Validation of Hemoglobin A1c as a Long‑Term Glycemic Biomarker Using Taconic Mouse Models, in Collaboration with Research Diets

Background

• Hemoglobin A1c (HbA1c) is a primary clinical measure of glycemic control in patients with diabetes, but is rarely measured in rodents. Glycation of hemoglobin is a non-enzymatic and irreversible process, occurs continuously and is only dependent on the concentration of glucose in the blood.
• HbA1c assays provide an estimated integrative measure of blood glucose homeostasis over the previous 5-6 weeks in mice, and 11-12 weeks in humans.
• Single-sample blood glucose measures are sensitive to stress and acute environmental stimuli, as observed during continuous glucose monitoring studies.
• Measurement of HbA1c in rodents may provide more information, and less stress than repeat mesaures of single point and GTT glucose assays in chronic studies of obesity and glucose homeostasis.
• Diet-Induced Obesity (DIO) mouse models are the most commonly used pre-clinical models of obesity, displaying increased body weight, elevated glucose, and insulin resistance. 
• Non-obese diabetic (NOD) mice are an inbred laboratory strain used extensively to study Type 1 Diabetes (T1D) and other autoimmune diseases, and serve as a positive control for elevated HbA1c in this study.

Objectives

• To validate the use of the A1CNow®+ assay in young pre-diabetic and diabetic NOD female mice, an immune deficiency and type 1 diabetes model, as a positive assay control.
• To survey a commercially available colony of age-matched DIO and chow-fed male mice to characterize HbA1c levels and their correlation to body weight and single-point glucose measures in a commonly used DIO model.

Materials and Methods

Animals:

³Non-Obese Diabetic Mice
NOD/MrkTac female mice were maintained ad libitum on NIH-31 chow and water at a cage density of 5/cage until either 8 weeks (n=5) or 22 weeks (n=7) of age. Mice of these ages were selected randomly from a commercial breeding/supply colony, and studies were performed by Taconic Biosciences, Rensselaer, NY.

Diet-Induced Obese Mice
70 C57BL/6NTac male mice were maintained ad libitum on NIH-31 chow and water at a cage density of 10/cage until 6 weeks of age (Taconic Biosciences) as part of a commercially available colony. At 6 weeks of age, animals are randomized for either continuation on NIH31 chow, or being fed a 60 kcal% fat diet, D12492 (Research Diets, Inc., New Brunswick, NJ) to induce DIO as a subset of a commercially available colony.

Whole Blood Assays
⁵Fasting blood glucose using Accu-Chek blood glucose meter, in duplicate. ⁴HbA1c using PTS Diagnostics test (A1CNow®+) in duplicate.

Procedure:
A subset of the DIO production lot was chosen, with age-matched diet cohorts selected to within a week of age, and sampled over two days. Half were assayed on the same day following a 2 2-4 hour fast. C57BL/6NTac mice fed either a high-fat diet (D12492, 60 kcal% fat) to induce diet-induced obesity or NIH-31 chow. Animals were assayed for blood glucose, followed by HbA1c via the same tail nick, and weighed, at 6, 14, 22, and 30 weeks of age (D12492 or chow fed for an additional 0, 8, 16, or 24 weeks). Eight week old NOD female mice and the age-matched male chow or DIO animals were assayed as above on day one after a 2 2-4 hour fast. The 22 week old NOD female animals were assayed on a separate day following a 2 2-4 hour fast. Animals were euthanized following the study.

Statistics:
Body weight, glucose, and HbA1c measures were assessed with a repeated measures two-way ANOVA with time and diet as variables. Scatterplots were analyzed using Pearson correlations.

Diet Composition

D12492 Purified Diet Induced Obesity 60kcal% Fat Diet and NIH-31 Grain-Based Chow Formulas

Summary and Follow Up

¹Obesity is a global health emergency, and is on the rise based on worldwide health data. Diet Induced Obesity (DIO) mouse models (D12492, 60kcal% fat and D12451,45 kcal% fat, Research Diets, Inc. New Brunswick, NJ) are the most commonly used pre-clinical models of obesity, displaying increased body weight, elevated glucose, and insulin resistance. ²In addition to obesity, these diets and other defined modifications are used in research in the comorbid diseases of pre-diabetes, cardiovascular disease, metabolic disease, MASH, cancer risk, immunology, neuroscience, and inflammation research. Diet compositions vary based on the target focus.

Glucose exposure causes biochemical changes at the proteome level as reflected in the accumulation of glycated proteins. A prominent example is HbA1c. HbA1c is a superior measure of long-term glycemic control than intermittent blood glucose measurements. However, very little literature exists on the use of HbA1c in mice, and most of the studies reporting HbA1c results do so without addressing the temporal dynamics, sensitivity, and the physiological range of HbA1c in this species. This study does not address these details.

The use of the ⁴A1CNOW+assay was validated using prediabetic NOD female mice as a negative control (8 weeks old) and emergent ³NOD female diabetic mice as a test cohort (22 weeks old). 8 week old animals did not register above the 4% lower limit of the HbA1c assay. 3/7 animals were positive for frank diabetes with greater than 600 mg/dl glucose and elevated HbA1c, with an average HbA1c of 9.35 +/ +/-1.2 (SEM), compared to non-NOD-diabetic but aged animals with HbA1c levels of 4.2 +/ +/-0.2 (SEM).

In the second study, the DIO group exhibited significantly elevated blood glucose, HbA1c, and body weight following 8 weeks of D12492 feeding from baseline (14 weeks old) compared to animals maintained on NIH-31 for 8 weeks from baseline (14 weeks old) These parameters’ significance continued after 16 weeks and 24 weeks of post baseline feeding. HbA1c showed a strong correlation with body weight (r² = 0.672; p < 0.0001). However, correlations between single point fasted blood glucose and both HbA1c (r² = 0.3) and body weight (r² = 0.02) were not significant.

We demonstrate that the A1CNow®+ assay provides an accurate and cost-effective integrative measure of HbA1c reflecting glycemic control in a commonly used model of DIO and mild hyperglycemia. These findings suggest that HbA1c is a reliable measurement of chronic hyperglycemia in mice, and its measurement could enhance studies of glycemic control through diet and therapeutic interventions in mice, and reduce the amount of handling and blood sampling needed for chronic monitoring of glycemic status.

The incorporation of periodic measures of HbA1c every 5-6 weeks during chronic DIO and therapeutic treatment studies may lessen the stress on animals during chronic treatment compared to monitoring via weekly glucose measures.

References

¹NCD Risk Factor Collaboration. Worldwide trends in underweight and obesity from 1990 to 2022: a pooled analysis of 3663 population-representative studies with 222 million children, adolescents, and adults. The Lancet 2024. 16;403(10431):1027 1027-1050. doi: 10.1016/S0140 S0140-6736(23)02750 02750-2

²de Moura e Dias, M., dos Reis, S.A., da Conceição, L.L. et al. Diet-induced obesity in animal models: points to consider and influence on metabolic markers. Diabetol Metab Syndr 13, 32 (2021). https://doi.org/10.1186/s13098 s13098-021-00647-2

³Mouse Models of Autoimmune Diabetes: The Nonobese Diabetic (NOD) Mouse Methods Mol Bio. 2020 2128: 87-92. doi:10.1007/978-1-0716-0385-7_6. NOD Mice Chen et al, 2021: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8253669

⁴HbA1c Assay: A1CNow®+ (PTS Diagnostics, www.ptsdiagnostics.com com)

⁵Fasting Blood Glucose (Accu Accu-Chek, www.accu accu-chek.com com)