1. Adipsin Is an Adipokine that Improves β Cell Function in Diabetes
James C. Lo, Sanda Ljubicic, Barbara Leibiger, Matthias Kern, Ingo B. Leibiger, Tilo Moede, Molly E. Kelly, Diti Chatterjee Bhowmick, Incoronata Murano, Paul Cohen, Alexander S. Banks, Melin J. Khandekar, Arne Dietrich, Jeffrey S. Flier, Saverio Cinti, Matthias Blüher, Nika N. Danial, Per-Olof Berggren, Bruce M. Spiegelman 
Cell 
Volume 158, Issue 1, Pages (July 2014)
DOI: /j.cell
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2. Cell  , 41-53DOI: ( /j.cell )
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3. Figure 1
Adipsin Regulates Adipose Tissue Inflammation and Protects against Diabetes
(A) WT and Adipsin−/− mice were fed a HFD for 4 months, starting at 1 month of age, and visceral adipose tissues were assayed for immune infiltration by qPCR.
(B and C) WT and Adipsin−/− adipose tissues were stained for crown-like structures (CLS). Representative pictures are shown (B), and numbers of CLS (C) were quantitated. Scale bar, 100 μm.
(D and E) WT and Adipsin−/− adipose tissues were stained with toluidine blue for mast cells. Representative pictures are shown (D), and numbers of mast cells (E) were quantitated. Scale bar, 25 μm.
(F) Glucose tolerance test (GTT) was performed on WT and Adipsin−/− mice fed a HFD for 16 weeks with measurement of blood glucose concentrations.
(G) Insulin tolerance test (ITT) was performed on WT and Adipsin−/− mice fed a HFD for 18 weeks with measurement of blood glucose concentrations. For GTT and ITT experiments, n = 12–16 per genotype.
Error bars represent SEM. ∗p < 0.05 and ∗∗p < See also Figures S1 and S2.
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4. Figure 2 Adipsin Regulates Insulin Secretion In Vivo and In Vitro
(A) WT and Adipsin−/− mice were fed a HFD diet for 4 months, starting at 1 month of age, and were challenged with i.p. glucose injections. Plasma insulin levels were assayed. n = 8–12 mice per genotype.
(B and C) Pancreata from WT and Adipsin−/− mice fed a HFD diet for 4 months were collected, and insulin immunohistochemistry staining was performed. Representative pictures are shown (B), and β cell area was quantitated (C). Scale bar, 400 μm.
(D) Glucose-stimulated insulin secretion assay on islets from WT and Adipsin−/− mice fed a HFD diet.
Error bars represent SEM. ∗p < 0.05 and ∗∗p < See also Figure S3.
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5. Figure 3
Restoration of Adipsin Improves Insulin Secretion and Glucose Homeostasis
(A) Diabetic db/db mice (3 months old) were treated i.v. with 2 × 109 IFU of control lacZ or adipsin adenovirus vectors, and 5 days later serum adipsin was assessed by western blot.
(B and C) Control lacZ- and adipsin-transduced mice were challenged by an i.p. GTT with measurements of blood glucose (B) and plasma insulin (C). n = 6 mice per group.
(D) Gluconeogenesis was determined by hepatic Pepck and G6pc gene expression.
Error bars represent SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < See also Figure S4.
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6. Figure 4 C3a Stimulates Pancreatic β Cells to Secrete Insulin
(A and B) Relative expression of Adipsin (A) and C3 (B) in liver, quadriceps muscle (quad), islets, epididymal (epi), and inguinal (ing) fat were quantitated by qPCR.
(C–E) Flow cytometry was performed on islet cells with antibodies to receptors C3aR1 (C), C5aR1 (D), and C5L2 (E).
(F and G) Islets from WT mice on a chow diet (F) or HFD (G) were subjected to a GSIS assay with recombinant C3a or C5a (100 nM) at the indicated concentrations of glucose.
(H and I) db/db mice (3 months old) transduced with lacZ or adipsin adenovirus were treated with vehicle or C3aR antagonist (SB ) and subjected to GTT with measurements of blood glucose (H) and plasma insulin (I). Data were pooled from two experiments. Statistics for GTT assays between groups: lacZ/vehicle versus adipsin/vehicle, p = 0.05; adipsin/vehicle versus adipsin/SB , p < 0.01; lacZ/SB versus adipsin/vehicle, p < n = 12–15 mice per group.
Error bars represent SEM. ∗p < See also Figure S5.
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7. Figure 5
C3a Stimulates Cytoplasmic Free Ca2+ ([Ca2+]i) and Oxygen Consumption in Islets
(A and B) Islets were treated with C3a (100 nM) in conjunction with 30 mM KCl (A) or 0.25 mM tolbutamide (B) and assayed for insulin secretion.
(C–E) [Ca2+]i was measured in islets treated with control or C3a and stimulated with 20 mM glucose, washed with 3 mM glucose, stimulated with KCl, and then washed with 3 mM glucose. The peak [Ca2+]i (D) and [Ca2+]i area under the curve (E) are quantified.
(F) Intracellular ATP levels were determined after treatment of islets with C3a at 3 or 20 mM glucose. Data were pooled from three experiments.
(G and H) Oxygen consumption rates (OCR) were measured in islets treated with C3a and the following treatments: 20 mM glucose, oligomycin, rotenone, and antimycin A. Statistical analysis was performed on traces prior to oligomycin. Data were pooled from three experiments.
(H) ATP-coupled respiration of islets is quantified.
Error bars represent SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p <
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8. Figure 6
Adipsin Is Decreased in Patients with Type II Diabetes Mellitus with β Cell Failure
(A–F) T2DM patients were classified according to those on oral metformin therapy (type II diabetes mellitus, T2DM) or T2DM patients with β cell failure (T2DM-βCF) for those on insulin therapy.
(A–C) Visceral and subcutaneous (SubQ) adipose tissue samples from T2DM and T2DM-βCF patients were analyzed for Adipsin (A), Adiponectin (B), and Leptin (C) mRNA levels.
(D–F) Circulating adipsin (D), adiponectin (E), and leptin (F) levels were measured from blood samples of T2DM and T2DM-βCF patients. Adiponectin and leptin are plotted according to male and female.
Error bars represent SEM. ∗p < See also Table S1.
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9. Figure S1
Circulating Adipsin Levels Wane with Obesity, Related to Figure 1
(A) WT mice were placed on a regular chow or a high fat diet starting at 1 month of age for the indicated number of weeks (w) and sera were collected for analysis of adipsin protein by Western blot.
(B) Adipsin expression in WT and Adipsin−/− mice on a regular diet at the corresponding ages, in months (m). Sera were collected for analysis of adipsin protein by Western blot.
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10. Figure S2
Glucose Homeostasis of Adipsin-Deficient Mice on Regular and Short-Term High-Fat Diets, Related to Figure 1
(A and B) Glucose tolerance test (GTT) was performed by intraperitoneal (i.p.) injection of glucose to WT and Adipsin−/− mice fed a (A) regular diet or a (B) HFD for 8 weeks starting at 1 month of age with measurement of blood glucose concentrations at the indicated times. Error bars represent SEM.
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11. Figure S3
Insulin Secretion in Adipsin−/− Mice and Islets from Regular and HFD-Fed Mice in Response to Glucose, Related to Figure 2
(A and B) WT and Adipsin−/− mice were fed a HFD diet starting at 1 month of age for 7 weeks (A) or 4 months (B) and challenged with i.p. glucose injections. Glucose (A) and (B) and plasma insulin (A) levels were assayed at 20 min postglucose challenge or the indicated times. N = 5-12 mice per genotype.
(C) The number of islet-associated macrophages from pancreata of WT and Adipsin−/− mice on a HFD diet for 4 months was analyzed by immunofluorescence staining of Mac-2 and insulin.
(D) Islets were isolated from WT and Adipsin−/− mice (3 month old) fed a regular diet and assessed by static glucose-stimulated insulin secretion (GSIS) assay at the indicated concentrations of glucose.
Error bars represent SEM.
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12. Figure S4
Hepatic Gene Expression in Mice Treated with Adipsin, Related to Figure 3
(A–E) Relative expression of Adipsin (A), Il1b (B), Il6 (C), Crp (D) and Tnf (E) from the livers of db/db mice (3 month old) treated with lacZ or adipsin adenovirus. Samples were quantitated by qPCR.
(F) Changes in weight of lacZ or adipsin-treated mice during the course of the experiment. Data are pooled from 2 experiments.
∗p < Error bars represent SEM.
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13. Figure S5
Relative Expression Pattern of Complement Factors and Their Role in Insulin Secretion, Related to Figure 4
(A–C) Relative expression of Complement factor B (A), C3ar1 (B), and Insulin (C) in liver, quadriceps muscle (quad), islets, epididymal (epi) and inguinal (ing) fat. Samples were quantitated by qPCR.
(D) Western blot for C3a/C3a-desArg in the sera of WT mice on regular diet. C3a standards with amounts (ng) as indicated and 1 μl of sera loaded.
(E) Islets were isolated from WT mice (3 month old) fed a regular diet and assessed by static glucose-stimulated insulin secretion (GSIS) assay with adipsin or C3 proteins at the indicated concentrations of glucose.
(F) Isolated islets from WT and Adipsin−/− mice fed a HFD diet for 5 months were subjected to a GSIS assay in the presence or absence of recombinant C3a (100 nM) and the indicated concentrations of glucose.
(G) db/db mice (3 month old) transduced with lacZ or adipsin adenovirus were treated with vehicle or C3aR antagonist (SB ) at a dose of 30 mg/kg bid for 3 days and subjected to an i.p. glucose challenge. Plasma was collected at time 0 and 20 min and assayed for insulin. The fold changes in insulin from time 0 to 20 min are plotted for each group.
Error bars represent SEM.
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