Effects of Chicken Serum Metabolite Treatment on the Blood Glucose Control and Inflammatory Response in Streptozotocin-Induced Type 2 Diabetes Mellitus Rats

Abstract

Chickens can live healthy without adverse effects despite high blood glucose levels. However, the blood biomolecules responsible for maintaining chronic hyperglycemia are unknown. Here, the effects of chicken serum metabolite treatment on blood glucose control and inflammatory response in streptozotocin (STZ)-induced Type 2 Diabetes Mellitus (T2DM) rats were investigated. First, chicken serum treatment reduced the advanced glycation end-products (AGEs) and blood glucose levels in STZ-induced T2DM rats. Second, insulin/glucose-induced acute hypoglycemic/hyperglycemic chickens and the blood biomolecules were screened via nontargeted ultra-performance liquid chromatography with mass spectroscopy (UPLC-MS), identifying 366 key metabolites, including DL-arginine and taurine, as potential markers for chronic hyperglycemia in chickens. Finally, DL-arginine functions for blood glucose control and inflammatory response were evaluated. We found that DL-arginine reduced the levels of blood glucose and AGEs in STZ-induced T2DM rats. In addition, DL-arginine treatment upregulated the glucose transporter type 4 (GLUT4) expression in the muscles and downregulated the advanced glycation end products receptor-1 (AGER1) expression in the liver and nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) expression in the pancreas and thymus tissues. Overall, these results demonstrate that serum metabolite of DL-arginine could maintain blood glucose homeostasis and suppress the inflammatory response in chickens. Therefore, DL-arginine may be a novel target for developing therapeutic agents to regulate hyperglycemia.

Keywords: DL-arginine; blood glucose; chicken; inflammatory response; streptozotocin-induced rats.

Figure 1

Streptozotocin (STZ)-induced type 2 diabetes mellitus (T2DM) rat model. Eight-week-old male Sprague-Dawley (SD) rats were fed high-fat diets for 4 weeks and administrated STZ (35 mg/kg BW) via intraperitoneal injection, 1 week after STZ infusion, and rats were assessed using the following tests. (A) Blood glucose levels were determined in venous blood samples obtained from alert fasted animals using a glucometer. (B) Pancreas histology was studied using serial 4 µm hematoxylin/eosin-stained (HE) sections, which were observed via light microscopy, focusing on islet structures (Scale bar = 50 μm). All values are presented as the mean ± SE of n = 6 rats per group, with *** p < 0.001, as determined by Student’s t-tests.

Figure 2

Acute hypoglycemia and hyperglycemia, induced by insulin/glucose, in chickens. (A) Acute hypoglycemic chickens using subcutaneous insulin injection (mmol/L). (B) Acute hyperglycemic chickens using oral glucose administration (mmol/L). (C) Morphology of pancreatic islets in different treatment groups stained with hematoxylin and eosin (HE) and β-cells were characterized by immunohistochemistry (IHC). Sections (5 μm) were cut from 4% paraformaldehyde-fixed tissue and treated with purified anti-mouse IRS1 antibody (Scale bar = 50 μm). The HE staining arrow refers to the morphology of the pancreatic islets, and the IHC arrow refers to the positive signal of the islet β cells (yellow). All values of (A,B) are mean ± SE n = 6 chickens per group, with *** p < 0.001, determined by one-way ANOVA. “ns” is “no significant”.

Figure 3

Effects of chicken serum treatment on feed intake, body weight, and blood glucose of rats. (A) The body weight of each group was determined every week. (B) The feed intake of each group was determined every week (C). Blood glucose levels of each group were after fasting for 3 h determined every week. All values of (A–C) are mean ± SE n = 6 rats per group, with ** p < 0.01, *** p < 0.001, determined by one-way ANOVA.

Figure 4

Principal component analysis of QC samples of serum metabolites from acute hyperglycemic/hypoglycemic chickens and control birds. (A) Acute hyperglycemic chickens and control birds PCA plot positive-ion pattern. (B) Acute hyperglycemic chickens and control birds PCA plot negative ion pattern. (C) Acute hypoglycemic chickens and control birds PCA plot positive ion pattern. (D) Acute hypoglycemic chickens and control birds PCA plot negative ion pattern. Abscissa PC1 and ordinate PC2 in the figure indicate the scores of principal components ranked first and second, respectively, and scatter in different colors indicates the samples of different experimental groups, with ellipses of 95% confidence intervals.

Figure 5

Differential metabolite Venn plots and Volcano plots of acute hyperglycemic/hypoglycemic chickens and control birds. (A) Differential metabolite Venn plots for acute hyperglycemic/hypoglycemic chickens and control model. (B) Differential metabolites Volcano plots for acute hyperglycemic/hypoglycemic chickens and control birds. Venn diagrams can visually compare common and unique differential metabolites between different groups and show the relationship between multiple groups of differential metabolites. The abscissa represents the fold change of difference (log2 Fold Change) of metabolites in different groups, the ordinate represents the significance level of difference (−log10 p-value), each point in the volcano plot represents a metabolite, significantly up-regulated metabolites are represented by red points, significantly down-regulated metabolites are represented by green points. The size of the dot represents VIP value.

Figure 6

KEGG Enrich scatterplot of acute hyperglycemic/hypoglycemic chickens and control birds. (A) Acute hyperglycemic chickens and control birds KEGG Enrich scatterplot positive ion pattern. (B) Acute hyperglycemic chickens and control birds KEGG Enrich scatterplot negative ion pattern. (C) Acute hypoglycemic chickens and control birds KEGG Enrich scatterplot positive ion pattern. (D) Acute hypoglycemic chickens and control birds KEGG Enrich scatterplot negative ion pattern. The abscissa in the figure is x/y (number of differential metabolites in the corresponding metabolic pathway/total number of metabolites identified in the pathway), and a larger value indicates a higher enrichment of differential metabolites in the pathway. The color of the dots represents the p-value value of the hypergeometric test, and the smaller the value, the greater and statistically significant the reliability of the test.

Figure 7

Differential metabolites of acute hyperglycemic/hypoglycemic chickens and control birds. (A) Peak area of representative key metabolites regulating blood glucose rise. (B) Peak area of representative key metabolites regulating blood glucose decline. All values of (A,B) are mean ± SE n = 6 chickens per group, with ** p < 0.01, *** p < 0.001, determined by Student’s t-test.

Figure 8

Effects of DL-arginine treatment on feed intake, body weight, and blood glucose of rats. (A) The body weight of each group was determined every week. (B) The feed intake of each group was determined every week. (C) Blood glucose levels of each group were after fasting for 3 h determined every week. All values of (A–C) are mean ± SE n = 6 rats per group, with ** p < 0.01, *** p < 0.001, determined by one-way ANOVA.

Figure 9

Effect of DL-arginine on rats’ glucose tolerance and insulin tolerance. (A) Rat glucose tolerance tests (OGTTs). Individual glucose tolerance was assessed by oral glucose tolerant tests. The fasted rats were oral administration of 2 g of glucose/kg body weight (BW). (B) The area under the curve for rat OGTTs. (C) Rat insulin tolerance tests (IPITTs). Individual insulin tolerance was evaluated by subcutaneous injection insulin tolerance tests by oral administration of 2 g glucose/kg BW immediately followed by subcutaneous insulin injection at a dose of 2 IU/kg BW; blood glucose levels were detected at 0, 30, 60, 90, and 120 min and compared to that at 0 min. (D) The area under the curve for rat IPITTs. (E) IR index of each group, HOMA-IR index = (FBG [in mmol/L] × FINS [in units/L])/22.5. All values are mean ± SE n = 6 rats per group, with * p < 0.05, ** p < 0.01, *** p < 0.001, determined by (B,D,E) by one-way ANOVA, determined with (A,C) by repeated ANOVA measurement.

Figure 10

Effects of DL-arginine on the content of glucose metabolism-related enzymes (GLUT2/4, PKM, CS, GCK, ICDH) in the liver and muscle of rats. (A) Expression of glucose transporters 2 (GLUT2), pyruvate kinase M (PKM), glucokinase (GCK), citrate synthase (CS), and isocitrate dehydrogenase (ICDH) mRNA in the liver was analyzed by quantitative RT-PCR. (B) Expression of GLUT4, PK, CS, and ICDH mRNA in muscle was analyzed by quantitative RT-PCR. All values of (A,B) are means ± SE; n = 3 per group, with * p < 0.05, ** p < 0.01, *** p < 0.001, determined by one-way ANOVA.

Figure 11

HE sections of the liver, kidney, and pancreas in different treatment groups of rats. (A) HE staining was used to detect the degree of injury and recovery in the liver sections. The arrow refers to the distribution of lipid droplet accumulation. (B) HE staining was used to detect the degree of injury and recovery in the kidney sections. The arrow refers to the distribution of lipid droplet accumulation glomeruli. (C) HE staining was used to detect the degree of injury and recovery in the pancreatic sections. The arrow refers to the distribution of pancreatic islets. (Scale bar = 50 μm).

Figure 12

Effects of DL-arginine on the expression of inflammatory genes (MAPK, NF-κB, AGER1) in rat tissues. (A) Expression of mitogen-activated protein kinase (MAPK), nuclear factor- κB (NF-κB), and AGE receptor 1 (AGER1) mRNA in the liver was analyzed by quantitative RT-PCR. (B) Expression of MAPK, NF-κB, and AGER1 mRNA in muscle was analyzed by quantitative RT-PCR. (C) Expression of MAPK, NF-κB, and AGER1 mRNA in the pancreas was analyzed by quantitative RT-PCR. (D) Expression of MAPK, NF-κB, and AGER1 mRNA in the thymus was analyzed by quantitative RT-PCR. All values of (A–D) are means ± SE; n = 3 per group, with * p < 0.05, ** p < 0.01, determined by one-way ANOVA.

Scheme 1

Time point of different treatments of animal models.