Hormonal activity of AIMP1/p43 for glucose homeostasis

Abstract

AIMP1/p43 is known as a cytokine working in the control of angiogenesis, inflammation, and wound healing. Here we report its enrichment in pancreatic alpha cells and glucagon-like hormonal activity. AIMP1 is secreted from the pancreas upon glucose starvation. Exogenous infusion of AIMP1 increased plasma levels of glucose, glucagon, and fatty acid, and AIMP1-deficient mice showed reduced plasma glucose levels compared with the wild-type mice under fasting conditions. Thus, AIMP1 plays a glucagon-like role in glucose homeostasis.

Fig. 1.

Enriched localization of AIMP1 in pancreatic α cells. (A) Tissue-dependent variations of protein levels were determined for AIMP1 and two tRNA synthetases, methionyl-tRNA synthetase and glutaminyl-tRNA synthetase, which are components of the multi-tRNA synthetase complex. Proteins extracted from each tissue were subjected to Western blot analysis with their respective antibodies. (B) AIMP1 was separated into the portions that were bound and unbound to the multi-tRNA synthetase complex by size exclusion chromatography. The proteins extracted from the lung and pancreas that contained low and high levels of AIMP1, respectively, were compared. The two other components for the complex, glutamyl-prolyl-tRNA synthetase and arginyl-tRNA synthetase, were used as indicators for the complex-bound portion. The eluted proteins were resolved by SDS/PAGE, and three proteins were detected with their respective antibodies. (C) Localization of AIMP1 in the pancreatic islet was determined by immunofluorescence staining. AIMP1 and nuclei were stained green with FITC-conjugated antibody and red with propidium iodide, respectively. (Magnification: ×20.) (D) Colocalization of AIMP1 (green) in the pancreatic α cells with glucagon (red) under a confocal microscope. In Upper, pancreatic β cells were stained with insulin antibody and rhodamine-conjugated secondary antibody. (Magnification: ×20.) (E) A mouse pancreas was dissected and fixed as described in Materials and Methods. Grids were reacted with anti-AIMP1 antibody and detected with colloidal gold-conjugated protein A. The labeled sections were observed under an electron microscope (JEOL) at 80 kV.

Fig. 2.

Low glucose levels induce pancreatic secretion of AIMP1. (A) Secretion of AIMP1 by pancreatic stimulation through the cardiac perfusion of the conditioned medium containing the indicated concentrations of glucose. After perfusion of the glucose solution for 5 min at a flow rate of 10 ml/min, pancreases were isolated and incubated as described in Materials and Methods. The secreted proteins were precipitated from the harvested medium by the addition of 10% TCA, separated by 10% SDS/PAGE, and blotted with anti-AIMP1 antibody. WCL, whole-cell lysates. (B) We also confirmed that the secretion of AIMP1 is induced under hypoglycemic conditions (75 mg/dl) from aTC1 clone 9 but not under hyperglycemic conditions (450 mg/dl). (C) Glucagon secretion was stimulated by AIMP1 (100 nM) from aTC1 clone 9. The secreted glucagon was quantified by using an RIA kit (LINCO Research). The values are means ± SD. ∗, P < 0.02.

Fig. 3.

The effect of AIMP1 on the blood levels of hormones and metabolites related to glucose metabolism. Sprague–Dawley rats were cannulated with AIMP1 for 2 h as described in Materials and Methods, blood was collected at the indicated time points, and plasma was obtained by centrifugation. We then measured the plasma levels of glucagon (A), insulin (B), glucose (C), lactate (D), free fatty acid (E), and glycerol (F) using their specific quantification kits (n = 4–5). The values are means ± SD. ∗, P < 0.07; ∗∗, P < 0.05; ∗∗∗, P < 0.02.

Fig. 4.

The effect of AIMP1 on glucose uptake, glycogenolysis, and lipolysis. We treated HepG2 with the indicated concentrations of AIMP1 and assayed the uptake rate of [U-¹⁴C]d-glucose (A) and [³H]palmitate (B). Glucagon was used as a positive control. We stimulated glycogen synthesis in HepG2 with 10 nM insulin, 25 mM d-glucose, and 2 μCi/ml [U-¹⁴C]d-glucose for 16 h, and we induced glycogenolysis by incubating cells with glucose-free DMEM containing glucagon or AIMP1 for 4 h. The culture medium (C) and cells (D) were harvested to quantify [U-¹⁴C]d-glucose. We cultured and differentiated 3T3-L1 into adipocytes as described in Materials and Methods. We induced the uptake of [³H]palmitate (1 μCi/ml) for 2 h and treated the cells with glucagon or AIMP1 for 7 h. The culture medium (E) and adipocytes (F) were harvested to measure [³H]palmitate as described in Materials and Methods. The values are means ± SD. ∗, P < 0.06; ∗∗, P < 0.01.

Fig. 5.

Genetic depletion of AIMP1 induces hypoglycemia. (A) AIMP1^+/+ and AIMP1^−/− mice were fasted for the indicated times, and blood glucose levels were measured (n = 9). (B) IPGTT in AIMP1^+/+ and AIMP1^−/− mice. Each group of mice was fasted for 14 h, and 2 g of glucose per kilogram of body weight was i.p. injected in a 20% sterile glucose solution. Blood was harvested at the indicated times, and glucose levels were analyzed (n = 9). Changes of plasma insulin (C) and glucagon (D) levels during IPGTT are shown. (E) Schematic representation of the hormonal activity of AIMP1 working on different organs for glucose homeostasis. AIMP1 released from the pancreatic α cells at low concentrations of glucose to induce glucagon secretion. AIMP1 inhibits glucose uptake, induces glycogenolysis in the liver, and stimulates lipolysis in adipocytes to increase blood levels of glucose. The values are means ± SD. ∗, P < 0.06; ∗∗, P < 0.05.