Asprosin, a Fasting-Induced Glucogenic Protein Hormone

Abstract

Hepatic glucose release into the circulation is vital for brain function and survival during periods of fasting and is modulated by an array of hormones that precisely regulate plasma glucose levels. We have identified a fasting-induced protein hormone that modulates hepatic glucose release. It is the C-terminal cleavage product of profibrillin, and we name it Asprosin. Asprosin is secreted by white adipose, circulates at nanomolar levels, and is recruited to the liver, where it activates the G protein-cAMP-PKA pathway, resulting in rapid glucose release into the circulation. Humans and mice with insulin resistance show pathologically elevated plasma asprosin, and its loss of function via immunologic or genetic means has a profound glucose- and insulin-lowering effect secondary to reduced hepatic glucose release. Asprosin represents a glucogenic protein hormone, and therapeutically targeting it may be beneficial in type II diabetes and metabolic syndrome.

Copyright © 2016 Elsevier Inc. All rights reserved.

Figures

Figure 1. NPS Mutations Reduce Plasma Insulin Levels while Maintaining Euglycemia in Humans

(A) Overnight fasted plasma glucose and insulin levels from two NPS patients (NPS) and four unaffected control subjects (WT). (B) FBN1 mutations and family pedigrees of the two NPS patients in (A). Standard pedigree symbols are used with affected status noted by filled symbols. (C) 3′ FBN1 mutations in seven NPS patients; two reported herein and five from published case reports. Patient #2 also has a heterozygous missense variant (c.8222T > C) in FBN1 that is predicted to be benign and is not indicated in the figure for clarity. (D) Schematic depicting the clustering of the NPS mutations at the 3′ end of the FBN1 gene. (E) All seven NPS mutations are clustered around the furin cleavage site (RGRKRR motif highlighted in yellow) and are predicted to result in heterozygous ablation of the 140-amino-acid C-terminal polypeptide (asprosin). Non-native amino acids due to a frameshift are shown in red. Patient #2, case 3, and case 5 have a mutation in a splice-donor site that has been predicted to produce the indicated mutant protein (Jacquinet et al., 2014). Data are represented as the mean ± SEM.

Figure 2. Asprosin, the C-Terminal Cleavage Product of Profibrillin, Is a Fasting-Responsive Plasma Protein

(A) Asprosin immunoblot on six individual human plasma samples (lanes 2–7). Bacterially expressed recombinant asprosin was used as a positive control (lane 8). The molecular weight marker is shown in lane 1. (B) Asprosin sandwich ELISA standard curve. (C) Sandwich ELISA was used to measure plasma asprosin levels in overnight fasted humans, mice, and rats (n = 7 in each group). (D) Sandwich ELISA was used to measure plasma asprosin levels in unaffected control subjects (WT), two patients with heterozygous FBN1 frameshift mutations 5′ to the threshold for mRNA nonsense-mediated decay (c.6769-6773del5, c.1328-23_c.1339del35insTTATTTTATT) (proximal truncation 1&2), and two NPS patients (distal truncation 1&2). (E) Sandwich ELISA was used to measure plasma asprosin every 4 hr from circadian C57Bl/6 mice entrained to total darkness (n = 5). The period of feeding is shaded. (F) Sandwich ELISA was used to measure plasma asprosin levels in ad libitum fed or overnight fasted humans, mice, and rats (n = 7 in each group). (G) FBN1 expression across all human tissues using the GTEx human RNaseq database. (H) Various WT C57Bl/6 mouse organs were assessed for Fbn1 mRNA expression by qPCR. (I) Plasma asprosin was assessed using sandwich ELISA on plasma from 13-week-old, 6-hr fasted male WT and Bscl2-null mice. (J) PPARg2 mRNA expression by qPCR and media asprosin by sandwich ELISA were assessed on cultured 3T3-L1 cells with or without exposure to an adipogenic cocktail for 7 days. Cells were washed with PBS and then exposed to glucose-free, serum-free media for 24 hr for assessment of secretion. (K) C3H10T1/2 cells were subjected to the same analysis as in (J). Data are represented as the mean ± SEM. See also Figures S1, S2, and S3.

Figure 3. Increase in Circulating Asprosin Is Associated with Elevated Blood Glucose and Insulin in Mice

(A) Profibrillin (350 kDa) immunoblot on liver lysates 10 days after WT mice were subjected to a onetime tail vein injection of 1011 viral particles of adenovirus carrying cDNA for FBN1 (lanes 3, 4, and 5) or GFP (lanes 1 and 2). Mice were subjected to a 2-hr fast for synchronization prior to sacrifice. (B) Sandwich ELISA was used to measure plasma asprosin levels from mice in (A) (n = 5 in each group). (C) Plasma glucose and insulin levels from mice in (A) (n = 5 in each group). (D) Plasma glucose and insulin levels were measured 10 days after WT mice were subjected to daily subcutaneous injection of 30 mg recombinant asprosin (validated to result in a 50 nM peak plasma level) or recombinant GFP for 10 days (n = 5 in each group). (E) Plasma glucose was measured at the indicated times after a single 30 μg dose of subcutaneous recombinant asprosin or GFP in mice that had been subjected to a 2-hr fast prior to injection (n = 6 in each group). Two-way ANOVA with Bonferroni post test was used to calculate the p value. (F) Plasma insulin was measured 15 min after injection from mice in (E) (n = 6 in each group). (G) Plasma glucose was measured at the indicated times after a single 30 μg dose of subcutaneous recombinant asprosin or GFP in mice that had been subjected to an overnight (~16 hr) fast prior to injection (n = 6 in each group). Two-way ANOVA with Bonferroni post test was used to calculate the p value. (H) Plasma insulin was measured 30 min after injection from mice in (G) (n = 6 in each group). (I) Plasma glucagon, catecholamines, and corticosterone were measured 15–20 min after a single 30 μg dose of subcutaneous recombinant asprosin or GFP in mice that had been subjected to a 2-hr fast prior to injection (n = 6 in each group). Data are represented as the mean ± SEM.

Figure 4. In a Cell-Autonomous Effect, Asprosin Targets the Liver to Increase Plasma Glucose

(A) A glucose tolerance test was performed 2 hr following a subcutaneous injection with 30 mg recombinant asprosin or GFP in WT mice fasted for 2 hr for synchronization prior to injection (n = 6 mice in each group). Two-way ANOVA with Bonferroni post test was used to calculate the p value. (B) An insulin tolerance test was performed 2 hr following subcutaneous injection with 30 μg recombinant asprosin or GFP in WT mice fasted for 2 hr for synchronization prior to injection (n = 6 mice in each group). Two-way ANOVA with Bonferroni post test was used to calculate the p value. (C) Basal (18 hr fasted) and clamped hepatic glucose production was measured using the hyperinsulinemic-euglycemic clamp 10 days after WT mice were subjected to a one-time tail vein injection of 1011 viral particles of adenovirus carrying cDNA for FBN1 or GFP (n = 7 mice in each group). (D) Glucose disposal rate was measured in mice from (C) (n = 7 mice in each group). (E) Media glucose accumulation was measured 2 hr after incubating mouse primary hepatocytes with 0, 4, 8, 16, 32, 64, 138, 275, 550, or 1,100 nM recombinant asprosin or GFP, 1 hr following isolation of cells from WT mice, without plating the cells. Data are represented as the mean ± SEM.

Figure 5. Asprosin Traffics to the Liver In Vivo and Binds the Hepatocyte Surface with High Affinity in a Saturable and Competitive Manner

(A) SPECT scans were performed 15 min after intravenous injection with 150 μCi I125-asprosin, boiled I125-asprosin, or free I125 in live, anesthetized mice previously injected with bismuth as a hepatic contrast agent. Three representative images are shown in axial and coronal planes. (B) Liver asprosin accumulation was measured as liver photon intensity from mice in (A). (C) Tissue radioactivity (normalized to tissue weight) was measured using a γ-counter after sacrificing mice from (A), 45 min following injection (n = 4 mice). (D) Sandwich ELISA was used to measure plasma His tag (recombinant asprosin contains an N-terminal His tag) in WT mice before injection and 15, 30, 60, and 120 min after injection with 30 μg recombinant asprosin. The time taken for peak signal to fall to half-maximal level is indicated by the arrow. (E) The level of biotin at the hepatocyte surface was measured using a colorimetric assay upon incubation of unplated mouse primary hepatocytes with increasing concentration of a recombinant asprosin-biotin conjugate, with (non-specific binding) or without (total binding) 100-fold excess recombinant asprosin in the media. Specific binding (shown in red) was calculated as the difference between the two curves. Data are represented as the mean ± SEM.

Figure 6. Asprosin Uses the cAMP Second-Messenger System and Activates PKA in the Liver

(A) Liver cAMP level was measured 15 min after a single 30-mg dose of subcutaneous recombinant asprosin or GFP in mice that had been subjected to a 2-hr fast prior to injection (n = 6 in each group). (B) Liver PKA activity was measured in mice from (A). (C) Immunoblot analysis for phosphorylated PKA catalytic subunit or for phosphorylated serine/threonine PKA substrate was performed on liver lysates from mice in (A). (D) Hepatocyte cAMP level was measured 10 min after incubating mouse primary hepatocytes with 50 nM recombinant asprosin, 1 hr following isolation of cells from WT mice, without plating the cells. (E) Hepatocyte PKA activity was measured in samples from (D). (F) Hepatocyte PKA activity was measured upon 2 hr of incubation of mouse primary hepatocytes with 0, 4, 8, 16, 32, 64, 138, 275, 550, or 1,100 nM recombinant asprosin or GFP, 1 hr following isolation of cells from WT mice, without plating the cells. (G) Media glucose accumulation was measured 2 hr after incubating mouse primary hepatocytes with 50 nM recombinant asprosin or GFP, with or without a G protein inhibitor (Suramin) (5 mM), 1 hr following isolation of cells from WT mice, without plating the cells. (H) Hepatocyte PKA activity was measured in samples from (G). (I) Media glucose accumulation was measured 2 hr after incubating mouse primary hepatocytes with 50 nM recombinant asprosin or GFP, with or without a competitive antagonist of cAMP-induced activation of PKA (cAMPS-Rp) (200 μM), 1 hr following isolation of cells from WT mice, without plating the cells. (J) Media glucose accumulation was measured 2 hr after incubating mouse primary hepatocytes with 50 nM recombinant asprosin or GFP, or 10 μg/ml glucagon, with or without a non-competitive antagonist of the glucagon receptor (L168,049) (1 μM) 1 hr following isolation of cells from WT mice, without plating the cells. (K) The same analysis was performed as in (J) using 100 μM epinephrine, with or without an antagonist of the β-adrenergic receptor (propranolol) (100 μM). The r GFP and r asprosin controls are common for (J) and (K). (L) Hepatocyte PKA activity was measured 2 hr after incubating mouse primary hepatocytes with 50 nM recombinant asprosin or GFP, with vehicle or 10 mg/l insulin, 1 hr following isolation of cells from WT mice, without plating the cells. (M) Hepatocyte glucose production was measured in samples from (L). Data are represented as the mean ± SEM.

Figure 7. Immunologic or Genetic Asprosin Loss of Function Reduces Hepatic Glucose Production, Plasma Glucose, and Plasma Insulin

(A) Sandwich ELISA was used to measure plasma asprosin levels in eight obese, insulin-resistant male human subjects and eight non-obese, sex- and age-matched control subjects. Pertinent physiological parameters are also presented. (B) Sandwich ELISA was used to measure plasma asprosin levels in male WT mice that had been subjected to a high-fat diet (60% of calories from fat) or normal chow for 12 weeks and from 5-week-old male Ob/+ or Ob/Ob mice upon 2 hr of fasting for synchronization (n = 5 mice in each group). (C) Sandwich ELISA was used to measure plasma asprosin levels at the indicated times after intraperitoneal injection of 500 μg of IgG or anti-asprosin monoclonal antibody, with ad libitum feeding following a 2-hr fast for synchronization, in male WT mice that had been subjected to a high-fat diet (60% of calories from fat) for 12 weeks (n = 6 mice in each group). (D) Plasma glucose was measured in mice from (C). (E) Plasma insulin was measured in mice from (C). (F) Plasma glucose was measured at the indicated times in 5-week-old male WT or Ob/Ob mice after intraperitoneal injection of 500 μg of IgG or anti-asprosin monoclonal antibody, with ad libitum feeding following a 2-hr fast for synchronization (n = 6 mice in each group). (G) Plasma insulin was measured in mice from (F). (H) Sandwich ELISA was used to measure plasma asprosin levels in male WT or homozygous MgR mice following a 2-hr fast for synchronization (n = 5 mice in each group). (I) Plasma glucose was measured in male WT or homozygous MgR mice following a 2-hr fast or following a 24-hr fast (n = 5–7 mice in each group). (J) Plasma insulin was measured in mice from (I). (K) Basal (18-hr fasted) and clamped hepatic glucose production was measured using the hyperinsulinemic-euglycemic clamp in 10-week-old WT or homo-zygous MgR mice (n = 6 mice in each group). (L) The glucose disposal rate was measured in mice from (K) (n = 6 mice in each group). (M) Plasma glucose and insulin were measured in WT or homozygous male MgR mice following an overnight fast, 30 min after subcutaneous injection of 30 μg recombinant asprosin or GFP (n = 5–7 mice in each group). Data are represented as the mean ± SEM. See also Figures S4, S5, S6, and S7.