hr fasting. e, Phosphorylation of STAT3 in hypothalamus of mice that were pre-fasted for 3 hr. f, Phospho-STAT3 immunostaining in the ARC, VMH and DMH of hypothalamus in ADKO mice and littermate controls that were fasted for 5 hr followed by leptin injection. Bar=200 μm. g, h, Daily food intake in male Asra ADKO (n=9) and littermate controls (n=12) (g) and male LKO (n=10) and littermate controls (n=10) (h) that were treated with leptin (0.5 μg/g body weight) twice a day. i, j, Food intake in male Asra ADKO (n=9) and littermate controls (n=12) (i) and male LKO (n=10) and littermate controls (n=10) (j) during an 8-hr cold challenge. k, l, Body weight loss after cold challenge. m, n, Body temperature at 0-hr and 8-hr cold challenge. o, p, Three-month-old male Asra ADKO mice (o) or four-month-old male Asra LKO mice (p) and littermate controls were either non-fasted (N) or fasted (F) for 12 hr, or were cold challenged for 8 hr. ASRA levels in CSF pooled from 8 mice per group were determined by Western blotting. 0.05 ng of rASRA protein (12 kD) was used as a standard.

Figure 5.. ASRA is a high affinity, orthosteric antagonist of LepR.

a, HEK293 cells were transfected with indicated plasmids and treated with leptin (10 nM) or vehicle along with rASRA at various concentrations for 24 hours. Luciferase activities were measured (n=3). Note, ASRA also inhibits basal LepR activity, *p<0.05 and ***p<0.001 (versus 0 nM ASRA). b, COS7 cells transfected with either vector or LepR plasmids were incubated with Cy5-labeled rASRA protein (300 nM) for 30 minutes. Scale bar = 100 μm. c, Comparison of the binding poses of the A-B loop of Leptin docked to LepR D3 domain (left), with the AF2.3-multimer-predicted complex of the ASRA core peptide bound to LepR D3 domain (right). d, Key contacts between Leptin’s A-B loop with LepR D3 domain (left), highlighting the sidechain of Gln55 fitting into a pocket defined by Trp367, the Cys413–418 disfulfide bridge, and His416 of LepR. ASRA Pro26 docked to the identical pocket of LepR’s D3 (right). e, Equivalent molar concentration of purified ASRA-Flag, ASRA-P26A-Flag, or Leptin-Flag was co-incubated with purified LepR ECD-His or vehicle for 1 hour with a small portion taken as input, followed by precipitation with Ni-NTA beads. After washing, levels of ASRA, leptin and LepR ECD were analyzed by Western blotting. f, HEK293 cells were co-transfected with either vector or LepR ECD-Flag plasmids along with HA-tagged wild-type ASRA, ASRA-P26A, or ²⁶PVL deleted ASRA plasmids. Co-immunoprecipitation was performed in conditioned medium. g, HEK293 cells were transfected with either vector or LepR plasmids. Cells in suspension were then incubated with FITC-labeled rASRA in PBS and flow cytometry was performed. h, Data of average fluorescence intensity per cell were obtained after deduction of background signals and were used to calculate the dissociation constant (Kd) of rASRA binding to LepR (n=3). i, HEK293 cells transfected with LepR plasmids were co-incubated with 10 nM FITC-labeled ASRA and 100 nM unlabeled ASRA or leptin. Flow cytometry was performed and average fluorescence intensity per cell was quantified (n=3).

Figure 6.. rASRA protein induces hyperleptinemia, and stimulates food intake in a leptin receptor signaling-dependent manner.

a, Three-month-old male mice were daily injected with rASRA protein (65 μg per mouse per day), and cumulative food intake was measured. n=15 per group. b, A single injection (65 μg) of rASRA purified from bacteria was ip injected, and cumulative food intake was measured. n=9 per group. c, Body weight of mice in (a). n=15 per group. d, Leptin levels of mice in (a) at day 10. Twelve serum samples per group were randomly picked. e, Blood glucose of mice in (a) at day 10. n=15 per group. f, Basal phospho-STAT3 in the hypothalamus of mice in (a) without fasting. g, Three-month-old male mice were daily injected with rASRA-P26A protein (65 μg per mouse per day), and cumulative food intake was measured. n=15 per group. h, Leptin levels were measured at day 10 from mice in (g). n=15 per group. i, Blood glucose of mice in (g) was measured at day 10. n=15 per group. j, Basal phospho-STAT3 in the hypothalamus of mice in (g) without fasting. k, Three-month-old male ob/ob mice were daily injected with rASRA protein (100 μg per mouse per day), and cumulative food intake was measured. n=15 per group. l, Blood glucose of mice in (k) was measured at day 10. n=15 per group. m, n, Fourteen-week-old male ob/ob mice were treated with rASRA (100 μg per mouse per day) or vehicle along with leptin (2 μg/g body weight per day) for 3 days. Phosphorylation of STAT3 in hypothalamus (m) and food intake (n) were measured. n=15 per group.