Skip to main content

In vivo magnetic resonance spectroscopy studies of muscle mitochondrial function in mice

Periodic Report Summary - INMARESS (In vivo magnetic resonance spectroscopy studies of muscle mitochondrial function in mice)

Alterations in muscle mitochondrial function have been implicated in the pathogenesis of numerous metabolic disorders, including insulin resistance, type 2 diabetes, obesity, and the deleterious effects of aging. However, the precise role for mitochondrial function in these processes remains to be established. In vivo magnetic resonance spectroscopy (MRS) is an effective technique that permits non-invasive investigation of skeletal muscle metabolism and transgenic mice are a novel tool for examining the effects of specific genes on mitochondrial function.

Transgenic mice overexpressing uncoupling protein 3 (UCP3+), a key-role protein in the regulation of the mitochondrial energetic activity, were studied in separate experiments to measure energy production and substrate oxidation. Energy production was assessed in the hindlimb of anesthetised control (WT) and UCP3+ mice by determining the rate of Pi --> ATP flux (Vatp) using 31P saturation-transfer MRS. Substrate oxidation via the tricarboxylic acid cycle (Vtca) was determined using a novel biopsy-based technique. Anesthetised mice were infused with 2-13C acetate for varying durations up to 90 minutes; plasma samples and the soleus-gastrocnemius muscle complex of each hindlimb were obtained at different intervals during the infusion from each mouse. Concentrations and 13C enrichments of the muscle metabolite pools were measured by 1H[13C]-MRS of the extracted tissue at 500 MHz. Vtca was calculated by metabolic modelling of the time-courses of enrichment of the muscle 13C4-glutamate, 13C3-glutamate and 13C4-glutamine pools.

MRS opens new scenarios in the investigation of metabolism, unveiling cellular functions at the molecular level and therefore contributing to the understanding of several etiopathogenetic mechanisms.

Introduction

Uncoupling protein 3 (UCP3) is a mitochondrial trans-membrane protein that is highly expressed in muscle (1, 2). It shares high homology with UCP1, which mediates thermogenesis in brown adipose tissue, and it has been proposed that UCP3 may also dissipate the proton electrochemical gradient across the inner mitochondrial membrane and 'uncouple' mitochondrial oxidation and ATP synthesis (2). Modulation of UCP3 expression alters oxygen consumption and proton leak in isolated mitochondria in vitro (3, 4), but its precise function in-vivo has yet to be fully elucidated. In fact, numerous roles for UCP3 have been proposed
(5), but its function in vivo remains unknown.

Aim

To examine the effects of muscle-specific over-expression of UCP3 in transgenic (TG) mice on mitochondrial metabolism in vivo.

Methods

- Substrate oxidation and energy production were determined in lightly anesthetised UCP3-TG and wild-type (WT) mice in separate experiments.
- Heart-rate, body temperature and respiration were monitored continuously throughout each protocol.
- Depth of anesthesia was regulated to maintain stable physiological function.

ATP synthesis (Vatp)

- Energy production was estimated using the 31P-saturation-transfer (ST) technique (6) to assess resting muscle Pi --> ATP flux ('ATP synthesis', Vatp). Experiments were performed on a 9.4T Bruker Biospec system. Mice positioned prone with a 15 mm diameter 31P surface coil located over the hindlimb muscles. 31P spectra were acquired using a custom-written adiabatic ST pulse sequence; T1'(Pi) was determined by an eight point inversion-recovery calibration. 31P metabolite concentrations were calculated from fully-relaxed 31P spectra; concentration of ATP [ATP] was determined from muscle extracts at 500 MHz.

TCA Cycle Flux

Substrate oxidation via the TCA cycle (Vtca) was assessed using a novel biopsy-based technique.
Mice were infused with [2-13C]-acetate for varying durations up to 90 minutes. Plasma samples and the soleus-gastrocnemius muscle complex of each hindlimb were obtained at different intervals during the infusion from each mouse. Metabolite concentrations and 13C enrichment were measured in extracted muscle by proton-observe / carbon-edited (POCE) MRS at 500 MHz.

Plasma acetate concentrations and enrichment were determined by GCMS.

Timecourses of enrichment of muscle [4-13C]- and [3-13C]-glutamate, [4-13C]-glutamine and plasma [2-13C]-acetate were fitted to a metabolic model of the TCA cycle using CWave software
(6).

Results

Resting rates of Pi --> ATP flux were reduced by approximately 20 % in the muscle of UCP3-TG mice, indicating decreased energy production. Monte-Carlo analyses of the modelling data revealed that Vtca was a significantly faster in UCP3-TG mice.

Discussion and conclusion

Overexpression of UCP3 in the muscle of TG mice increased rates of mitochondrial substrate oxidation and decreased energy production in vivo, signifying reduced mitochondrial efficiency. These data are consistent with UCP3 mediating the uncoupling of oxidative phosphorylation in these mice.

References

1. O. Boss et al., FEBS Lett 412: 111 - 114, 1997
2. A. Vidal-Puig et al., BBRC 235: 79 - 82, 1997
3. A. Vidal-Puig et al., JBC 275: 16 258 - 16 266, 2000
4. S. Cadenas et al., JBC 277: 2773 - 2778, 2002
5. M. E. Harper, Biochem Soc Trans 26: 768 - 73, 2001
6. D. E. Befroy et al., Meth Enzymol 457: 373 - 393, 2009