Preclinical modeling of metabolic syndrome to study the pleiotropic effects of novel antidiabetic therapy independent of obesity

Abstract

Cardiovascular-kidney-metabolic health reflects the interactions between metabolic risk factors, chronic kidney disease, and the cardiovascular system. A growing body of literature suggests that metabolic syndrome (MetS) in individuals of normal weight is associated with a high prevalence of cardiovascular diseases and an increased mortality. The aim of this study was to establish a non-invasive preclinical model of MetS in support of future research focusing on the effects of novel antidiabetic therapies beyond glucose reduction, independent of obesity. Eighteen healthy adult Beagle dogs were fed an isocaloric Western diet (WD) for ten weeks. Biospecimens were collected at baseline (BAS1) and after ten weeks of WD feeding (BAS2) for measurement of blood pressure (BP), serum chemistry, lipoprotein profiling, blood glucose, glucagon, insulin secretion, NT-proBNP, angiotensins, oxidative stress biomarkers, serum, urine, and fecal metabolomics. Differences between BAS1 and BAS2 were analyzed using non-parametric Wilcoxon signed-rank testing. The isocaloric WD model induced significant variations in several markers of MetS, including elevated BP, increased glucose concentrations, and reduced HDL-cholesterol. It also caused an increase in circulating NT-proBNP levels, a decrease in serum bicarbonate, and significant changes in general metabolism, lipids, and biogenic amines. Short-term, isocaloric feeding with a WD in dogs replicated key biological features of MetS while also causing low-grade metabolic acidosis and elevating natriuretic peptides. These findings support the use of the WD canine model for studying the metabolic effects of new antidiabetic therapies independent of obesity.

Keywords: Cardiorenal metabolic diseases; Metabolic syndrome; One health; Western diet.

Fig. 1

Molecular bases for the interrelationship between cardiovascular, renal, and metabolic diseases. Adjusted and simplified from Kadowaki et al. .

Fig. 2

Temporal changes in standard clinical chemistry parameters after ten weeks of feeding with a high-fat, high-monosaccharide, low-fiber western diet. No notable alterations were observed in liver-related chemical parameters, such as ALT, ALP, albumin, and total protein, when comparing BAS1 to BAS2. Dogs at BAS2 had decreased levels of serum bicarbonates, phosphorus, and potassium, but increased levels of chloride. There was also a reduction in BUN at BAS2, along with an elevation in serum creatinine levels. Box plots represent the 25th, 50th and 75th percentile of the data ± 1.5 IQR (interquartile range). •: 0.01 < P ≤ 0.05; ••: 0.001 < P ≤ 0.01; •••: P ≤ 0.001. Summary box plots produced using the ggplot2 package in R version 4.2.2.

Fig. 3

Temporal changes in fasting blood glucose and glucagon after ten weeks of feeding with a high-fat, high-monosaccharide, low-fiber western diet. The WD resulted in a significant 16.5% increase in fasting blood glucose, approaching the upper physiological limit. This was accompanied by a trend towards lower serum glucagon levels which did not reach statistical significance. Box plots represent the 25^th, 50^th and 75^th percentile of the data ± 1.5 IQR (interquartile range). •••: P ≤ 0.001. Summary box plots produced using the ggplot2 package in R version 4.2.2.

Fig. 4

Serum insulin time-course post-glucose tolerance test. Dogs received an oral dose of 5 g of dextrose per kg in the form of a solution containing 1 g of dextrose powder per mL of water. Blood samples were collected at 30, 60, 90, 120, and 180 min after oral administration of the glucose solution. Compared to BAS1, dogs fed an isocaloric WD for ten weeks showed a trend towards a higher peak concentration of insulin, with an increase of approximately 35% in AUC_ins(0–90) at BAS2. Time-course data are presented as mean ± S.E. Scatter plots produced using the ggplot2 package in R version 4.2.2.

Fig. 5

Temporal changes in systolic blood pressure (A) and NT-proBNP (B) after ten weeks of feeding with a high-fat, high-monosaccharide, low-fiber western diet. (A) Dogs fed a WD for ten weeks had significantly higher blood pressure measurements compared with baseline (BAS1). Measures were taken by a certified cardiologist using a Doppler device. To avoid bias in the recordings, these measurements were consistently taken before any blood was collected during each study period. To follow the ACVIM consensus panel guidelines for assessing hypertension and ensure accuracy, five consecutive and consistent SBP measurements were obtained from each subject. These values were then averaged to calculate an individual estimate of SBP. (B) NT-proBNP concentrations significantly increased at BAS2, with two dogs presenting values exceeding 900 pmol/L, a level commonly associated with structural heart disease in canines^,. Box plots represent the 25^th, 50^th and 75th percentile of the data ± 1.5 IQR (interquartile range). •: 0.01 < P ≤ 0.05; •••: P ≤ 0.001. Summary box plots produced using the ggplot2 package in R version 4.2.2.

Fig. 6

Temporal changes in total cholesterol, HLD-cholesterol and LDL-cholesterol after ten weeks of feeding with a high-fat, high-monosaccharide, low-fiber western diet. Circulating levels of cholesterol were significantly increased (+ 44.2%) after ten weeks of feeding with the isocaloric WD. Notably, this change was accompanied by a significant reduction in HDL-cholesterol and a 26.8% elevation in LDL-cholesterol. Box plots represent the 25th, 50th and 75th percentile of the data ± 1.5 IQR (interquartile range). •••: P ≤ 0.001. Summary box plots produced using the ggplot2 package in R version 4.2.2.

Fig. 7

Temporal changes in antioxidant (A) and oxidant (B) stress markers after ten weeks of feeding with a high-fat, high-monosaccharide, low-fiber western diet. The WD had mild effects on antioxidant markers, with no significant changes in CUPRAC, FRAP, TEAC, and Thiol values. However, PON-1 levels significantly decreased at BAS2. The impact of the WD on oxidative stress parameters was more consistent, with total oxidant status significantly increasing at BAS2. The increase extended to reactive oxygen metabolites (d-ROMs). Conversely, there was a decrease in POX-Act post-WD, but no notable effects on AOPP. Box plots represent the 25^th, 50^th and 75^th percentile of the data ± 1.5 IQR (interquartile range). •: 0.01 < P ≤ 0.05; ••: 0.001 < P ≤ 0.01; •••: P ≤ 0.001. Summary box plots produced using the ggplot2 package in R version 4.2.2.

Fig. 8

PCA score plots (General Metabolism) of (A) Urine, (B) Stool, and (C) Serum before feature selection using the PLS_Toolbox software (Version 9.0; Eigenvector Research, Manson, WA).

Fig. 9

PCA score plots (General Metabolism) of (A) Urine, (B) Stool, and (C) Serum after feature selection using the PLS_Toolbox software (Version 9.0; Eigenvector Research, Manson, WA).

Fig. 10

PCA score plots (Complex Lipids) of (A) Urine, (B) Stool, and (C) Serum before feature selection using the PLS_Toolbox software (Version 9.0; Eigenvector Research, Manson, WA).

Fig. 11

PCA score plots (Complex Lipids) of (A) Urine, (B) Stool, and (C) Serum after feature selection using the PLS_Toolbox software (Version 9.0; Eigenvector Research, Manson, WA).

Fig. 12

PCA score plots (Biogenic Amines) of (A) Urine, (B) Stool, and (C) Serum before feature selection using the PLS_Toolbox software (Version 9.0; Eigenvector Research, Manson, WA).

Fig. 13

PCA score plots (Biogenic Amines) of (A) Urine, (B) Stool, and (C) Serum after feature selection using the PLS_Toolbox software (Version 9.0; Eigenvector Research, Manson, WA).