Implementation of FAST-MRS in mouse permits the rapid assessment of muscle ATP synthesis in-vivo

Introduction Conventional saturation-transfer (CST) has proven to be a useful technique for measuring the rates of metabolic exchange reactions in a variety of preparations. We have used it effectively in both rats and humans to investigate muscle energy production in-vivo, and found that rates of ATP synthesis (VATP) are modulated by pharmacological uncoupling of the mitochondrion (1), aging (2) and in insulin-resistant offspring of type 2 diabetic patients (3,4). The existence of numerous transgenic mouse models with modulated metabolism offers scope for many new avenues of investigation. To date, the application of CST in mouse has been restricted due to the small amount of muscle tissue available for MRS, necessitating very long experimental durations (~8 hours) or that time-consuming elements (eg T1\u2019 calibration) of the CST experiment are eliminated (5). The Four-Angle Saturation Transfer (FAST) technique (6) has been proposed as an alternative to CST and may permit more rapid assessment of VATP in mouse muscle. This technique dispenses with an independent T1\u2019 calibration by acquiring saturation-transfer spectra at two different pulse-angles \u2013 the T1 of a metabolite of interest can be calculated indirectly from the ratio of it\u2019s signal at the two pulse-angles, precise knowledge of those pulse angles and the TR. Here, we demonstrate that FAST is a valid alternative to CST for measuring VATP in mouse muscle in-vivo. Methods Experiments were performed on a 9.4T Magnex magnet interfaced to a Bruker Biospec console. Mice were anesthetized with isoflurane and underwent continuous physiological monitoring. The hindlimb muscles were positioned directly under a 15mm double-turn 31P surface coil; scout images and shimming were performed using 25mm diameter quadrature 1H coils. 31P spectra for CST were acquired using a custom-written pulse sequence with frequency-selective saturation of the \u3b3ATP peak (M\u2019) or with saturation at a downfield frequency equidistant from Pi (M0), using the following parameters: 1msec AHP excitation pulse (centered between Pi and \u3b3ATP), 10sec \u2018soft\u2019 saturation pulse, sweep width = 8kHz, 1024 complex points, effective TR = 10sec, 64 transients. FAST spectra were acquired using 1msec adiabatic BIR4 pulses (7) for excitation (\u3b1 = 90o, \u3b2 = 30o) and a 2sec pulse for \u3b3ATP or symmetric saturation. The T1 of Pi under conditions of \u3b3ATP saturation (T1\u2019) was measured in each animal using an 8-point inversion-recovery calibration with \u3b3ATP saturation prior to and during the inversion delay. Each block of CST or FAST spectra was repeated twice prior to, and twice after the T1\u2019 calibration. Fully-relaxed 31P spectra (AHP excitation, TR = 25 sec, 32 transients) were obtained to determine metabolite concentrations in-vivo, absolute [ATP] was determined by high resolution 31P-MRS of freeze clamped tissue extracts. Results A cross sectional mouse hindlimb image is shown in Fig 1; typical muscle 31P saturation-transfer spectra (90o AHP excitation) are shown in Fig 2. Rates of ATP synthesis obtained using either CST or FAST were equivalent (Table 1)

Effects of UCP3 overexpression on mouse muscle mitochondrial function in-vivo

Uncoupling protein 3 (UCP3) is a mitochondrial trans-membrane protein that is highly expressed in muscle. 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 \u2018uncouple\u2019 mitochondrial oxidation and ATP synthesis. Modulation of UCP3 expression alters oxygen consumption and proton leak in isolated mitochondria in vitro, but its precise function in-vivo has yet to be fully elucidated. The aim of this study was to determine whether muscle-specific overexpression of UCP3 in transgenic (TG) mice alters the coupling of mitochondrial oxidative phosphorylation in vivo. Energy production was assessed in the hindlimb of anesthetized control (WT) and UCP3-TG mice by determining the rate of Pi \u2192 ATP flux (VATP) using 31P saturation-transfer MRS. In a separate series of experiments, substrate oxidation via the tricarboxylic acid cycle (VTCA) was determined using a novel biopsy-based technique. Anesthetized 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 500MHz. VTCA was calculated by metabolic modeling of the time-courses of enrichment of the muscle 13C4-glutamate, 13C3-glutamate and 13C4-glutamine pools. VATP was decreased by 19% in the muscle of UCP3-TG mice compared to WT mice (4.55 \ub1 0.29 vs 5.65 \ub1 0.27 \u3bcmol/(g-min), P=0.02). In contrast, Monte-Carlo analysis of the metabolic modeling data indicated that VTCA was increased by 22% in the muscle of UCP3-TG mice with respect to WT mice (120.3 \ub1 9.9 vs 94.3 \ub1 5.8 nmol/(g-min), P=0.014). 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

Publisher Correction: Deletion of the diabetes candidate gene <em>Slc16a13</em> in mice attenuates diet-induced ectopic lipid accumulation and insulin resistance.

The original published version of the Article contained an error in the abstract whereby the words “loss of” were inadvertently omitted from the following sentence: “We show that loss of Slc16a13 increases mitochondrial respiration in the liver, leading to reduced hepatic lipid accumulation and increased hepatic insulin sensitivity in high-fat diet fed Slc16a13 knockout mice.” The error has been corrected in the HTML and PDF versions of the Article

Deletion of the diabetes candidate gene <em>Slc16a13</em> in mice attenuates diet-induced ectopic lipid accumulation and insulin resistance.

Genome-wide association studies have identified SLC16A13 as a novel susceptibility gene for type 2 diabetes. The SLC16A13 gene encodes SLC16A13/MCT13, a member of the solute carrier 16 family of monocarboxylate transporters. Despite its potential importance to diabetes development, the physiological function of SLC16A13 is unknown. Here, we validate Slc16a13 as a lactate transporter expressed at the plasma membrane and report on the effect of Slc16a13 deletion in a mouse model. We show that Slc16a13 increases mitochondrial respiration in the liver, leading to reduced hepatic lipid accumulation and increased hepatic insulin sensitivity in high-fat diet fed Slc16a13 knockout mice. We propose a mechanism for improved hepatic insulin sensitivity in the context of Slc16a13 deficiency in which reduced intrahepatocellular lactate availability drives increased AMPK activation and increased mitochondrial respiration, while reducing hepatic lipid content. Slc16a13 deficiency thereby attenuates hepatic diacylglycerol-PKCε mediated insulin resistance in obese mice. Together, these data suggest that SLC16A13 is a potential target for the treatment of type 2 diabetes and non-alcoholic fatty liver disease

Selective Chemical Inhibition of PGC-1 alpha Gluconeogenic Activity Ameliorates Type 2 Diabetes

Type 2 diabetes (T2D) is a worldwide epidemic with a medical need for additional targeted therapies. Suppression of hepatic glucose production (HGP) effectively ameliorates diabetes and can be exploited for its treatment. We hypothesized that targeting PGC-1α acetylation in the liver, a chemical modification known to inhibit hepatic gluconeogenesis, could be potentially used for treatment of T2D. Thus, we designed a high-throughput chemical screen platform to quantify PGC-1α acetylation in cells and identified small molecules that increase PGC-1α acetylation, suppress gluconeogenic gene expression, and reduce glucose production in hepatocytes. On the basis of potency and bioavailability, we selected a small molecule, SR-18292, that reduces blood glucose, strongly increases hepatic insulin sensitivity, and improves glucose homeostasis in dietary and genetic mouse models of T2D. These studies have important implications for understanding the regulatory mechanisms of glucose metabolism and treatment of T2D. © 2017 Elsevier Inc.1