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.

We have discovered a protein hormone that regulates glucose homeostasis. It is the C-terminal cleavage product of profibrillin (encoded by FBN1). Its absence in humans results in a unique pattern of metabolic dysregulation that includes partial lipodystrophy, accompanied by reduced plasma insulin, while maintaining euglycemia. We name it Asprosin, after the Greek word for white (ασπροσ), because of the reduction in subcutaneous white adipose tissue that is displayed by asprosin-deficient patients and because white adipose tissue appears to be a source of plasma asprosin.

Hormones, their receptors, and the associated signaling pathways make compelling drug targets because of their wide-ranging biological significance (). Protein hormones, as a subclass, have defining characteristics. They usually (but not always) result from cleavage of a larger proprotein and, upon secretion, traffic via the circulation to a target organ. There they bind a target cell using a cell-surface receptor, displaying high affinity, saturability, and ability to be competed off. They stimulate rapid signal transduction using a second-messenger system, followed by a measurable physiological consequence. Given the brain’s strict dependence on glucose as a fuel, plasma glucose levels are precisely regulated by an array of hormones (). Some are secreted in response to nutritional cues, while others respond to glucose itself, producing highly coordinated and precise regulation of circulating glucose levels. Perturbations in this system can cause pathological alteration in glucose levels, often with severe consequences.

Finally, a single subcutaneous administration of asprosin in overnight-fasted MgR mice was sufficient to completely rescue the insulin deficiency displayed by these mice ( Figure 7 M). This result demonstrates that the insulin deficiency displayed by MgR mice is entirely due to a deficiency in circulating asprosin, and not to some indirect effect of their decreased expression of functional fibrillin protein.

To validate immunologic sequestration as a legitimate loss-of-function strategy, we tested FBN1 hypomorphic mice (homozygous MgR mice), which express only ∼20% of the WT FBN1 transcript (). MgR mice displayed a 70% decrease in circulating asprosin ( Figure 7 H). Upon 2 hr of fasting, MgR mice displayed a 2-fold deficit in plasma insulin, while maintaining euglycemia (similar to what we observed with immunologic sequestration of asprosin in ad-libitum-fed mice) ( Figures 7 I and 7J). However, upon 24 hr of fasting, a physiologic situation that eliminates insulin from the circulation of mice ( Figure 7 J), MgR mice displayed fasting hypoglycemia ( Figure 7 I), suggesting that insulin’s buffering effect needs to be eliminated (via a long fast) to unmask the reduction in plasma glucose induced by asprosin loss of function. To confirm this, we performed a hyperinsulinemic-euglycemic clamp study on MgR mice that had been fasted for ∼18 hr (basal). Under such conditions, we found an acute deficit in hepatic glucose production (HGP) in MgR mice compared with WT mice ( Figure 7 K). This result is consistent with clamp results showing an increase in HGP upon asprosin gain of function ( Figures 4 C and 4D). Expectedly, neither clamp study demonstrated a change in whole-body glucose disposal (insulin sensitivity) ( Figures 4 D and 7 L), suggesting that asprosin’s effect on glucose homeostasis is limited to serving as a stimulator of HGP ( Figure S4 ), and any change in plasma insulin levels is indirect and downstream of the change in HGP.

Schematic depicting asprosin action at the hepatocyte surface, leading to use of cAMP as a second messenger, a burst of PKA activity, and glucose release into the circulation, which in turn leads to an insulin response that in time normalizes the plasma glucose.

We found that plasma asprosin levels are pathologically elevated in human subjects with insulin resistance ( Figure 7 A). Similar elevations were seen in two independent mouse models of insulin resistance (diet-induced obesity and Ob mutation) ( Figure 7 B). Intraperitoneal injection of a single dose of an asprosin-specific monoclonal antibody was sufficient to acutely drop plasma asprosin levels at 3 and 6 hr post-injection, with recovery to normal levels at 24 hr ( Figure 7 C). Both ad-libitum-fed (following a 2-hr fast for synchronization) models of mouse insulin resistance showed an acute reduction in plasma insulin levels (while maintaining euglycemia), concurrent with plasma asprosin depletion ( Figures 7 D–7G). To directly test the effect of loss of asprosin on hepatocyte glucose production without the potential insulin compensatory effect, we treated mouse primary hepatocytes with the asprosin-specific antibody prior to incubating them with asprosin. As expected, the asprosin-specific antibody blocked asprosin-mediated hepatocyte glucose release, while a non-specific control antibody had no effect ( Figure S3 D).

(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).

(K) Basal (18-hr fasted) and clamped hepatic glucose production was measured using the hyperinsulinemic-euglycemic clamp in 10-week-old WT or homozygous MgR mice (n = 6 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).

(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).

(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).

(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).

(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).

(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.

Exposing mice to a single 30-μg dose of recombinant asprosin for 20 min (validated to result in a 50-nM peak level) was sufficient to increase liver cyclic AMP (cAMP) and protein kinase A (PKA) activity ( Figures 6 A–6C). Identical results were obtained upon incubating mouse primary hepatocytes with recombinant asprosin for 10 min ( Figures 6 D and 6E). Hepatocyte PKA activity increased in a dose-responsive manner upon addition of recombinant asprosin ( Figure 6 F), similar to what we observed with hepatocyte glucose release ( Figure 4 E). The effects of asprosin on both hepatocyte glucose release and PKA activation were blocked by suramin, a general heterotrimeric G protein inhibitor ( Figures 6 G and 6H). In addition, asprosin-mediated hepatocyte glucose release could be blocked by using cAMPS-Rp, a competitive antagonist of cAMP binding to PKA ( Figure 6 I). These results demonstrate that asprosin increases hepatocyte glucose release by employing the G protein-cAMP-PKA axis in vivo and in vitro. Because glucagon and catecholamines also employ the same intracellular signaling axis, we tested the impact of inhibiting the glucagon receptor or the β-adrenergic receptor on the ability of asprosin to enhance hepatocyte glucose release. While the respective inhibitors completely blocked the effects of glucagon or epinephrine, they had no impact on the ability of asprosin to influence hepatocyte glucose release ( Figures 6 J and 6K). This suggests that asprosin uses a cell-surface receptor system that is distinct from those used by glucagon and catecholamines. Since insulin is known to induce a reduction in intracellular cAMP (via activation of the Gpathway), we tested whether insulin would oppose asprosin’s effect on hepatocyte PKA activation and glucose release, which is demonstrated to be due to an increase in intracellular cAMP. Indeed, we found that insulin suppressed asprosin-mediated hepatocyte PKA activation ( Figure 6 L) and glucose release ( Figure 6 M).

(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.

(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).

(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.

(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.

(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 μM), 1 hr following isolation of cells from WT mice, without plating the cells.

(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.

(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.

(C) Immunoblot analysis for phosphorylated PKA catalytic subunit or for phosphorylated serine/threonine PKA substrate was performed on liver lysates from mice in (A).

(A) Liver cAMP level was measured 15 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).

To examine specific binding of asprosin by hepatocytes, we incubated mouse primary hepatocytes with an increasing amount of an asprosin-biotin conjugate, washed with PBS, and measured the relative level of biotin at the hepatocyte surface. Asprosin bound the hepatocyte surface in a dose-responsive and saturable manner ( Figure 5 E). Repeating the same procedure in the presence of 100-fold excess unconjugated asprosin abolished the effect, suggesting competition for potential receptor binding sites ( Figure 5 E).

We labeled recombinant asprosin with iodine-125 (I) and injected it intravenously in mice, followed by single-photon emission computerized tomography (SPECT) scans to identify sites of accumulation. An equivalent amount of free Ior I-Asprosin that was boiled for 5 min (to induce loss of the asprosin tertiary structure) was used as a control. In contrast to the accumulation patterns for free Iand boiled I-Asprosin, SPECT scans in coronal and axial planes ( Figure 5 A), and mean liver photon intensity ( Figure 5 B), both showed that I-Asprosin trafficked primarily to the liver and that asprosin’s tertiary structure was essential for its liver recruitment. In accord with liver trafficking, gamma counting of blood and viscera showed that recombinant blood asprosin levels decrease in concert with the increased liver levels ( Figure 5 C). To measure plasma half-life, we used a sandwich ELISA system targeting the N-terminal His-tag on the recombinant asprosin protein at 15, 30, 60, and 120 min following subcutaneous injection. Consistent with our results using IV infusion of I-Asprosin, plasma His-tagged asprosin showed a half-life of approximately 20 min and a peak level of 50 nM that was achieved 20 min post-injection ( Figure 5 D).

(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.

(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.

(A) SPECT scans were performed 15 min after intravenous injection with 150 μCi I 125 -asprosin, boiled I 125 -asprosin, or free I 125 in live, anesthetized mice previously injected with bismuth as a hepatic contrast agent. Three representative images are shown in axial and coronal planes.

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

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

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

Glucose and insulin tolerance tests in mice exposed to a single dose of recombinant asprosin showed little evidence of altered glucose uptake (in response to insulin) in peripheral organs, such as muscle or fat (unchanged slope of glucose disposal), but showed altered peak glucose levels, again implicating the liver ( Figures 4 A and 4B ). To confirm the liver as the site of asprosin action, we performed the hyperinsulinemic-euglycemic clamp. This test unequivocally showed that elevated plasma asprosin results in increased hepatic glucose production ( Figure 4 C), but has no impact on the ability of peripheral organs to take up glucose in response to insulin ( Figure 4 D). To test whether the effect of asprosin on the liver is cell autonomous, we exposed isolated primary mouse hepatocytes to increasing concentrations of recombinant asprosin or GFP for 2 hr. Media from cells exposed to asprosin showed an increase in glucose concentration in a dose-dependent manner, demonstrating a direct effect of asprosin on hepatocytes ( Figure 4 E).

(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.

(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 10 11 viral particles of adenovirus carrying cDNA for FBN1 or GFP (n = 7 mice in each group).

(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.

(A) A glucose tolerance test was performed 2 hr following a 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.

In order to understand acute responses, we injected a single dose of recombinant asprosin subcutaneously in mice that had been subjected to a preceding 2-hr fast, and measured plasma glucose at 15, 30, 60, and 120 min post-injection. Mice were denied access to food through the length of the experiment. A single asprosin dose resulted in an immediate spike in blood glucose levels ( Figure 3 E). This resulted in compensatory hyperinsulinemia (measured at the 15-min time point) ( Figure 3 F), which normalized blood glucose levels by 60 min post-injection ( Figure 3 E). Similar results were obtained in mice that were subjected to a preceding overnight fast, although the rate of the resultant blood glucose spike was somewhat slower, likely due to fasting-induced depletion of glucogenic substrates ( Figures 3 G and 3H). These results implicated the liver as the target organ for asprosin due to its role as the primary site for stored glucose (as glycogen), which is rapidly released into the circulation during fasting. Interestingly, asprosin treatment had no effect on plasma levels of catabolic hormones (glucagon, catecholamines, and glucocorticoids), known to induce hepatic glucose release ( Figure 3 I).

We employed ectopic overexpression of full-length FBN1 using an adenovirus in the hope that the transduced organ (in this case, the liver, which normally shows low endogenous FBN1 expression [ Figures 2 G and 2H] and is the primary target of adenoviral infection) would process the resultant profibrillin and secrete asprosin into the circulation. This strategy showed robust overexpression of profibrillin protein in the liver and a 2-fold elevation in plasma asprosin ( Figures 3 A and 3B ). The second strategy involved daily subcutaneous injection of bacterially expressed asprosin (validated to result in a 50 nM peak level 20 min after injection; Figure 5 D) or recombinant GFP as a control. 10 days of exposure to increased plasma asprosin in either a continuous (adenoviral overexpression) or pulsatile fashion (daily recombinant asprosin injection) resulted in elevated glucose and insulin levels in 2-hr fasted mice using both experimental strategies ( Figures 3 C and 3D). This result demonstrated that bacterially expressed recombinant asprosin retains the biological activity displayed by its endogenously expressed counterpart and that elevation of circulating asprosin is sufficient to increase blood glucose and insulin levels.

(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).

(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.

(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.

(D) Plasma glucose and insulin levels were measured 10 days after WT mice were subjected to daily subcutaneous injection of 30 μg recombinant asprosin (validated to result in a 50 nM peak plasma level) or recombinant GFP for 10 days (n = 5 in each group).

(A) Profibrillin (350 kDa) immunoblot on liver lysates 10 days after WT mice were subjected to a one-time tail vein injection of 10 11 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.

We examined the FBN1 mRNA profile across all human tissues using the Genotype-Tissue Expression Project (GTex) RNaseq dataset and found that adipose tissue demonstrated the highest FBN1 mRNA expression across all tissues ( Figure 2 G). To confirm this in mice, we assessed the Fbn1 expression profile across various metabolically important organs. Consistent with the human profile, we found that white adipose tissue displayed the highest Fbn1 mRNA expression ( Figure 2 H). Given that white adipose tissue is a well-known endocrine organ (), we examined whether it could serve as a source of circulating asprosin. We assessed plasma levels of asprosin in mice that had been subjected to genetic ablation of adipose tissue. We used Bscl2−/− mice for this purpose. BSCL2 deficiency results in Berardinelli-Seip congenital lipodystrophy in humans (knockout mice mimic this phenotype) with a 60%–70% reduction in adipose tissue (). In such mice we detected a ∼2-fold reduction in plasma asprosin ( Figure 2 I). The next experimental strategy we employed was to assess whether adipocytes in culture were capable of generating and secreting asprosin. For this, we differentiated two distinct adipogenic cell lines, 3T3-L1 and a mesenchymal stem cell line (C3H10T1/2), into mature adipocytes ( Figures 2 J and 2K) and subjected the cell culture media to asprosin protein analysis. We found robust accumulation of asprosin in serum-free culture media from mature adipocytes, but not from preadipocytes ( Figures 2 J and 2K), suggesting that adipocytes are capable of generating and secreting asprosin.

To assess daily fluctuations in circulating asprosin concentrations, mice were kept in a 12-hr light/12-hr dark cycle for 7 days to establish entrainment and were subsequently kept in constant darkness. Plasma was then isolated from these mice at 4-hr intervals and subjected to asprosin ELISA analysis. We found that plasma asprosin displays circadian oscillation with an acute drop in levels coinciding with the onset of feeding ( Figure 2 E). In the opposite direction, overnight fasting in humans, mice, and rats resulted in increased circulating asprosin ( Figure 2 F).

To measure circulating asprosin levels, we developed a sandwich ELISA ( Figure S3 A). We constructed a standard curve using recombinant asprosin and used it to calculate plasma and media levels ( Figure 2 B). As expected, the asprosin sandwich ELISA displayed high specificity using media from WT and Fbn1−/− cells ( Figure S3 C). Asprosin was found to be present in plasma at consistent nanomolar levels in humans, mice, and rats ( Figure 2 C). Interestingly, NPS patients displayed a greater reduction in circulating asprosin level than predicted from their heterozygous genotype, compared not only with WT control subjects but also when compared with patients that have heterozygous truncations of profibrillin sufficiently N-terminal so as to undergo mRNA nonsense-mediated decay ( Figure 2 D). This suggests that the mutant profibrillin that is predicted to be expressed in NPS cells (due to escape from mRNA NMD) exerts a dominant-negative effect on secretion of asprosin from the WT allele. We tested this concept by overexpressing the truncated, mutant version of profibrillin in WT cells and found that this interfered with the ability of those cells to secrete asprosin into the media, compared with overexpression of an irrelevant protein, such as GFP ( Figures S3 E and S3F).

Data are represented as the mean ± SEM. For evaluation of statistical significance, unpaired Student’s t test was used. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

(F) Asprosin was assessed by sandwich ELISA on media from (E). Cells were grown in regular media, washed with PBS and then exposed to serum-free media for 24 hr for assessment of secreted proteins.

(E) FBN1 mRNA expression by qPCR in WT human dermal fibroblasts transfected with CMV driven vectors encoding GFP or C-terminal truncated human profibrillin (carrying the human NPS mutation c.8206_8207InsA that induces a frame-shift, premature stop codon, and ablation of 136 of the 140 C-terminal profibrillin amino acids). Monensin, a pharmacological secretion blocker, was used as a negative control.

(D) Media glucose accumulation was measured 2 hr after incubating mouse primary hepatocytes with 50 nM recombinant asprosin or GFP, following 1 hr of pretreatment with 50 ug IgG or anti-asprosin monoclonal antibody, 1 hr following isolation of cells from WT mice, without plating the cells.

(C) Asprosin sandwich ELISA on serum-free media from WT and Fbn1 null mouse embryonic fibroblasts. Cells were grown in regular media, washed with PBS and then exposed to serum-free media for 24 hr for assessment of secreted proteins.

(A) Sequence of human recombinant asprosin expressed in E.coli. The N-terminal his tag is shown in yellow and the capture antibody and detection antibody epitopes (for sandwich ELISA) are bolded and underlined.

Development and Validation of the Asprosin Sandwich ELISA and Its Use for Assessment of the Dominant-Negative Effect of Mutant Profibrillin on Asprosin Secretion, Related to Figure 2

Asprosin is encoded by the ultimate two exons of FBN1. Exon 65 encodes 11 amino acids, while exon 66 encodes 129 amino acids. Together, those two exons display a somewhat higher vertebrate evolutionary conservation score compared with the rest of the profibrillin coding sequence ( Figures S1 A and S1B). We developed an asprosin-specific monoclonal antibody and validated its specificity for asprosin using Fbn1 wild-type (WT) and null cells ( Figure S1 C). Immunoblotting human plasma with the anti-asprosin antibody shows a single protein running on SDS-PAGE at ∼30 kDa, while bacterially expressed recombinant asprosin runs at ∼17 kDa ( Figure 2 A). Asprosin is predicted to have three N-linked glycosylation sites and potentially other post-translational modifications that are lacking in bacteria ( Figures S1 D and S1E). This likely explains the difference in molecular weight between mammalian and bacterially expressed asprosin. Indeed, using mammalian cells for expression of asprosin produced a protein that was secreted into the media and ran on SDS-PAGE at the same molecular weight (∼30 kDa) () as we observed in human plasma, cell lysates and media from mouse embryonic fibroblasts, and cell/tissue lysates from cultured adipocytes and mouse white adipose tissue ( Figures 2 A, S1 C, S2 A, and S2B ).

(B) Asprosin and profibrillin immunoblots on cultured 3T3-L1 cells with and without exposure to an adipogenic cocktail for 7 days. Adipogenesis was confirmed by visualization of lipid droplets (not shown) and expression of the adipogenic master gene – PPARg2 ( Figure 2 J).

(J) PPARγ2 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.

(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).

(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.

(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).

(C) Sandwich ELISA was used to measure plasma asprosin levels in overnight fasted humans, mice, and rats (n = 7 in each group).

(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) Base-pair conservation using the PhyloP tool across 100 vertebrate species is depicted for FBN1 exons 1-64, exon 65-66 which encode asprosin and exon 66 alone (which contributes 129 out of the 140 asprosin amino acids). Exon 66 contains the 3′ UTR that was excluded from the analysis.

(A) Human FBN1 gene and its evolutionary conservation across 100 vertebrate species is depicted using the UCSC genome browser. The asprosin coding region is boxed.

Mammalian Asprosin Is Evolutionarily Well Conserved, Has a Molecular Weight of ∼30 kDa, and Is Predicted to Contain 3 N-Linked Glycosylation Sites, Related to Figure 2

Profibrillin is translated as a 2,871-amino-acid long proprotein, which is cleaved at the C terminus by the protease furin (). This generates a 140-amino-acid long C-terminal cleavage product, in addition to mature fibrillin-1 (an extracellular matrix component). All seven NPS mutations are clustered around the cleavage site, resulting in heterozygous ablation of the C-terminal cleavage product (asprosin) ( Figure 1 E), whose fate and function were unknown.

Whole-exome and Sanger sequencing identified de novo, heterozygous 3′ truncating mutations in FBN1 in both patients ( Figures 1 B and 1C). Upon reaching the genetic diagnosis, we searched the literature for similar cases and discovered five single-patient case reports of NPS associated with FBN1 3′ truncating mutations (). All seven subjects, including the two reported herein, were diagnosed with NPS, and all have truncating mutations within a 71-bp segment at the 3′ end of the FBN1 coding region, displaying tight genotype-phenotype correlation ( Figure 1 D). All seven mutations occur 3′ to the last 50 nt of the penultimate exon and are therefore predicted to escape mRNA nonsense-mediated decay (NMD), leading to expression of a mutant, truncated profibrillin protein ( Figure 1 E).

Progeroid facial features and lipodystrophy associated with a novel splice site mutation in the final intron of the FBN1 gene.

Marfan syndrome with neonatal progeroid syndrome-like lipodystrophy associated with a novel frameshift mutation at the 3′ terminus of the FBN1-gene.

Further evidence for a marfanoid syndrome with neonatal progeroid features and severe generalized lipodystrophy due to frameshift mutations near the 3′ end of the FBN1 gene.

Neonatal progeroid syndrome (NPS) was first described in 1977 (OMIM: 264090 ) and is characterized by congenital, partial lipodystrophy, predominantly affecting the face and extremities (). Although NPS patients appear progeroid because of facial dysmorphic features and reduced subcutaneous fat, the term is a misnomer as the patients do not display accelerated aging. We identified two unrelated individuals with NPS. We examined their glucose and insulin homeostasis status, since both partial and generalized lipodystrophic disorders are frequently associated with insulin resistance (). Contrary to this notion, overnight-fasted plasma insulin levels from our NPS patients were 2-fold lower than unaffected subjects, while maintaining euglycemia ( Figure 1 A).

(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 ().

(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.

(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.

Discussion

Whether circulating asprosin concentration is experimentally decreased (genetic depletion in NPS patients, genetic depletion in MgR mice, or acute removal via immunologic sequestration in mice) or increased (adenovirus-mediated overexpression or direct recombinant protein injection), the result is a corresponding change in plasma glucose and insulin. With asprosin loss of function, hypoglycemia is only unmasked upon elimination of a β-cell-mediated corrective action, by fasting mice long enough to drive insulin levels close to zero, leaving little room for β cells to further decrease insulin secretion and normalize plasma glucose.

It seems surprising that a nutritionally responsive hormone that displays circadian oscillation ( Figure 2 E) would be derived from what would seem a relatively “static” structural/ECM protein. This led us to examine the profile of the FBN1 transcript using a publicly available circadiomics database ( http://circadiomics.igb.uci.edu ). Interestingly, the Fbn1 transcript displays robust daily circadian oscillation in several tissues, such as the heart, adrenal, lung, white fat, and kidney. The notion that fibrillin-1 is a static, structural molecule may need further examination. Another pertinent question relates to the primary tissue of origin of asprosin. We demonstrate adipose to be one of the sources of plasma asprosin. This observation is consistent with the known function of adipose as an endocrine organ and a sensor/modulator of energy homeostasis. However, it is worth noting that organs besides adipose could also serve as sources of plasma asprosin, given the fairly high expression of FBN1 in several organs.

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Saint-Hillier Y. Acute oral calcium load decreases parathyroid secretion and suppresses tubular phosphate loss in long-term renal transplant recipients. Figure S5 White Adipose Tissue-Mediated Secretion of Asprosin Is Suppressed by Glucose in a Negative-Feedback Loop, Related to Figure 7 Show full caption (A) PPARg2 mRNA expression by qPCR, was assessed on cultured 3T3-L1 cells that had been exposed to an adipogenic cocktail for 7 days. (B) Media asprosin by sandwich ELISA was assessed on cultured 3T3-L1 cells that had been exposed to an adipogenic cocktail for 7 days. Cells were washed with PBS and then exposed to either glucose-free or glucose-containing serum-free media for 24 hours for assessment of secretion. (C) PPARg2 mRNA expression by qPCR, was assessed on cultured C3H10T1/2 cells that had been exposed to an adipogenic cocktail for 7 days. (D) Media asprosin by sandwich ELISA was assessed on cultured C3H10T1/2 cells that had been exposed to an adipogenic cocktail for 7 days. Cells were washed with PBS and then exposed to either glucose-free or glucose-containing serum-free media for 24 hours for assessment of secretion. (E) Asprosin immunoblot on cells lysates from cultured 3T3-L1 and C3H10T1/2 cells with or without exposure to an adipogenic cocktail for 7 days. Mature adipocytes were exposed to serum-free media with or without glucose for 24 hr. Preadipocytes were only exposed to serum-free media without glucose for the same duration. (F) Asprosin was assessed by sandwich ELISA on plasma from 12-week old, male, WT C57Bl/6 mice injected with saline or streptozotocin i.p. (injected three times over the course of 2 weeks until blood glucose values by a handheld glucometer were > 600 mg/dl). Mice were subjected to a 2 hr fast for synchronization prior to sacrifice. Data are represented as the mean ± SEM. For evaluation of statistical significance, unpaired Student’s t test was used. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Since asprosin functions to increase plasma glucose levels, and circulating asprosin levels are increased by fasting (a baseline glucose condition) ( Figure 2 F) and decreased by feeding (a high glucose condition) ( Figure 2 E), we hypothesized that glucose could serve as a suppressor of plasma asprosin levels in a negative-feedback loop. To determine this, we subjected mature adipocytes in culture to high glucose levels and found that this treatment was sufficient to strongly inhibit the accumulation of asprosin in media, compared with adipocytes subjected to glucose-free conditions ( Figures S5 B and S5D). We detected no decrease in intracellular asprosin protein with glucose addition ( Figure S5 E), suggesting that glucose-mediated downregulation of extracellular asprosin levels does not occur at the level of transcription, biosynthesis, or processing. To confirm this result in vivo, we subjected WT mice to streptozotocin (STZ) treatment, which is known to ablate pancreatic β cells, resulting in high blood glucose. In such mice plasma asprosin was found to be far lower than in mice with normal blood glucose ( Figure S5 F). Together, these in vitro and in vivo results are consistent with the notion that glucose serves as a negative influencer of plasma asprosin levels in a negative-feedback loop and is consistent with the regulation of other major hormones (for example, calcium suppresses parathyroid hormone secretion and glucose suppresses glucagon secretion) ().

Lönnqvist et al., 1998 Lönnqvist L.

Reinhardt D.

Sakai L.

Peltonen L. Evidence for furin-type activity-mediated C-terminal processing of profibrillin-1 and interference in the processing by certain mutations. Nickel, 2003 Nickel W. The mystery of nonclassical protein secretion. A current view on cargo proteins and potential export routes. Figure S6 Intracallular Asprosin Is Capable of Secretion Despite Absence of an N-Terminal Signal Peptide, Related to Figure 7 Show full caption (A) The human asprosin coding sequence (driven by a CMV promoter) or an empty vector were transfected into Fbn1 null mouse embryonic fibroblasts. 48 hr later, asprosin-transfected cells were washed with PBS and then exposed to glucose free or glucose containing serum-free media for 24 hr for assessment of secretion. Empty vector transfected cells were only exposed to glucose free serum-free media for the same duration. Human FBN1 exon 66 (which encodes 129 of the 140 asprosin amino acids) expression was determined by qPCR. (B) Asprosin was assessed by sandwich ELISA on serum-free media from S6A. Cells were grown in regular media, washed with PBS and then exposed to serum-free media with or without glucose for 24 hr for assessment of secreted proteins. Cells transfected with empty vector were only exposed to serum-free media without glucose. Data are represented as the mean ± SEM. For evaluation of statistical significance, unpaired Student’s t test was used. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Generally, protein hormones are processed via endoplasmic reticulum and Golgi pathways and stored in intracellular granules, followed by secretion in response to appropriate cues. Consistent with this, we detected processed asprosin intracellularly in cultured fibroblasts, mouse white adipose tissue, and cultured adipocytes ( Figures S1 C, S2 A, and S2B). Asprosin has been shown to retain the ability to be secreted from the cell, despite the lack of a signal peptide. This was demonstrated by overexpressing just the asprosin coding exons in mammalian cells, followed by detection of asprosin in the media (). We repeated this assay by overexpressing asprosin-encoding cDNA in Fbn1−/− cells (to prevent contamination from endogenous asprosin), and asprosin secretion was detected in the media ( Figures S6 A and S6B). Furthermore, asprosin secretion could be suppressed by glucose ( Figure S6 B), consistent with the phenomenon observed in cultured adipocytes ( Figures S5 B and S5D). Several extracellular proteins, such as FGF-1, FGF-2, and IL-1β, lack an N-terminal signal peptide and are secreted using non-classical or leaderless secretion (), as demonstrated by asprosin.