NMR-based metabolomics and breath studies show lipid and protein catabolism during low dose chronic T(1)AM treatment

Abstract

Objective: 3-Iodothyronamine (T1 AM), an analog of thyroid hormone, is a recently discovered fast-acting endogenous metabolite. Single high-dose treatments of T1 AM have produced rapid short-term effects, including a reduction of body temperature, bradycardia, and hyperglycemia in mice.

Design and methods: The effect of daily low doses of T1 AM (10 mg/kg) for 8 days on weight loss and metabolism in spontaneously overweight mice was monitored. The experiments were repeated twice (n = 4). Nuclear magnetic resonance (NMR) spectroscopy of plasma and real-time analysis of exhaled (13) CO2 in breath by cavity ring down spectroscopy (CRDS) were used to detect T1 AM-induced lipolysis.

Results: CRDS detected increased lipolysis in breath shortly after T1 AM administration that was associated with a significant weight loss but independent of food consumption. NMR spectroscopy revealed alterations in key metabolites in serum: valine, glycine, and 3-hydroxybutyrate, suggesting that the subchronic effects of T1 AM include both lipolysis and protein breakdown. After discontinuation of T1 AM treatment, mice regained only 1.8% of the lost weight in the following 2 weeks, indicating lasting effects of T1 AM on weight maintenance.

Conclusions: CRDS in combination with NMR and (13) C-metabolic tracing constitute a powerful method of investigation in obesity studies for identifying in vivo biochemical pathway shifts and unanticipated debilitating side effects.

Figure 1. Comparison of weight loss in T₁AM treated and control mice

Decrease in weight is shown as a % of body weight in T₁AM treated mice (light gray bars) and sham treated mice (dark gray bars) over 21 day study period. Error bars reflect the standard error of the mean (SEM). Statistical significance (p < 0.05) compared to the vehicle treated group indicated by asterisk.

Figure 2. Exhaled breath analysis measured by CRDS reveals lipid breakdown

Daily stable isotope breath analyses of T₁AM treated animals (n=4) are reported as the average daily δ¹³C values. The minimum average values are at 80 minutes (δ¹³C_80min) post injection for each treatment day. Statistical significance with p < 0.05 are indicated by asterisks.

Figure 3. Analysis of plasma by NMR-based metabolomics reveals an increase in the lipid oxidation and protein break down in non-fasting condition

A) A significant increase in 3-hydroxybutyrate at day 7 of T₁AM treatment. The concentration of 3-hydroxybutyrate is shown as light gray bars for T₁AM treated mice (n=3) and dark gray bars for control mice (n=4) (p= 0.03). B) Increases in the plasma amino acids glycine (glucogenic) and valine (branched and ketogenic/glucogenic) in post T₁AM treatment regime. Plasma samples were collected in the following two weeks after discontinuation of T₁AM treatment and were two hours post [U-¹³C]-glucose injection. The concentration of glycine (p=0.05) and valine (p=0.001) is shown as light gray bars for T₁AM treated mice and dark gray bars for sham mice. T₁AM associated increases in both of these amino acids indicate a shift in energy metabolism following glucose administration. C) Increase in the plasma acetate concentration (lipid intermediate) at day 7 of T1AM post treatment. Light gray bars are for T₁AM treated mice and dark gray bars represent control mice. Error bars represent the SEM. Statistical significance (p < 0.05) compared to the sham treated group are indicated by asterisks.

Figure 3. Analysis of plasma by NMR-based metabolomics reveals an increase in the lipid oxidation and protein break down in non-fasting condition

A) A significant increase in 3-hydroxybutyrate at day 7 of T₁AM treatment. The concentration of 3-hydroxybutyrate is shown as light gray bars for T₁AM treated mice (n=3) and dark gray bars for control mice (n=4) (p= 0.03). B) Increases in the plasma amino acids glycine (glucogenic) and valine (branched and ketogenic/glucogenic) in post T₁AM treatment regime. Plasma samples were collected in the following two weeks after discontinuation of T₁AM treatment and were two hours post [U-¹³C]-glucose injection. The concentration of glycine (p=0.05) and valine (p=0.001) is shown as light gray bars for T₁AM treated mice and dark gray bars for sham mice. T₁AM associated increases in both of these amino acids indicate a shift in energy metabolism following glucose administration. C) Increase in the plasma acetate concentration (lipid intermediate) at day 7 of T1AM post treatment. Light gray bars are for T₁AM treated mice and dark gray bars represent control mice. Error bars represent the SEM. Statistical significance (p < 0.05) compared to the sham treated group are indicated by asterisks.

Figure 3. Analysis of plasma by NMR-based metabolomics reveals an increase in the lipid oxidation and protein break down in non-fasting condition

A) A significant increase in 3-hydroxybutyrate at day 7 of T₁AM treatment. The concentration of 3-hydroxybutyrate is shown as light gray bars for T₁AM treated mice (n=3) and dark gray bars for control mice (n=4) (p= 0.03). B) Increases in the plasma amino acids glycine (glucogenic) and valine (branched and ketogenic/glucogenic) in post T₁AM treatment regime. Plasma samples were collected in the following two weeks after discontinuation of T₁AM treatment and were two hours post [U-¹³C]-glucose injection. The concentration of glycine (p=0.05) and valine (p=0.001) is shown as light gray bars for T₁AM treated mice and dark gray bars for sham mice. T₁AM associated increases in both of these amino acids indicate a shift in energy metabolism following glucose administration. C) Increase in the plasma acetate concentration (lipid intermediate) at day 7 of T1AM post treatment. Light gray bars are for T₁AM treated mice and dark gray bars represent control mice. Error bars represent the SEM. Statistical significance (p < 0.05) compared to the sham treated group are indicated by asterisks.