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 that associates with autophagosomes.

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 regular diet (RD) (n=11) or three week high-fat diet (HFD) (n=10)58. 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 (TG) mice (n=12) and littermate controls (n=13) on a chow diet. b, Fat mass and lean mass of 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) ASRA TG mice and littermate controls. d, Body weight of male ASRA TG mice (n=5) and littermate controls (n=7) on a high fat diet. e, Cumulative food intake of ASRA TG mice (n=10) and littermate controls (n=11). f, g, Circulating leptin (f) and glucose (g) levels in 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 ASRA TG mice (n=8) and ad libitum-fed littermate controls (n=6). j, Phosphorylation of STAT3 at Tyr705 in hypothalamus of ASRA TG mice and littermate controls that were pre-fasted for 3 hr. k, Phospho-STAT3 immunostaining in ASRA TG mice and littermate controls that were pre-fasted for 5 hr and then injected with leptin. Shown are representative images from 3 mice per group. Bar=200 μm. l, m, Circulating leptin (l) (n=4/group) and glucose levels (m) (n=6/group) of mice injected with either GFP or ASRA adenoviruses. n, Phosphorylation of STAT3 in mice that were pre-fasted for 3 hr. o, Phospho-STAT3 immunostaining in mice that were pre-fasted for 5 hr and then injected with leptin. Shown are representative images from 3 mice per group. 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 HFD. n=19–37/group. d, Body weights of a second cohort of male ASRA ADKO mice (n=17) and littermate controls (n=20) on HFD. 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 HFD. i, Body weights of a second cohort of male ASRA LKO mice (n=15) and littermate controls (n=18) on a HFD. 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 control littermates (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 mice that were pre-fasted for 5 hr and then injected with leptin. Shown are representative images from 3 mice per group. 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 and littermate controls (n=9–12/group) (i) and male LKO and littermate controls (n=10/group) during an 8-hr cold challenging. k, l, Body weight loss after cold challenging. m, n, Body temperature at 0-hr and 8-hr cold challenging.

Figure 5.. rASRA protein binds to leptin receptor and directly antagonize leptin action in cell culture.

a, Phospho-STAT3 immunostaining in COS7 cells transfected with the human leptin receptor. Cells were treated with rASRA (300 nM) for 30 min and leptin (100 nM) was added for another 30 min. Bar=100 μm. b, Western blot analysis of phospho-STAT3 of HEK293 cells treated with rASRA and leptin as described in (a). c, COS7 cells transfected with vector or the human leptin receptor (LepRb) were pre-incubated with vehicle, leptin (100 nM) or rASRA protein (300 nM) for 30 min, and then cy5-labeled rASRA protein (300 nM) was added for another 30 min. Arrows indicate remaining residual binding in the competition assays. Bar=100 μm. d, COS7 cells transfected with human leptin receptor or point mutations were incubated with cy5-labeled rASRA protein (300 nM) for 30 min. Bar=100 μm.

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

a, Three-month-old male mice were ip injected with His-tagged rASRA protein (5.4 nmole/mouse). The presence of rASRA in CSF was detected with a His-tag antibody. b, Three-month-old male mice were tail-vein injected with Cy5 dye (24 nmole/mouse) or Cy5-ASRA (5.4 nmole/mouse). Two-hour post injection, the binding of Cy5-ASRA along with leptin receptor immunostaining was examined in hypothalamic slices. c, 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. d, A single injection (65 μg) of rASRA purified from bacteria was ip injected, and cumulative food intake was measured. n=9 per group. e, Body weight of mice in (c). n=15 per group. f, Twelve serum samples per group at Day 10 were randomly picked up from mice in (c) and leptin level was measured. g, Blood glucose of mice in (c) was measured at Day 10. n=15 per group. h, Basal phospho-STAT3 in the hypothalamus of mice in (c). i, 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. j, Blood glucose of mice in (i) was measured at Day 10. n=15 per group. k, Three-month-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 was examined. l, Daily food intake of mice in (k). n=15 per group. m, Analysis of ASRA mRNA expression in subcutaneous WAT of 770 men in relation to BMI.