Plasma-based proteomics analysis of molecular pathways in canine diabetes mellitus after astaxanthin supplementation

Abstract

The hyperglycemic state in diabetes mellitus induces oxidative stress and inflammation, contributing to diabetic tissue damage and associated complications. Astaxanthin, a potent antioxidant carotenoid, has been investigated for its potential to prevent and manage diabetes across various species; however, its effect on client-owned dogs remains poorly studied. This study explored the impact of astaxanthin supplementation on canine diabetes mellitus using a proteomics approach. A total of 18 client-owned dogs were enrolled: 6 dogs with diabetes mellitus and 12 clinically healthy dogs. The diabetic dogs received their standard treatment regimen along with daily oral supplementation of 12 mg of astaxanthin (1.5-2.4 mg/kg) for 90 days. Plasma samples were collected at the beginning and end of the study period for proteomics analysis. After astaxanthin supplementation, significant alterations in the expression of proteins associated with the complement system, coagulation cascade, JAK-STAT signaling, and protein kinase C signaling (all of which contribute to inflammation and oxidative stress) were observed. Astaxanthin exhibited potential for reducing diabetes-associated complications, such as insulin resistance, vascular dysfunction, nephropathy, and cardiac issues, even though it did not affect clinical parameters (hematology, plasma biochemistry, blood glucose, and serum fructosamine). These findings suggest that astaxanthin may be a valuable complementary therapy for managing diabetes-related complications in canines.

Fig 1. Venn diagram of protein detection (Canis spp.).

Fig 2. Principal component analysis (PCA).

Healthy adult dogs (green dots) and diabetic dogs before (DM D0, blue dots) and after (DM D90, red dots) astaxanthin supplementation.

Fig 3. Volcano plots.

(A) Diabetic dogs before astaxanthin supplementation (DM D0) vs. healthy adult dogs; (B) DM D0 vs. diabetic dogs after astaxanthin supplementation (DM D90). Each dot represents a protein. The red dots indicate significantly downregulated proteins (P < 0.05, fold-changes < 0.5). The green dots indicate significantly upregulated proteins (P < 0.05, fold-changes > 2).

Fig 4. Gene Ontology annotation.

Gene Ontology annotation of differentially expressed proteins pertaining to (A) biological processes, (B) molecular function, and (C) cellular components between diabetic dogs before (DM D0) and after (DM D90) astaxanthin supplementation and healthy adult dogs.

Fig 5. STRINGS network analysis of protein–protein interactions.

Analysis of differentially regulated proteins in diabetic dogs (DM D0) vs. healthy controls and diabetic dogs after astaxanthin supplementation (DM D90) (red: complement and coagulation cascade; blue: JAK–STAT signaling pathway; green: insulin secretion; yellow: PI3K–Akt signaling pathway; pink: glycoprotein metabolic process). Each node represents a unique protein, whereas the edges connecting the nodes indicate known or predicted interactions.

Fig 6. Bar chart comparing the relative intensity of proteins in the complement and coagulation cascades across the three groups.

(A) C1S, (B) C7, (C) FGA, (D) FGG, (E) F3, (F) F7, and (G) F12. *Statistically significant at P < 0.05, **statistically significant at P < 0.01, ***statistically significant at P < 0.001, ****statistically significant at P < 0.0001.

Fig 7. Bar chart comparing the relative intensity of proteins in JAK–STAT signaling pathway and insulin secretion across the three groups.

(A) IL2RB, (B) IL20RB, (C) IL23A, and (D) PRKCA **Statistically significant at P < 0.01, ***statistically significant at P < 0.001, ****statistically significant at P < 0.0001.

Fig 8. Bar chart comparing the relative intensity of proteins in PI3K–Akt signaling pathway and glycoprotein metabolic process across the three groups.

(A) PRKCSH and (B) MDM2 ***Statistically significant at P < 0.001, ****statistically significant at P < 0.0001.

Fig 9. Proteomics pathways involved in diabetes and the effects of astaxanthin supplementation in dogs.