High Expression of Thyroid Hormone Receptors and Mitochondrial Glycerol-3-phosphate Dehydrogenase in the Liver Is Linked to Enhanced Fatty Acid Oxidation in Lou/C, a Rat Strain Resistant to Obesity*

Besides its well recognized role in lipid and carbohydrate metabolisms, glycerol is involved in the regulation of cellular energy homeostasis via glycerol-3-phosphate, a key metabolite in the translocation of reducing power across the mitochondrial inner membrane with mitochondrial glycerol-3-phosphate dehydrogenase. Here, we report a high rate of gluconeogenesis from glycerol and fatty acid oxidation in hepatocytes from Lou/C, a peculiar rat strain derived from Wistar, which is resistant to age- and diet-related obesity. This feature, associated with elevated cellular respiration and cytosolic ATP/ADP and NAD+/NADH ratios, was linked to a high expression and activity of mitochondrial glycerol-3-phosphate dehydrogenase. Interestingly, this strain exhibited high expression and protein content of thyroid hormone receptor, whereas circulating thyroid hormone levels were slightly decreased and hepatic thyroid hormone carrier MCT-8 mRNA levels were not modified. We propose that an enhanced liver thyroid hormone receptor in Lou/C may explain its unique resistance to obesity by increasing fatty acid oxidation and lowering liver oxidative phosphorylation stoichiometry at the translocation of reducing power into mitochondria.

Obesity has been widely studied in subjects obese or prone to obesity, but the mechanisms underlying a resistance to obesity have received little attention. Here, we studied an inbred strain derived from Wistar rats, which display a remarkable resistance to obesity (1). Indeed, Lou/C rats exhibit a lower body mass and percentage of fat mass throughout their lives (2)(3)(4), in contrast to the well reported age-related obesity of their Wistar counterparts (5). Moreover, these animals also maintain their body mass in response to a high fat diet (6). Such features are only partly related to a spontaneous lower caloric intake (7,8) because Lou/C rats have lower fat deposits but a higher percentage of carcass proteins and muscle mass as compared with pair-fed Wistar rats (4), even when subjected to a high fat diet (6). This peculiar metabolic phenotype has recently been investigated (9), showing lower blood glucose together with liver and muscle glycogen content, whereas insulin sensitivity was higher. These animals also exhibit a high basal activity, associated with a high capacity for long term exercise (9). Interestingly, when submitted to 60 min of exercise, these rats maintain blood glucose with no depletion of liver or skeletal muscle glycogen, in marked contrast to Wistar rats (9) and in accordance with a reported preference for fat oxidation as compared with carbohydrate (10). Preliminary experiments investigating liver gluconeogenesis revealed that hepatocytes from 24-h fasted Lou/C exhibited lower gluconeogenesis rates from most precursors (lactate-pyruvate, fructose, dihydroxyacetone, alanine), whereas glycerol was a remarkable exception with a higher rate of glucose production as compared with Wistar rats (9).
Glycerol is an important gluconeogenic substrate in liver and kidney because of high glycerol kinase activity (EC 2.7.1.30). However, muscle, brain, and other tissues also contain glycerol kinase (11,12). Glycerol also has important implications via L-glycerol-3-phosphate (G3P), 3 which plays a key role in both lipid metabolism and energy homeostasis (11,13). This latter effect is related to the transport of reducing equivalents across the inner mitochondrial membrane via the FAD-dependent mitochondrial glycerol-3-phosphate dehydrogenase (mGPdH, EC 1. 1.99.5), and thanks to the presence of cytoplasmic NADdependent glycerol-3-phosphate dehydrogenase (cGPdH, EC 1.1.1.8), recycling occurs between G3P and dihydroxyacetone phosphate (DHAP). This leads to a net transfer of reducing equivalents from cytosolic NADH to mitochondrial matrix (14) by reducing the quinone pool directly from mGPdH-linked FADH 2 (15). In the malate-aspartate carrier system, NADH is the final electron donor to the respiratory chain, whereas in the DHAP-G3P shuttle, NADH is converted to FADH 2 , resulting in a substantial disparity between the two pathways regarding oxidative phosphorylation stoichiometry: three coupling sites with NADH versus two coupling sites with FADH 2 . Hence, due to its role in the regulation of the respective flux through these two shuttles, G3P metabolism and its modulation by mGPdH activity play an important role in cellular energetic homeostasis. Indeed, knock-out mice for both mitochondrial and cytosolic GPdH died a few days after birth (16). mGPdH is present in most tissues of various animal species, but its activity varies from very high (brown adipose tissue and muscle) to very low (the heart and liver) (17). Thyroid hormones are potent activators of mGPdH transcription, especially in the liver, kidney, heart, skeletal muscle, diaphragm, and adipose tissue, but not in the brain, lungs, spleen, stomach, or small intestine (11,17). Furthermore, steroid hormones (18), a low protein/high sucrose diet (19), long fasting, and acute cold exposure (20) also increase liver mGPdH. In addition to transcriptional regulation, calcium increases liver mGPdH activity by increasing enzyme affinity toward G3P (21).
In this study, we have investigated glycerol metabolism in freshly isolated liver cells from Lou/C and Wistar rats. A higher rate of glycerol metabolism was observed in Lou/C in comparison with Wistar. The main steps involved in this effect were glycerol phosphorylation and G3P oxidation, depending on the presence or not of fatty acids. We failed to find any significant change in glycerol kinase and cGPdH activities or mRNAs or in aquaglyceroporin 9 mRNA. By contrast, mGPdH activity, protein content, and transcripts were significantly higher in Lou/C. Moreover, we found significantly higher amounts of liver thyroid hormone receptor TR␣1 protein and mRNAs, whereas circulating thyroid hormones were slightly lower in Lou/C. Higher octanoate metabolism and oxidative capacity are also reported. We proposed that the higher liver mGPdH activity represents an important feature of the metabolic phenotype of Lou/C FIGURE 1. Gluconeogenesis from glycerol in isolated hepatocytes from Lou/C and Wistar rats, activation at the glycerol kinase step. Hepatocytes (220 mg of dry cells) isolated from liver of 24-h-starved Wistar (closed symbols) and Lou/C rats (open symbols) were perifused with increasing concentrations of glycerol. The total metabolic flux from glycerol (A, J2GLP) was expressed as three-carbon equivalents and calculated from the sum of 2glucose ϩ lactate ϩ pyruvate. The rates of gluconeogenesis (B, J Glucose ) and glycolysis (JLϩP, panel C) were calculated from the glucose and lactate-plus-pyruvate concentrations in the perifusate, respectively. Lactate-to-pyruvate ratio represents the cytosolic redox potential (D). Cytosolic ATP (E) and ATP-to-ADP (F) were measured by HPLC. At each steady state (20 min), samples of cell suspension were removed from the perifusion chamber and centrifuged through an oil layer into HClO 4 to separate the intracellular from the extracellular compartment and the cytosolic from the mitochondrial compartment. Intracellular fructose 6-phosphate (F6P) (G), DHAP (H), and G3P (I) were measured enzymatically in the neutralized intracellular fraction and plotted against J Glucose . The results are expressed as means Ϯ S.E. for five rats in each condition. *, p Ͻ 0.05 between Wistar and Lou/C. strain, which may explain, at least in part, their resistance to obesity. The well known relationship between thyroid hormone and mGPdH transcription, associated with the present finding of higher liver thyroid hormone receptor mRNA in Lou/C, led us to propose it as a possible mechanism.

EXPERIMENTAL PROCEDURES
Isolation of Hepatocytes-Hepatocytes were prepared from male Wistar (250 -350 g) and male Lou/C rats (220 -280 g) aged 14 weeks according to the method of Berry and Friend (22) and modified by Groen et al. (23). 1 mg of dry hepatocytes equals ϳ465,000 cells.
Perifusion of Hepatocytes-Isolated hepatocytes (200 -220 mg of dry cells) were perifused according to the method of Van Der Meer and co-workers (44) and modified by Groen (23) with increasing amounts of glycerol (from 0.15 to 9.6 mM), in the presence or not of 0.4 mM octanoate. At each steady state, perifusate and cell samples were collected. Glucose, lactate, pyruvate, 3-hydroxybutyrate, and acetoacetate were determined spectrophotometrically (24). Metabolite flux results were expressed in mol⅐min Ϫ1 ⅐dry cells Ϫ1 . Cell samples were collected from the perifusion chamber to separate the intracellular from the extracellular space by centrifugation through a layer of silicon oil (Rhodorsil 640 V 100) into HClO 4 ϩ EDTA (10% mass/volume, 25 mM) (25), and intracellular metabolites (fructose 6-phosphate, DHAP, and G3P) were determined (24). Mitochondrial and cytosolic compartments were separated by digitonin fractionation (26) for subsequent determination of adenine nucleotide content with the HPLC method.
Determination of Cellular Oxygen Consumption Rate-Hepatocytes (7.5 mg of dry cells⅐ml Ϫ1 ) were incubated in a shaking bath at 37°C in closed vials containing 3.2 ml of Krebs Ringer bicarbonate calcium buffer without any substrates or after the addition of 20 mM glycerol, or 4 mM octanoate. Oxygen consumption rate (JO 2 ) was determined polarographically at 37°C with a Clark electrode. JO 2 was measured before and after the addition of oligomycin (6 g⅐ml Ϫ1 ) and then DNP (50 M) and myxothiazol (3.8 M).
Determination of Enzymatic Activities-Glycerol Kinase and cytosolic GPdH activity were measured spectrophotometrically in the supernatant of sonicated isolated hepatocytes (24). Mitochondrial GPdH activity was measured on the supernatant of isolated mitochondria after three cycles of freezing and thawing. Mitochondria were extracted according to the method of Klingenberg and Slenczka (27)   ured spectrophotometrically at 600 nm at 37°C and is expressed as mol⅐min Ϫ1 ⅐mg of proteins Ϫ1 .
Western Blot Analysis-Samples of isolated hepatocytes were lysed, and proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Expression of mGPdH and eukaryotic elongation factor 2 (EF2) were monitored by SDS-PAGE immunoblots using mGPdH (gift from Dr. J. Weitzel), EF2 (Santa Cruz Biotechnology), and TR␣1-specific antibodies linked to horseradish peroxidase. Blots were developed using chemiluminescence (Roche Applied Science).
RNA Purification and Reverse Transcription-coupled PCR-RNA was extracted from isolated hepatocytes with the Tripure RNA isolation reagent (Roche Diagnostics). Total RNA (1 g) was reverse-transcribed, and quantitative real-time PCR was then performed with SYBR Green Core kit on a MyIQ thermal cycler (Bio-Rad). mRNA contents were normalized for actin mRNA and expressed relative to that of Wistar using the 2 Ϫ⌬⌬CT method (28). MCT-8, THR␣1, and THR␤1 were normalized to RSP12 as an internal standard.
Confocal Microscopy-Images of isolated hepatocytes were acquired with a Nikon TE 200 microscope equipped for epifluorescent illumination (xenon light source, 75 watts), associated with a 12-bit digital-cooled charged-coupled device camera (SPOT-RT, Diagnostic Instruments). The autofluorescence of NADH and FAD ϩ was obtained after the incubation of isolated hepatocytes without any substrates, with glycerol (10 mM), octanoate (2 mM), or DNP (75 M) for 5 min.
Statistics-The results are expressed as mean Ϯ S.E. Statistically significant differences were assessed by analysis of vari- The rates of gluconeogenesis (A, J Glucose ) and ketogenesis (B, J acetoacetate (AA) ϩ ␤-hydroxybutyrate (␤OH)) were calculated from the glucose and acetoacetate ϩ ␤-hydroxybutyrate concentration in the perifusate, respectively. The mitochondrial and cytosolic redox potentials were calculated from the ratio of the ␤-hydroxybutyrate-to-acetoacetate (C, ␤-hydroxybutyrate/acetoacetate) and lactate-to-pyruvate (D) and the ratios, respectively. Cytosolic ATP-to-ADP (E) and mitochondrial AMP (F) were measured by HPLC. At each steady state (20 min), samples of cell suspension were removed from the perifusion chamber. They were then centrifuged through an oil layer into HClO 4 . Intracellular DHAP (G) and G3P (H) were measured enzymatically in the neutralized acid-soluble fraction and plotted as J Glucose . The results are expressed as means Ϯ S.E., n ϭ 5. *, p Ͻ 0.05 between Wistar and Lou/C. ance followed by Fisher's protected least significant post hoc test (Statview 5.0.1. software).

Activation of Glycerol Metabolism in Lou/C as Compared
with Wistar-Successive steady states of glycerol metabolism, as estimated by the sum of three-carbon products (Fig. 1A, 2ϫ glucose ϩ lactate ϩ pyruvate (2GLP)), were obtained by the infusion of increasing glycerol concentrations. A higher glycerol metabolism was evidenced in Lou/C, resulting from higher gluconeogenesis (Fig. 1B), because lactate plus pyruvate production was similar in both strains (Fig. 1C). Interestingly, at saturating concentration (i.e. higher than 1.2 mM glycerol), a slight decline in the maximal glucose flux was observed despite a further increase in substrate concentration (see below). Because glycerol metabolism is mainly controlled by cellular redox state (29), increased glycerol metabolism is expected to be associated with an elevated NAD ϩ /NADH ratio (30,31). However, we found here that higher glycerol metabolism in Lou/C coexisted with a reduced cytosolic compartment, i.e. a decrease in the NAD ϩ /NADH ratio, as indicated by a higher lactate-to-pyruvate ratio (L/P ratio, Fig. 1D) (32). This indicates that this higher rate is not due to enhanced oxidative capacity but to higher glycerol phosphorylation ability.
To investigate this apparent discrepancy, we took advantage of our experimental model and allowed a succession of true steady states; each rate of substrate infusion generates an equal flux through every step, which is the rate of glucose formation (J Glucose ). Therefore, by determining J Glucose at each steady state, we can infer this value to each step of the pathway. From the data presented in Fig. 1, G and H, it appears that the steps located downstream of DHAP were not the location of any difference between the two strains because we found a single relationship between substrate concentration and related flux in both groups. Considering G3P oxidation (Fig. 1I), a unique relationship was also observed, confirming that the difference in glycerol metabolism observed in Lou/C was not due to a redox effect at the G3P oxidation step. Another interesting observation was that the relationship between G3P concentration and its metabolism exhibited a biphasic shape; the rate of G3P oxidation declined at G3P concentrations higher than 15 mol⅐g of dry cells Ϫ1 . This is probably the consequence of glucose 6-phosphatase (EC 3.1.3.9) inhibition by G3P (33).
Because gluconeogenesis from glycerol requires a stoichiometric utilization of ATP and NAD ϩ , higher J Glucose in Lou/C must be associated with a higher mitochondrial oxidative phosphorylation rate. Indeed, cellular oxygen consumption rate was significantly higher in hepatocytes of Lou/C as compared with Wistar rats with endogenous substrate ( Fig. 2A) and with glycerol (Fig. 2B). The difference between the strains was found at basal (physiological) state, upon oligomycin addition (an inhibitor of ATP synthesis) and in respiration uncoupled by 2,4dinitrophenol. However, when mitochondrial respiration was fully inhibited by myxothiazol, a complex 3 inhibitor, no difference was observed, indicating that the mitochondrial respiratory chain is the location of the increased oxygen consumption in Lou/C. Cytosolic ATP (Fig. 1E) and ATP-to-ADP ratio (ATP/ADP ratio) (Fig. 1F) were significantly higher in Lou/C. Taken together, these results indicate that a higher gluconeogenesis rate from glycerol in Lou/C hepatocytes is accompanied by a higher respiration rate, L/P ratio, and cytosolic ATP/ADP ratio.
Glycerol and Octanoate Metabolism in Lou/C, the Effect of High Reducing Power-Fatty acid metabolism dramatically decreases gluconeogenesis from glycerol because of the reducing effect (30,31). Therefore, the combination of glycerol and fatty acid, a physiologically relevant situation, represents a suitable metabolic condition to further characterize the metabolic relationship between cytosolic redox state and hepatic glycerol metabolism in Lou/C. As expected, octanoate addition resulted in a dramatic inhibition of gluconeogenesis from glycerol in both strains (compare Fig. 3A with Fig. 1A). Indeed, the lowering effect of octanoate metabolism on J Glucose is significantly less in Lou/C (p Ͻ 0.005) as compared with Wistar. Octanoate metabolism was higher in Lou/C, as indicated by higher rates of ketogenesis (Fig. 3B) and oxygen consumption (Fig. 2C). Moreover, a unique relationship was found between DHAP and J Glucose (Fig. 3G) regardless of the strain, whereas two different relationships were evidenced between G3P and J Glucose (Fig.  3H), contrasting with the finding obtained with glycerol alone (Fig. 1I). Because these two relationships characterize glycerol pathways up-and downstream of DHAP, respectively, this result pointed to the step of G3P oxidation as the main target for the difference between Lou/C and Wistar in these conditions of glycerol and fatty acids. Indeed, an effect located upstream of the G3P dehydrogenase step leads to a single relationship between J Glucose and G3P, whereas an effect located downstream of DHAP leads to a double relationship between J Glucose and DHAP. As evidenced in Fig. 3D and the inset, octanoate metabolism markedly reduced the cytosolic compartment, as indicated by the rise in lactate-to-pyruvate ratio. This effect is significantly more pronounced in the control Wistar in comparison with Lou/C (p Ͻ 0.05 between the two strains). The L/P ratio decreased progressively, whereas glycerol concentration increased in the Wistar group; however, the ratio was barely affected in the Lou/C group, and it was identical in both groups above 4 mM glycerol. The mitochondrial compartment was also reduced by octanoate as shown by the change in the 3-hydroxybutyrate-to-acetoacetate ratio (Fig. 3C and the inset) without a significant difference between the two strains. ATP/ADP ratios were slightly higher in both cytosol and mitochondria in Lou/C, but the difference did not reach a significant level (Fig. 3E and the inset). In addition, octanoate substantially increased mitochondrial AMP due to the intra-matricial activation of octanoate in liver cells by the medium chain fatty acyl-CoA synthetase (EC 6.2.1.2) (34, 35) ( Fig. 3F  and the inset). Interestingly, the effect of octanoate on mitochondrial AMP concentration was significantly more pronounced in Lou/C than in Wistar, another indication of higher fatty acid metabolism in this strain as compared with Wistar (see oxygen consumption ( Fig. 2C) and ketones production (Fig. 3B)). Hence, in the condition where both glycerol and octanoate were provided to liver cells, the higher glucose production in Lou/C appeared to be related to a redox effect located at the G3P oxidation step, contrasting with the finding above in which glycerol was the unique exogenous substrate.
In Situ Hepatocyte Redox State Assessment by Nucleotide Autofluorescence-Assessment of cytosolic and mitochondrial redox state is not accurately evaluated from direct determination of reduced and oxidized nucleotide contents because the ratio between free and bound metabolites is not accessible; therefore, an indirect method based on the measurement of lactate-to-pyruvate or ␤-hydroxybutyrate-to-acetoacetate ratios was proposed (32). However, by taking advantage of the specific autofluorescence of NADH and FAD (see "Experimental Procedures" and Fig. 4), an assessment of the cellular redox state could also be achieved in freshly isolated liver cells by confocal microscopy. NADH fluorescence (blue) decreases while oxidized to NAD, whereas flavin fluorescence (green) increases when oxidized (FAD); therefore, a decrease in the blue-togreen ratio reflects a more oxidized state. The decrease in the blue/green fluorescence ratio following uncoupling by DNP (shown in Fig. 4) indicated the physiological relevance of this assessment of the global cellular redox state, which included nicotinamide and flavin cofactors. The fluorescence ratio was significantly lower in Lou/C in comparison with Wistar, reflect-

FIGURE 5. Determination of expression and activity of mitochondrial glycerol-3-phosphate dehydrogenase and several other proteins involved in liver glycerol pathway, a comparison between Lou/C and Wistar rats.
A, cytosolic GPdH and glycerol kinase (GlyK) activities were measured in the supernatant of hepatocyte suspensions after cell disruption by sonication. Results (n ϭ 5) are expressed as mol⅐min Ϫ1 ⅐mg of proteins Ϫ1 . B, mGPdH activity was measured spectrophotometrically on isolated mitochondria. Results (n ϭ 9) are expressed as mol⅐min Ϫ1 ⅐mg of proteins Ϫ1 . C and G, mGPdH (C) and TR␣1 (G) protein contents were assessed in hepatocytes from Wistar and Lou/C rats by Western blot using EF2 as loading controls (C). Results are representative of six different cell preparations. D and F, hepatocyte RNAs were extracted, and quantitative real-time PCR on cDNA was performed for mGPdH, aquaglyceroporin (AQ9), and peroxisome proliferatoractivated receptor-␣ (PPAR␣) (D) and for MCT-8, TR␣1, TR␤1, deiodinase 1, Spot 14 (S14), cytochrome oxidase (COX), F1-ATP synthase, and mitochondrial transcription factor A (mTFA) (F). mRNA contents (n ϭ 6) were normalized to RSP12 mRNA and expressed as relative values using the 2 Ϫ⌬⌬CT method. F, circulating thyroid hormones were measured by radioimmunoassay in the serum (n ϭ 12 in each group). All results are expressed as means Ϯ S.E.; * denotes a significant difference (p Ͻ 0.05) between Wistar and Lou/C. Dio1, deodinase 1. FEBRUARY 13, 2009 • VOLUME 284 • NUMBER 7

TH Receptors and mGPdH in Lou/C
ing an apparently higher oxidized state. Indeed, this difference persisted after the successive addition of glycerol and octanoate, but not of 2,4 DNP, when both cells were fully oxidized.
Molecular Targets Involved in the Modification of Glycerol Metabolism in Lou/C Rats-As commonly recognized, and accordingly to the data presented above, the main controlling steps of gluconeogenesis from glycerol are glycerol phosphorylation and G3P oxidation; however, glycerol transport across the plasma membrane might also play a role. The membranebound aquaglyceroporins are recognized as glycerol carriers, and aquaglyceroporin 9 has been proposed as being specific to the liver (36). Similar transcript levels were found in both strains (Fig. 5D), indicating that glycerol transport across the cellular membrane was probably similar in both strains. Potentially, G3P oxidation depends on both cGPdH and mGPdH, with activity of the latter being very low in the liver (29). Glycerol kinase (9.8 Ϯ 0.5 versus 9.2 Ϯ 0.2 mol⅐min Ϫ1 ⅐g of dry cells Ϫ1 for Wistar and Lou/C, respectively, n ϭ 5) and cGPdH (70.25 Ϯ 4.78 versus 65.42 Ϯ 9.84 mol⅐min Ϫ1 ⅐mg of proteins Ϫ1 for Wistar and Lou/C, respectively, n ϭ 5) activities and transcripts (Fig. 5A) were similar in both strains. By contrast, mGPdH activity (ϩ70%, p Ͻ 0.05, Fig. 5B), protein, and transcript contents (ϩ100%, p Ͻ 0.01) were markedly higher in Lou/C. The transcription of peroxisome proliferator-activated receptor-␣ (PPAR␣), a factor involved in the expression of several genes involved in glycerol metabolism including mGPdH (37), was no different between the two strains, excluding its implication in the higher expression of mGPdH in Lou/C (Fig. 5D).
THs are known as potent activators of mGPdH transcription in the liver. However, plasma concentrations of T3 and T4 were slightly but significantly lower in Lou/C rats, whereas TSH was higher (Fig. 5E), a finding already reported in this strain (38). Interestingly, the assessment of TH receptors TR␣1 and TR␤1 in livers from Wistar and Lou/C revealed significantly higher TR␣1 protein (Fig. 5G) and TR␣1 and TR␤1 mRNA levels in Lou/C (Fig. 5F). Several other targets dependent on TH receptor activation (Fig. 5F, Spot 14 (S14), cytochrome oxidase (COX), F1-ATPase, mitochondrial transcription factor A (mTFA), and deodinase 1 (Dio1)) were also modified, indicating a general effect (Fig. 5F). In addition, we have investigated in vivo the effect of an exogenous load of T3 on mGPdH transcription level in the two strains Wistar and Lou/C. It was found that basal as well as T3-stimulated levels of mGPdH transcripts were significantly higher in Lou/C, whereas plasma T3 levels were similar (data not shown).

DISCUSSION
Higher TH receptors associated with a higher mGPdH activity in the liver from the Lou/C strain represent the main findings of the present work. This feature appears to have a substantial effect on hepatic fatty acid oxidation and glycerol metabolism. This may, at least to some extent, explain the key physiological characteristics of this peculiar strain. Indeed, modulating the pathway of electron supply to the respiratory chain has considerable implications on whole body energy homeostasis.
In comparison with Wistar, Lou/C strain is characterized by a spontaneous lower body mass and food intake. However, FIGURE 6. Supply of reducing power to the respiratory chain, the respective roles of malate-aspartate shuttle (MAS) and G3P-DHAP shuttle (GDS), and implication in fatty acidoxidation control. Glycerol metabolism in the liver is mainly controlled by two consecutive steps: glycerol phosphorylation by glycerol kinase and G3P oxidation, either via cGPdH or via mGPdH. These two enzymes are crucial for the translocation of the reducing power (indicated as NADH/NAD ϩ ) into the mitochondrial (mit) matrix. The malate-aspartate shuttle carries NADH into the matrix at the expense of mitochondrial membrane potential because the glutamate carrier is energy-dependent. The G3P-DHAP shuttle is not energy-dependent; it translocates reducing equivalents by converting NADH to FADH 2 . Therefore, electrons are provided to the quinone pool, i.e. downstream of complex 1, which is a less efficient way for ATP synthesis in comparison with NADH because two coupling sites (complexes 3 and 4) are involved instead of three with NADH. In liver mitochondria, complex 1 is an important controlling step of the respiratory chain, and the fact that this complex provides downstream electrons allows a higher respiratory flux if the rate of electron supply is sufficient, as is the case with succinate as mitochondrial substrate. However, in the liver, mGPdH activity is physiologically very low, and the flux through the G3P-DHAP shuttle is low despite the absence of the control exerted by complex 1. However, when mGPdH activity is high, as shown here in the Lou/C strain, the flux through the G3P-DHAP shuttle is also high, and the net result is a lower efficiency at a higher rate of respiration and ATP synthesis. This delicate balance is finely tuned in physiological conditions by the level of the thyroid hormones T3 because it has a powerful effect on mGPdH transcription. The higher level of thyroid hormone receptors reported here in the liver of Lou/C rats could explain this "hyperthyroid metabolic phenotype," whereas the thyroid status phenotype is on the hypothyroid side because of the low circulation levels of T4 and T3 and the high TSH. However, these changes, albeit significant, are not very marked as compared with those found in hypothyroidism. 1, glutamate carrier; 2, respiratory chain complex 1; 3, mitochondrial glycerol-3-phosphate dehydrogenase; 4, succinate dehydrogenase; 5, respiratory chain complex 3; 6: respiratory chain complex 4; 7, ATP synthase; 8, adenine nucleotide translocator; 9, glycerol transport, aquaporin 9; 10, glycerol kinase; 11, cytosolic glycerol-3-phosphate dehydrogenase; 12, glucose transport-GLUT 3. cyt, cytoplasmic.
unlike pair-fed animals, muscle mass is proportionally higher, whereas fat mass is lower, indicating disturbances other than food intake. Impressive resistance to diet-and age-related obesity with hyperactivity associated with an exceptional ability for long term physical exercise represent the most characteristic features of this animal (6,39). The reported increase in mGPdH activity is most likely a transcriptional effect because both transcripts and protein were higher. A local (hepatic) increase in TH effects, due to higher levels of TR␣1 and TR␤1, probably explains such an effect occurring despite low circulating T3 and T4 levels.
The higher gluconeogenic rate from glycerol in Lou/C is due to an effect located upstream of G3P oxidation and associated with a higher cytosolic ATP/ADP ratio. The absence of any difference in aquaglyceroporin 9 transcripts does not point to an effect on glycerol transport. The flux of glycerol phosphorylation is higher in Lou/C despite the fact that it has a similar glycerol kinase content, activity, and transcription. This is probably explained by the higher cytosolic ATP/ADP ratio (11,15). The association of a higher mGPdH activity with a high ATP/ADP ratio in LouC is interesting because the opposite was expected in light of the decreased oxidative phosphorylation stoichiometry resulting from a higher G3P shuttle. ATP levels and the ATP/ADP ratio depend on both the rate of ATP synthesis and the utilization of ATP rather than on oxidative phosphorylation efficiency. In fact, we have shown that mitochondrial adaptation to various cellular energy demands is based on a permanent compromise between rate and efficiency; the highest rate is achieved at the lowest efficiency and vice versa (40). On one hand, bypassing complex 1 by mGPdH lowers ATP-to-oxygen stoichiometry (ATP/O) as complex 1, i.e. the first coupling site of the respiratory chain, is excluded from the pathway. However, on the other hand, due to the absence of any flux controlling effect of complex 1, a higher respiratory rate is achieved, explaining the higher rate of ATP synthesis despite lower efficiency (40,41). Octanoate addition to glycerol further reduces cytosolic compartment (compare Figs. 1D and 3D), whereas glucose production and ATP/ADP ratio decrease (compare Figs. 1, A and F, and 3, A and E) in both strains. However, the lowering effect of octanoate on gluconeogenesis and its reducing effect are less pronounced in Lou/C as compared with Wistar. This feature probably results from a higher rate of NADH oxidation via the FAD-dependent G3P shuttle activation in Lou/C. Thus, as proposed above for glycerol metabolism, in the case of glycerol and octanoate, higher mGPdH activity in Lou/C might also explain an increased fatty acid oxidation in this strain.
The present work emphasizes the role of a reducing-power translocation pathway across the mitochondrial inner membrane in the regulation of mitochondrial oxidative phosphorylation and fatty acid oxidation. Two pathways carry reducing power into the mitochondrial matrix: the malate-aspartate and the G3P-DHAP shuttles (Fig. 6). Although the former is thermodynamically controlled by mitochondrial membrane potential because of the DP m -dependent glutamate transport, the latter is kinetically controlled in the liver by a very low mGPdH activity (17). When electrons are carried by the malate-aspartate shuttle, high oxidative phosphorylation stoichiometry (three coupling sites) is achieved at a limited rate of respiration and ATP synthesis because of the control exerted at complex 1. By contrast, G3P shuttle provides electrons from cytosolic NADH to the quinone pool, i.e. downstream of complex 1. This results in a lower stoichiometry (two coupling sites only) but at a higher rate of respiration and ATP synthesis. Because malateaspartate shuttle flux is dependent on the mitochondrial membrane potential ⌬P m , it is sensitive to any process affecting the protonmotive force, such as uncoupling. This is not the case for G3P shuttle flux because it does not depend on ⌬P m but mostly on mGPdH activity in the liver. Therefore, uncoupling would favor FAD-linked substrate oxidation (42,43).
The high rate of fatty acid oxidation in Lou/C, which was related to the high mG3PdH activity, may explain the resistance to obesity. This metabolic feature appears to be related to a "hyperthyroid status" limited to the liver, resulting from the high TH receptor transcription. The mechanism that leads to overexpression of TH receptors in this intriguing strain remains to be clarified, but this finding opens a new direction in the field of obesity and energy homeostasis.