Recent investigations have associated consumption of high fat diets (HFD) with inflammation in the hypothalamus, the main cerebral regulator of energy balance in the brain, reporting processes as astroglisosis and neuronal injury at a local level¹. The implementation of a technique capable of detecting in vivo the changes in metabolites, neurotransmitters and metabolic fluxes in the hypothalamus, could represent an excellent strategy with which to investigate the physiologic and pathologic conditions of the mouse brain. Thus, the aim of this study was to follow the incorporation of ¹³C-labeled glucose into its metabolites in the hypothalamus of mice fed regular diet or high fat diet using ¹H-[¹³C] MRS methods, and investigate the HFD induced effects on glucose metabolites and neuroglial metabolic fluxes.C57BL/6 mice (12weeks old, n=3, 28.5±1.2g) were fed with a 60% kcal fat diet (http://www.researchdiets.com) during two months or with regular food chow (13 weeks old, n=5, 28.6±1g). Animals were fasted overnight (12h) before experiments, and body weight and blood glucose levels were controlled in each experimental session. All MR measurements were performed in a horizontal 14.1T/26cm Varian Magnet, using a homemade ¹H quadrature surface coil (12mm diameter) combined with a linear ¹³C coil (11 mm diameter). The hypothalamus voxel (1.8x2.7x1.8 mm³) was selected based on anatomical MR images and using a mouse brain atlas as a reference² and field homogeneity was improved using FASTMAP methods³. Indirect ¹H-[¹³C] detection was achieved using the full signal intensity NMR sequence BISEP-SPECIAL⁴combined with OVS and VAPOR water supression⁵. Hypothalamic glucose (Glc) metabolism was evaluated during the infusion of [1,6-¹³C₂] glucose solution (20% w/v , bolus= 9mL/kg, infusion rate= 15mL/kg/h). To increase the SNR, spectra of all animals’ from the same group were added together every 13 min of acquisition, and metabolites were quantified using LCModel. Non-edited 1H MR spectra acquired with ¹³C decoupling but without ¹³C editing contain ¹H resonances coupled to both ¹²C and ¹³C and thus can be quantified with a standard basis set for neurochemical profiles of mouse hypothalamus. The ¹³C-editing spectra were quantified using another simulated basis set as previously4. The fraction of isotope enrichment (FE) in lactate (Lac), glutamate (Glu), glutamine (Gln) and g-amino-butyrate (GABA) were estimated. Time course of Glu C4, Gln C4 and Glx C3 was fitted to a one compartment model of glucose metabolism using MATLAB nonlinear regression methods, and TCA rate (Vtca), transmitocondrial flux (Vx), neurotransmission rate (Vnt), dilution factor (Kdil) and composite flux (Vgt) were estimated. The errors of all adjusted metabolic fluxes were evaluated by Monte–Carlo simulation⁶.Body weight of HFD fed and control animals before the MRS measurements (fasting state) (45.8±2.6g, 25.6±1g) were significantly different (p<0.005, t-test) as well as their fasting blood glucose levels (13±1.2mmol, 5.25±1.2 mmol). Figure 1 shows representative non-edited and edited hypothalamic spectra in control animals after 160 min of infusion of labeled glucose: note the high levels of GABA, a typical feature of the mouse hypothalamus⁷. SNR of the edited spectra was >5 for all cases, as measured from LCModel, and linewidths of total creatine at 3.0ppm <20Hz. Figure 2 depicts the time course of the ¹³C label accumulation in the same animals, and the calculated FE for HFD and control animals is illustrated in Figure 3. FE of Lac C3, which depicts a similar turnover in both groups, was used as an input function for the metabolic fluxes quantification. The total pool size of the metabolites was also measured along the glucose infusion, and values are summarized in Figure 4. Glc and Asp show higher values in the HFD group and GABA and Lac higher in the control group (p<0.001 in all cases, t-test). Finally, derived metabolic fluxes are illustrated in Figure 5. Calculated values show similar Vtca rates for both animal groups, higher Vx and Vgt for the HFD animals and higher Vnt and Kdil for the control group.Indirect ¹H-[¹³C] detection revealed the incorporation of ¹³C-labeled glucose in vivo into a number of metabolites of biological importance in the hypothalamus. We show for the first time, in vivo reliable measurements of the turnover of Lac C3, Glu C4, Gln C4, Glu C3, Gln C3, GABA C4, GABA C3 in the mouse hypothalamus. HDF animals, which show prediabetic fasting blood glucose levels, display increased Glc and Asp levels and decreased Lac and GABA levels in the hypothalamus, as compared to regular fed animals. Moreover, the increased Vx and decreased Vnt and Kdil highlight putative alterations in glucose metabolism.