The ability for the brain to sense peripheral fuel availability is mainly accomplished within the hypothalamus, which detects ongoing systemic nutrients and adjusts food intake and peripheral metabolism as needed. Here, we hypothesized that mitochondrial reactive oxygen species (ROS) could trigger sensing of nutrients within the hypothalamus. For this purpose, we induced acute hypertriglyceridemia in rats and examined the function of mitochondria in the hypothalamus. Hypertriglyceridemia led to a rapid increase in the mitochondrial respiration in the ventral hypothalamus together with a transient production of ROS. Cerebral inhibition of fatty acids–CoA mitochondrial uptake prevented the hypertriglyceridemia-stimulated ROS production, indicating that ROS derived from mitochondrial metabolism. The hypertriglyceridemia-stimulated ROS production was associated with change in the intracellular redox state without any noxious cytotoxic effects, suggesting that ROS function acutely as signaling molecules. Moreover, cerebral inhibition of hypertriglyceridemia-stimulated ROS production fully abolished the satiety related to the hypertriglyceridemia, suggesting that hypothalamic ROS production was required to restrain food intake during hypertriglyceridemia. Finally, we found that fasting disrupted the hypertriglyceridemia-stimulated ROS production, indicating that the redox mechanism of brain nutrient sensing could be modulated under physiological conditions. Altogether, these findings support the role of mitochondrial ROS as molecular actors implied in brain nutrient sensing.

The long-term maintenance of the body’s energy homeostasis is a complex biological process achieved by numerous complementary mechanisms that imply sensor systems of fuel availability located in both peripheral and central sites. The hypothalamus in the central nervous system is a primary site of integration of nutritional information, which includes neural inputs as well as circulating metabolic signals, i.e., glucose or fatty acids (1,2). In turn, the hypothalamus elicits appropriate behavioral and metabolic responses to counterbalance any changes in the energy status.

Previous studies have already shown that an overload of systemic lipids could stimulate activity of specific hypothalamic neurons (3) and modulate expression of neuropeptides, the key effectors of the hypothalamus (36), which leads to rapid or more delayed changes in peripheral metabolism. Moreover, chronic elevation of lipids may alter the hepatic sensitivity to insulin through their direct effect on the central nervous system (7). Recent findings (8) indicate that circulating lipids can directly act as signaling molecules, informing the hypothalamus about the body’s metabolic status. Accordingly, it has been shown (9) that intracerebroventricular administration of long-chain fatty acids inhibited food intake and stimulated peripheral energy storage.

One of the current challenges is to define the way fatty acids control neural activity within the hypothalamus. Growing compelling evidence points out the role of cellular metabolism in hypothalamic nutrient sensing (8,1012). For instance, elevation of cytosolic long-chain fatty acid–CoAs in the arcuate nucleus of the hypothalamus decreases food intake and whole-body glucose production (13). Moreover, defects in the cellular metabolism of fatty acids in the brain that prevent hypothalamic esterification of long-chain fatty acids can disrupt peripheral glucose homeostasis (14). Sensing the abundance of the intracellular pool of malonyl-CoA is another way to monitor the fuel availability, and a persistent decrease in hypothalamic malonyl-CoA is sufficient to stimulate food intake and to induce obesity (15). Additionally, drugs that increase the malonyl-CoA levels in the hypothalamus, such as C75, mainly by inhibition of the fatty acid synthesis, stimulate energy expenditure and could be used as appetite suppressants (16,17).

A recent study (18) reported that β-oxidation in the brain is required for the effects of fatty acids on glucose-induced pancreatic insulin secretion. Moreover, the anorectic effect of intracerebroventricularly infused fatty acids depends on their chain length, suggesting that changes in the rate of mitochondrial β-oxidation could signal nutrient availability to the hypothalamus (9). The intensity of the mitochondrial uptake of fatty acids represents another checkpoint in cerebral nutrient sensing. Accordingly, molecular or pharmacological inhibition of hypothalamic CPT-1, a mitochondrial transporter of fatty acids, decreases food intake (13). Thus, within the hypothalamus, fatty acids may accomplish multiple physiological effects through intracellular mechanisms depending on mitochondrial activity, i.e., the mitochondrial ability to import fatty acids, and its rate of β-oxidation.

Several observations indicate that reactive oxygen species (ROS) could be one of the mitochondrial effectors implied in the nutrient sensing. First, ROS can be produced by electron leakage during mitochondrial metabolism (19), and the rate of ROS production is enhanced as mitochondrial metabolism increases (2022). Second, ROS can function as signaling molecules to regulate signaling pathways (23,24), and recent evidence (25,26) supports the view that the subtle rise of the steady-state ROS concentration is sufficient to have a fundamental physiological role. Third, ROS can contribute to nutrient sensing at the cellular level. In particular, it has been shown that an increase in pyruvate supply induces a ROS production by a mitochondria-dependant metabolism of this substrate, which finally accounts for the increase in the glycogen synthase activity (27). The subsequent energy-storage improvement reveals an efficient cellular nutrient sensing dependent on mitochondrial ROS (28). Thus, ROS can act as intracellular messengers between the mitochondria and the cytosol in response to nutrient influx.

Here, we tested whether a lipid overload can stimulate a ROS-dependent signaling pathway within the hypothalamus to regulate energy homeostasis. For this purpose, we first measured ROS production in distinct brain areas in an experimental rat model of hypertriglyceridemia and evaluated the mitochondria as a potential source of ROS. Then, we measured food intake in hypertriglyceridemic or control rats intracerebroventricularly injected with reduced glutathione ethyl-ester (GSH-EE), a potent ROS scavenger that is particularly effective in restoring mitochondrial glutathione (29).

Animals.

All procedures involving rats were in strict accordance with the European Communities Council Directive (86/609/EEC) and were reviewed by our local committee for care and use of laboratory animals. This study was performed on male Wistar rats (275/325 g; Charles River Laboratories). They were housed under a 12-h light/dark cycle (light on at 0600) on an average ambient temperature of 23°C. They had free access to standard laboratory chow and water. Food was removed 2 h before each experiment. For tissue collection, rats were killed by cervical dislocation. Brains were quickly removed and placed in cold-buffered solution (5 mmol/l HEPES in PBS) to dissect hypothalami. Whole hypothalamus explants were cut according to procedure of Glowinski and Iversen (30).

Surgery.

Under anesthesia with pentobarbital, rats underwent stereotaxic surgery to implant a chronic stainless steel cannula (Plastics One). The third ventricle was targeted using the following coordinates: −0.8 mm posterior to the bregma, +1.4 mm lateral to the sagittal suture, and −3.5 mm below the skull surface. The cannula was fixed to the skull using dental cement. Rats were finally housed individually and were allowed 1 week for recovery before experiment.

Drug administration.

Rats received a single intraperitoneal injection of 5 ml intralipid 20% (IL; Sigma) starting at 1000 h. Control rats received intraperitoneal injection of saline (NaCl 0.9%). In some cases, rats were further subjected to intracerebroventricular treatment. Before these experiments, animals were handled daily and were adapted to intracerebroventricular procedures by giving them at least two mock trials in which the injection needle was inserted with no injection. Intracerebroventricular treatment was performed in conscious rats; the infusion (10 μl for 2 min) consisted of 1 μmol/l GSH-EE (Sigma), 0.08 μmol/l Trolox (Sigma), or 0.5 μmol/l Etomoxir (Sigma), dissolved in PBS and adjusted at pH 6.8. For these experiments, control rats received intracerebroventricular infusion of PBS.

Measurement of food intake.

Food was removed 2 h before experiment. Rats first received one intracerebroventricular infusion, and after 20 min, one intraperitoneal injection. After another 20 min, food (25 g) was presented to rats (t = 0), and food intake was measured at t = 1, 2, and 4 h.

Blood biochemistry.

Blood samples obtained from the tail were used for measurements of metabolites and hormones. Blood glucose was measured using a glucose analyzer (GlucoTrend). Plasma triglycerides and free fatty acids were determined by colorimetric assays (TG PAP, Biomerieux, and NEFA-C test, Wako). Insulin and leptin plasmatic levels were determined using ELISA kits (Linco Research). During blood puncture, rats did not receive any food.

ROS detection.

The 2′,7′-dichlorofluorescein diacetate (H2DCFDA) is a cell-permeable dye that is oxidized to fluorescent 2′,7′-dichlorofluorescein by H2O2 and can therefore be used to monitor intracellular generation of ROS. Tissues were harvested in cold-buffered medium (5 mmol/l HEPES in PBS) and immediately frozen in liquid nitrogen to improve the following probe diffusion. After rapid thawing, medium was discarded. Samples were exposed to 8 μmol/l H2DCFDA (Molecular Probes) dissolved in 400 μl fresh medium and were incubated at 37°C for 30 min under agitation. Medium was then removed, and samples were further incubated in a lysis buffer (0.1% SDS, Tris-HCl, pH 7.4) for 15 min at 4°C. After homogenization, samples were centrifuged at 6,000g for 20 min at 4°C. Supernatants were collected and subjected to fluorescence analysis at 520 nm under excitation at 485 nm using a microplate reader (Victor; Wallac). Dichlorofluorescein fluorescence corresponds to the ROS production generated during the ex vivo sample incubation. In addition, although the tissue preparation caused sensible reduction of total mitochondrial activity, the main bioenergetic properties of mitochondria were preserved after freezing, as evidenced by oxygraphy (Table 1). Notably, antimycin-sensitive mitochondrial respiration represented 60% of the total O2 consumption, in either fresh or frozen/thawed tissues as well. The oligomycin-inhibited coupled respiration was also conserved. This procedure was thus suitable to detect ROS production derived from mitochondria.

O2 consumption measurement.

O2 consumption was measured using a respirometer (Oxygraph, Oroboros). Measurements were performed at 37°C with continuous stirring in 2 ml Dulbecco’s modified Eagle’s medium containing 5 mmol/l glucose without pyruvate (Invitrogen). Before each O2 consumption measurement, the medium in the chambers was equilibrated with air for 30 min, and freshly dissected whole hypothalami were placed in the medium for 15 min. Then, biopsies were transferred into the respirometer glass chambers and positioned on a nylon membrane to avoid homogenization of the tissues by stirrers. After stabilization of the initial O2 consumption, successive inhibition of complex V and complex III was achieved with 12 μg/ml oligomycin (Sigma) and 5 μmol/l antimycin (Sigma), respectively. O2 consumption was calculated using DataGraph software (Oroboros). The difference between initial- and oligomycin-O2 consumption indicates the coupled mitochondrial respiration. The difference between oligomycin- and antimycin-O2 consumption indicates the uncoupled mitochondrial respiration. The resting O2 consumption corresponds to the nonmitochondrial respiration.

Western blot analysis of UCP2 expression.

Tissues were fractionated to extract mitochondria. Briefly, fresh tissues were dissociated in a buffered solution (PBS 5 mmol/l HEPES) and centrifuged at 1,000g for 10 min at 4°C. Homogenates were incubated at 4°C for 15 min in an hypotonic buffer (240 mmol/l sucrose, 10 mmol/l KCl, 2 mmol/l EDTA, 0.5 mmol/l dithiothreitol, and 10 mmol/l HEPES, pH 7.4) supplemented by a cocktail of proteases inhibitors (Complete Mini; Roche). After cell lysis, samples were centrifuged at 1,500g for 10 min at 4°C. Supernatants were further centrifuged at 12,000g for 10 min at 4°C, and pellets were resuspended in 20 μl of distilled water. Proteins (10 μg per lane) were separated by 10% SDS-PAGE and transferred onto an Hybond membrane (Amersham). Blocking was achieved for 1 h in 5% nonfat dry milk prepared in Tris-buffered saline with Tween. Membranes were then probed with 2.5 μg/ml of rabbit anti-UCP2 antibody (α-diagnostics) overnight at 4°C under agitation. The blot was detected by using goat anti-rabbit peroxidase–conjugated secondary antibody (Amersham) and developed with an enhanced chemiluminescence kit (Amersham). Finally, the blots were exposed to autoradiographic films. After revelation, equality in the protein loading was checked using Coomassie blue staining of transferred membrane.

Cytochrome c oxidase activity measurement.

Fresh hypothalami were homogenized in 5 ml cold buffer (0.25 mol/l sucrose, 5 mmol/l TES, pH 7.2). Measurements of COX activity were carried out as previously described (31). Briefly, homogenates (50 μl) were mixed in a 1-ml volume reaction of a buffered solution containing 100 mmol/l reduced cytochrome c, 32 mmol/l phosphate, 0.1 mmol/l sodium ascorbate, 0.4 mmol/l aluminum chloride, and 5 mg lubrol. Reaction was followed from absorbance at 550 nm for 5 min at 37°C.

Glutathione assay.

Frozen hypothalami were homogenized in a lysis saline solution (3 mmol/l EDTA, 150 mmol/l KCl, pH 7.4). Aliquots of the homogenates (50 μl) were mixed with 450 μl of 5% metaphosphoric acid. After vortexing, samples were centrifuged at 1,500g for 10 min at 4°C. Supernatants were collected and stored at −80°C until use. Glutathione assay was performed by reverse-phase high-performance liquid chromatography (HPLC) as previously described (32).

Lipid hydroperoxides assay.

Fresh hypothalami were homogenized in 3 mmol/l EDTA solution, and the lipid hydroperoxides were immediately extracted and then stored at −80°C. Extraction and assay of lipid hydroperoxides were performed using the Lipid Hydroperoxides Assay Kit (Cayman Chemical Company) as described by the manufacturer.

Protein assay.

Protein concentration of samples was determined using the DC Protein Assay Kit (Bio-Rad SA) according to the manufacturer’s instructions.

Statistical analysis.

Data are means ± SE. Comparisons of groups were made using a nonpaired Student’s t test or a Mann-Whitney U test, as appropriate after ANOVA, and differences among groups were considered significant when P < 0.05.

Characterization of the experimental model of hypertriglyceridemia.

Intraperitoneal intralipid administration gradually increased plasma triglycerides and free fatty acids (Table 2). Changes in plasma triglycerides were rapid, substantial at 30 min postinjection (+15%), and reached 2.88 ± 0.39 g/l 4 h postinjection, corresponding to a twofold increase compared with controls. The rise in plasma free fatty acids was delayed compared with that of triglycerides and was detected only 1 h postinjection. Rise of plasma free fatty acids reached 0.35 g/l 4 h postinjection, corresponding to a threefold increase compared with control. In contrast, intralipid administration did not modify glucose, insulin, or leptin levels over the time course of the experiment. Thus, this model mimicked an acute hypertriglyceridemia with limited hormonal modification.

Hypertriglyceridemia induces a local, rapid, and transient ROS production in the brain.

Cerebral ROS production was monitored using the fluorogenic redox-sensitive dye H2DCFDA loaded in dissected brain explants. Intralipid injection provoked a significant increase in ROS production in the hypothalamus (+32%) as soon as 30 min postinjection (Fig. 1). ROS production remained elevated up to 2 h at least in the hypothalamus of intralipid-injected rats and returned to basal value 4 h postinjection. The increase in hypothalamic ROS production was mainly focused in the ventral part, whereas only a punctual increase was observed in the dorsal part (+25%, 1 h postinjection). No change in ROS production in the cortex of hypertriglyceridemic rats was detected over the time course of the experiment.

Hypertriglyceridemia enhances the hypothalamic mitochondrial activity and provokes release of ROS.

O2 consumption was assessed by oxygraphy in fresh hypothalamus explants of intralipid-injected or control rats (Fig. 2A). At 30 min postintralipid injection, O2 consumption in hypothalamus increased by 15%. The oligomycin-inhibited part of O2 consumption, corresponding to the mitochondrial coupled respiration, was significantly higher in hypertriglyceridemic than in control rats. On the contrary, the antimycin-inhibited part of the resulting O2 consumption, corresponding to the mitochondrial uncoupled respiration, was not affected after intralipid injection. Likewise, no modification of the resting nonmitochondrial O2 consumption was observed in hypothalamus of intralipid-injected rats.

To further characterize the mitochondrial function, we measure maximal cytochrome c oxidase activity in whole hypothalamus at 30 min in intralipid-injected or control rats (Fig. 2B). We did not find any difference between the two conditions, suggesting that total mitochondrial oxidative capacities in the hypothalamus were not affected by the intralipid challenge.

Next, we examined whether the mitochondrial activity could trigger the intralipid-stimulated ROS production. Etomoxir, a CPT-1 inhibitor, was centrally delivered into the third lateral ventricle to block the mitochondrial fatty acid uptake in the hypothalamus before the induction of hypertriglyceridemia. Etomoxir pretreatment fully prevented the intralipid-stimulated ROS production (Fig. 2C). No significant effect of Etomoxir infusion was observed concerning basal ROS production.

Hypothalamic ROS elevation modifies the cellular redox state but does not promote an oxidative stress.

Impact of the intralipid-stimulated ROS production on the cellular redox state was then assessed by HPLC analysis of oxidized glutathione (GSSG) and reduced glutathione (GSH) levels, respectively. Glutathione redox state, defined as GSSG/GSH ratio, was determined in hypothalamus homogenates of intralipid-injected or control rats (Fig. 3A). At 30 min postintralipid injection, the glutathione redox state in the ventral hypothalamus was greatly enhanced by 81%. This modification was entirely due to a drop in reduced glutathione level (32.5 ± 3.4 nmol/mg protein to 26.2 ± 0.8 nmol/mg protein), whereas level of oxidized glutathione was unchanged (1.2 ± 0.3 nmol/mg protein vs. 1.3 ± 0.1 nmol/mg protein). The glutathione redox state returned to basal value as soon as 1 h postintralipid injection. In the dorsal hypothalamus, the glutathione redox state remained stable, regardless of the timepoint considered.

We further investigated the impact of the stimulated ROS production in the hypothalamus by evaluating the lipid peroxidation level as a marker of oxidative stress. Amount of lipid hydroperoxide was measured in hypothalamus homogenates from intralipid-injected or control rats (Fig. 3B). No variation in hydroperoxide levels could be detected in the ventral or dorsal portion of hypothalamus of intralipid-injected rats over the time course of analysis.

Hypothalamic ROS production is involved in the regulation of food intake.

We then designed experiments to examine the physiological relevance of the hypertriglyceridemia-induced hypothalamic ROS production in the brain nutrient sensing. For this purpose, we assessed food intake in response to acute hypertriglyceridemia in combination with a prior central administration of reduced GSH-EE, a potent ROS scavenger (Fig. 4A).

To test the ability of intracerebroventricular infusion of GSH-EE to inhibit the formation of ROS, we first observed the hypothalamic ROS response to intralipid injection after an intracerebroventricular infusion of GSH-EE. GSH-EE central delivery was effective in fully preventing intralipid-stimulated ROS production without affecting the basal ROS production in control rats (Fig. 4B).

We then measured the food intake in intralipid-injected rats. As expected, intralipid injection inhibited food intake as soon as 1 h after injection (Fig. 4C). Moreover, the inhibitory effect of intralipid injection on cumulative food intake increased over the time. Intracerebroventricular infusion of GSH-EE produced a full recovery of the basal food intake in intralipid-injected rats. The GSH-EE treatment alone did not significantly modified basal food intake in control rats.

An additional study based on intracerebroventricular infusion of Trolox, a water soluble vitamin E analog, reproduced these results. Trolox pretreatment contained the food intake decline evoked by intralipid, confirming that pharmacological ROS modulation is sufficient to alter brain lipid sensing (Fig. 5).

Modulation of the hypothalamic ROS response under physiological conditions.

We then questioned whether the hypothalamic ROS response to lipids could be modulated by changes in the whole body’s energy status. For this purpose, rats were subjected to an 18-h fasting before the intralipid challenge. In fasted rats, intralipid challenge did not promote any elevation in ROS production within the hypothalamus at 30 min postinjection (Fig. 6A).

Respirometry analysis revealed a higher basal O2 consumption in the hypothalamus of fasted rats (+23%) compared with that of fed rats (Fig. 6B). This increase was due to both higher coupled and uncoupled mitochondrial respiration rates. Interestingly, intralipid injection did not stimulate any source of O2 consumption when rats were fasted.

Assay of the maximal cytochrome c oxidase activity indicated no difference between fed and fasted rats (Fig. 6C). Intralipid injection did not modify this value. This finding indicated that changes in respiratory rate could be attributed to functional adaptation rather than modification of the mitochondrial mass.

Immunoblotting analysis of hypothalamus homogenates from fed and fasted rats showed that the uncoupling protein UCP2 was overexpressed after fasting the hypothalamus (Fig. 6D). Therefore, UCP2 upregulation may account for the higher uncoupling respiratory rate in fasted rats.

Recent studies (13,18) strongly support the involvement of mitochondria in the hypothalamic nutrient sensing. Here, we report that acute hypertriglyceridemia promotes subtle ROS elevation within the hypothalamus, which was derived from an increase in mitochondrial activity related to the fat overload. Based on our results, we suggest that a ROS-sensitive signaling pathway is implied in brain lipid sensing. We demonstrate for the first time the physiological relevance of this concept in conscious animals.

Here, we used the established (3) intralipid model to mimic a high fuel state. Intralipid injection is sufficient to produce rapid hypertriglyceridemia in normal weight rats, consistent with postprandial conditions. However, plasma glucose and hormones levels remain stable in this model by opposition of that which can be encountered with high-fat diet paradigms. Thus, the intralipid model is very suitable to investigate the effect of a fat flow toward the hypothalamus without any other metabolic and hormonal inputs. In addition, the intralipid model is characterized by a reduced free food intake. Numerous studies have already shown that lipid influx could inhibit food intake regardless of the route of administration, i.e., intraduodenal or intracerebral (9,33). Nevertheless, impact of fatty acids on feeding and hypothalamic activity differs from their length and degree of unsaturation (9,33,34). The anorexigenic effect of fatty acids could depend on the rate of mitochondrial activity, since long-chain fatty acids produce higher inhibition rather than short-chain ones. Linoleic acid (C18:2) is a potent anorexigen fatty acid and constitutes major component of intralipid-triglycerides. It is therefore not surprising that intralipid administration provoked a marked inhibition of food intake.

Chronic excess of energy, as observed during diabetes or obesity, is frequently associated with an elevation of steady-state ROS production in a number of tissues, such as adipose tissue, muscle, and vascular walls (20,35). Recently, it has been shown (36) that high dietary fat induced oxidative stress in rat cerebral cortex. Here, we show that hypothalamus is a site affected by a rise of ROS during hypertriglyceridemic state. Moreover, we provide evidence for a rapid and transient, likely physiological, induction of ROS caused by acute hypertriglyceridemia between 30 and 240 min after lipid loading. This time course fits with those of previous works (3739), which reported early ROS production within 1 or 2 h after postprandial hypertriglyceridemia or after fatty acid in vitro exposure in circulating leukocytes or in lymphocyte-derived and pancreatic cell lines, respectively. Thus, lipids can rapidly stimulate elevation of intracellular ROS levels in numerous cell populations.

Based on our findings, we suggest that systemic lipid overload induced hypothalamic ROS production through a mitochondrial-dependent mechanism. First, we found that ROS production was concomitant with an increase in hypothalamic mitochondrial respiration. Second, the hypertriglyceridemia-induced ROS production was abolished when mitochondrial fatty acid uptake was inhibited. Third, the hypothalamic ROS response to hypertriglyceridemia was lost in fasted animals, which are characterized by high uncoupling. The importance of the mitochondria in cellular ROS production stimulated by lipid influx has been fully emphasized (3941). Mitochondrial ROS are produced by electron leakage at complex I, when the oxidative phosphorylation (OXPHOS) process is activated by lipids as substrates (42). Another potent source for ROS induced by lipid influx is the NADPH oxidase system. However, NADPH oxidase involvement in lipid-induced ROS production is particularly evident only after long-term exposure (35,43). As the hypertriglyceridemia-induced ROS production was rapid and fully inhibited by etomoxir pretreatment, we did not investigate the implication of NADPH oxidase pathway in our model. Moreover, the comparison between fed and fasted rats suggest that a coupling of OXPHOS is required for the hypertriglyceridemia-induced ROS production. This is consistent with recent observations (44) showing that fat intake increased mitochondrial ROS production in skeletal muscle, in dependence on both activity of complex I and coupling of OXPHOS.

We observed that the hypertriglyceridemia-induced ROS response was attenuated after fasting. The loss of response is related to a higher resting respiration in the hypothalamus compared with that of fed rats. During fasting, persistent increase in plasma nonesterified fatty acid due to lipolysis is thought to induce overexpression of UCP proteins, which is driven under perioxisome proliferator-activated receptor-α activation by intracellular elevation of fatty acids (45). We observed that UCP2 was upregulated in hypothalamus of fasted rats, probably in relation with the 18 h-lasting threefold increase in plasma nonesterified fatty acid (data not shown). Therefore, it is conceivable to propose that persistent elevation of circulating lipids can provoke uncoupling in hypothalamus via UCP2 overexpression. Similar observations (46,47) have been previously reported from chronic hypertriglyceridemic rats, as the liver and heart of such animals displayed high mitochondrial resting respiration and functional uncoupling, respectively. Thus, lipid loading in hyperlipemic rats might fail to stimulate mitochondrial ROS production in hypothalamus because of a high uncoupling. In this way, previous works (48,49) have clearly demonstrated the ability of UCP2 to modulate ROS mitochondrial generation. Finally, these results suggest that the ROS-sensitive pathway that allows brain lipid sensing could be modulated under physiological conditions. The physiological relevance of such adaptation has been recently discussed (50,51). Uncoupling improves fatty acids export from mitochondrial matrix and dissipates the proton motive force. Thus, the increase in mitochondrial uncoupling that occurs during a chronic high-fuel state may represent a simple feedback mechanism to avoid persistent and cytotoxic ROS production.

Hypertriglyceridemia increases the GSSG/GSH ratio in the hypothalamus, suggesting modification of intracellular redox state. This is consecutive to a drop in GSH without any concomitant GSSG increase. GSH depletion can obviously reflect a ROS-mediated oxidation (52). However, we can exclude neither GSH efflux by mannosylrentinyl phosphate-1 (53), although GSH transport is low (Km = 5–10 mmol/l), nor a diminution in GSH synthesis. The latter could be achieved by high-fat diet or linoleic acid through a NO dependant pathway (52). Stable GSSG levels can result from the efflux by MRP1 of neoformed GSSG (Km = 100 μmol/l) (54). However, the most documented mechanism is the S-glutathionylation of proteins, by which GSSG induces reversible thiol oxidation of cysteines to mixed disulfides (55). During oxidative stress, glutathionylation is thought to protect proteins from ROS damage (56), while GSSG export could prevent excessive shifting in GSSG/GSH ratio in association with GSSG reductase activity (57). Together, these events represent early cellular responses to a mild oxidative environment and are associated with redox regulation (58,59).

Finally, our results strongly support the involvement of a ROS-sensitive signaling pathway in the control of food intake. In this study, we did not identify the ROS targets involved in the hypertriglyceridemia-induced satiety. However, recent findings lead us to consider some potent candidates, such as AMPK or ATP-sensitive K+ channels, as redox-regulated systems involved in hypothalamic control of energy homeostasis (60,61). In addition, an elegant study has clearly shown that the cellular redox state influenced exocytose process in pancreatic β-cells, a model of neuroendocrine cells (62). We found that inhibition of ROS production completely abolished the behavioral response to hypertriglyceridemia. This result suggests that activation of ROS-sensitive mechanisms could be sufficient to promote satiety. However, in view of the many redundant mechanisms of hypothalamic control of food intake, this mechanism would probably not be exclusive. In conclusion, identification of the ROS-sensitive molecular actors, which trigger the hypothalamic sensing of fat, may provide new targets in the management of hyperphagia related to metabolic diseases such as obesity or diabetes.

FIG. 1.

Transient production of mitochondrial ROS in the hypothalamus during acute hypertriglyceridemia. ROS levels were assessed in distinct brain areas by oxidation of H2DCFDA probe at different times after intralipid (IL) or saline intraperitoneal injection. Data are means ± SE (n = 6–8). *Significant differences vs. controls, P < 0.05. DCF, dichlorofluorescein.

FIG. 1.

Transient production of mitochondrial ROS in the hypothalamus during acute hypertriglyceridemia. ROS levels were assessed in distinct brain areas by oxidation of H2DCFDA probe at different times after intralipid (IL) or saline intraperitoneal injection. Data are means ± SE (n = 6–8). *Significant differences vs. controls, P < 0.05. DCF, dichlorofluorescein.

FIG. 2.

Activation of mitochondrial activity in the hypothalamus and subsequent release of ROS during acute hypertriglyceridemia. A: Cellular respiration in hypothalamic explants was assessed by oxygraphy at 30 min after intralipid (IL) or saline intraperitoneal injection. B: Maximal cytochrome c oxidase activity in hypothalamic homogenates at 30 min after IL or saline intraperitoneal injection. C: Effect of cerebral inhibition of fatty acid-CoA mitochondrial uptake by intracerebroventricular infusion of Etomoxir (Eto) on hypertriglyceridemia-induced hypothalamic ROS production. ROS levels were assessed in ventral hypothalamus after 30 min as described. Data are means ± SE (n = 6). *Significant differences vs. controls, P < 0.05.

FIG. 2.

Activation of mitochondrial activity in the hypothalamus and subsequent release of ROS during acute hypertriglyceridemia. A: Cellular respiration in hypothalamic explants was assessed by oxygraphy at 30 min after intralipid (IL) or saline intraperitoneal injection. B: Maximal cytochrome c oxidase activity in hypothalamic homogenates at 30 min after IL or saline intraperitoneal injection. C: Effect of cerebral inhibition of fatty acid-CoA mitochondrial uptake by intracerebroventricular infusion of Etomoxir (Eto) on hypertriglyceridemia-induced hypothalamic ROS production. ROS levels were assessed in ventral hypothalamus after 30 min as described. Data are means ± SE (n = 6). *Significant differences vs. controls, P < 0.05.

FIG. 3.

Transient change in the intracellular redox state in the hypothalamus during acute hypertriglyceridemia. A: GSH and GSSG levels were measured by HPLC in hypothalamic homogenates at different times after intralipid (IL) or saline intraperitoneal injection. B: Lipid hydroperoxides levels were determined by colorimetric assay in the hypothalamic homogenates at different time after IL or saline intraperitoneal injection. Data are means ± SE (n = 5). *Significant differences vs. controls, P < 0.05.

FIG. 3.

Transient change in the intracellular redox state in the hypothalamus during acute hypertriglyceridemia. A: GSH and GSSG levels were measured by HPLC in hypothalamic homogenates at different times after intralipid (IL) or saline intraperitoneal injection. B: Lipid hydroperoxides levels were determined by colorimetric assay in the hypothalamic homogenates at different time after IL or saline intraperitoneal injection. Data are means ± SE (n = 5). *Significant differences vs. controls, P < 0.05.

FIG. 4.

Hypothalamic ROS production is required to restrain food intake during hypertriglyceridemia. A: Experimental procedures of food intake measurement. B: Effect of GSH-EE cerebral delivery on hypertriglyceridemia-induced ROS production. GSH-EE or vehicles were infused in the third lateral ventricle before intraperitoneal injection of either intralipid (IL) or saline. ROS levels were assessed in ventral hypothalamus 30 min after intraperitoneal injection as described above. Data are means ± SE (n = 6). *Significant differences between vehicle and controls, P < 0.05. ‡Significant differences between vehicle- and intralipid-treated rats, P < 0.05. C: Effect of GSH-EE cerebral delivery on the hypertriglyceridemia-induced satiety. Food intake was assessed by weighing residual chow at different times. Data are means ± SE (n = 6). *Significant differences between vehicle and controls, P < 0.05. ‡Significant differences between vehicle- and intralipid-treated rats, P < 0.05. icv, intracerebroventricular; IL, intralipid.

FIG. 4.

Hypothalamic ROS production is required to restrain food intake during hypertriglyceridemia. A: Experimental procedures of food intake measurement. B: Effect of GSH-EE cerebral delivery on hypertriglyceridemia-induced ROS production. GSH-EE or vehicles were infused in the third lateral ventricle before intraperitoneal injection of either intralipid (IL) or saline. ROS levels were assessed in ventral hypothalamus 30 min after intraperitoneal injection as described above. Data are means ± SE (n = 6). *Significant differences between vehicle and controls, P < 0.05. ‡Significant differences between vehicle- and intralipid-treated rats, P < 0.05. C: Effect of GSH-EE cerebral delivery on the hypertriglyceridemia-induced satiety. Food intake was assessed by weighing residual chow at different times. Data are means ± SE (n = 6). *Significant differences between vehicle and controls, P < 0.05. ‡Significant differences between vehicle- and intralipid-treated rats, P < 0.05. icv, intracerebroventricular; IL, intralipid.

FIG. 5.

Pharmacological ROS inhibition is sufficient to modulate the lipid-induced suppression of food intake. Effects of Trolox cerebral delivery was tested on the 4-h food intake modulation by intralipid (IL). Data are means ± SE (n = 6). *Significant differences between vehicle and controls, P < 0.05. ‡Significant differences between vehicle- and IL-treated rats, P < 0.05.

FIG. 5.

Pharmacological ROS inhibition is sufficient to modulate the lipid-induced suppression of food intake. Effects of Trolox cerebral delivery was tested on the 4-h food intake modulation by intralipid (IL). Data are means ± SE (n = 6). *Significant differences between vehicle and controls, P < 0.05. ‡Significant differences between vehicle- and IL-treated rats, P < 0.05.

FIG. 6.

Modulation of hypertriglyceridemia-induced mitochondrial-ROS signaling in hypothalamus by fasting. A: Absence of any stimulated ROS production in hypothalamus of fasted rats in response to intralipid (IL) intraperitoneal injection. ROS levels were assessed at 30 min after intraperitoneal injection as described above. Cx, cortex; DH, dorsal hypothalamus; VH, ventral hypothalamus. B: Cellular respiration in hypothalamic explants assessed by oxygraphy in hypothalamus after fasting. Data are means ± SE (n = 6). ‡Significant differences with fed rats, P < 0.05. NS, nonsignificant. C: Maximal cytochrome c oxidase activity in hypothalamic homogenates in fasted rats at 30 min after IL or saline intraperitoneal injection. Data are means ± SE (n = 6). D: Comparison of UCP2 expression in hypothalamus between fed and fasted rats (n = 3).

FIG. 6.

Modulation of hypertriglyceridemia-induced mitochondrial-ROS signaling in hypothalamus by fasting. A: Absence of any stimulated ROS production in hypothalamus of fasted rats in response to intralipid (IL) intraperitoneal injection. ROS levels were assessed at 30 min after intraperitoneal injection as described above. Cx, cortex; DH, dorsal hypothalamus; VH, ventral hypothalamus. B: Cellular respiration in hypothalamic explants assessed by oxygraphy in hypothalamus after fasting. Data are means ± SE (n = 6). ‡Significant differences with fed rats, P < 0.05. NS, nonsignificant. C: Maximal cytochrome c oxidase activity in hypothalamic homogenates in fasted rats at 30 min after IL or saline intraperitoneal injection. Data are means ± SE (n = 6). D: Comparison of UCP2 expression in hypothalamus between fed and fasted rats (n = 3).

TABLE 1

O2 consumption from fresh and frozen/thawed hypothalamic tissues in PBS-HEPES measured by oxygraphy (pmol · s−1 · mg−1)

TissuePlus oligomycinPlus antimycin
Fresh    
    Raw data 2.90 ± 0.04 2.09 ± 0.06 1.09 ± 0.10 
    Percentage 100 ± 3 72 ± 2 38 ± 4 
Frozen/thawed    
    Raw data 0.91 ± 0.03 0.68 ± 0.07 0.37 ± 0.03 
    Percentage 100 ± 4 75 ± 8 41 ± 4 
TissuePlus oligomycinPlus antimycin
Fresh    
    Raw data 2.90 ± 0.04 2.09 ± 0.06 1.09 ± 0.10 
    Percentage 100 ± 3 72 ± 2 38 ± 4 
Frozen/thawed    
    Raw data 0.91 ± 0.03 0.68 ± 0.07 0.37 ± 0.03 
    Percentage 100 ± 4 75 ± 8 41 ± 4 

Data are means ± SE (n = 3 per condition).

TABLE 2

Time course of plasmatic parameters after administration of intralipid

030 min1 h2 h4 h
TG (g/l)      
    NaCl 1.35 ± 0.20 1.28 ± 0.10 1.07 ± 0.29 1.07 ± 0.29 1.33 ± 0.23 
    IL 1.45 ± 0.21 1.65 ± 0.12* 2.24 ± 0.34* 2.12 ± 0.29* 2.88 ± 0.39* 
FFA (g/l)      
    NaCl 0.10 ± 0.01 0.11 ± 0.02 0.14 ± 0.01 0.14 ± 0.02 0.11 ± 0.02 
    IL 0.12 ± 0.02 0.11 ± 0.01 0.21 ± 0.03* 0.27 ± 0.04* 0.35 ± 0.05* 
Glucose (mmol/l)      
    NaCl 7.0 ± 0.1 6.7 ± 0.3 7.0 ± 0.1 6.8 ± 0.1 6.6 ± 0.2 
    IL 7.0 ± 0.1 6.8 ± 0.3 6.9 ± 0.2 7.0 ± 0,2 6.5 ± 0.2 
Insuline (mmol/l)      
    NaCl 59 ± 06 74 ± 23 68 ± 05 72 ± 07 74 ± 10 
    IL 60 ± 10 51 ± 07 57 ± 16 58 ± 06 61 ± 07 
Leptine (mmol/l)      
    NaCl 5.7 ± 0.7 ND 3.8 ± 0.3 5.4 ± 0.9 4.7 ± 1.0 
    IL 5.4 ± 1.1 ND 3.0 ± 0.3 4.6 ± 0.9 3.8 ± 0.8 
030 min1 h2 h4 h
TG (g/l)      
    NaCl 1.35 ± 0.20 1.28 ± 0.10 1.07 ± 0.29 1.07 ± 0.29 1.33 ± 0.23 
    IL 1.45 ± 0.21 1.65 ± 0.12* 2.24 ± 0.34* 2.12 ± 0.29* 2.88 ± 0.39* 
FFA (g/l)      
    NaCl 0.10 ± 0.01 0.11 ± 0.02 0.14 ± 0.01 0.14 ± 0.02 0.11 ± 0.02 
    IL 0.12 ± 0.02 0.11 ± 0.01 0.21 ± 0.03* 0.27 ± 0.04* 0.35 ± 0.05* 
Glucose (mmol/l)      
    NaCl 7.0 ± 0.1 6.7 ± 0.3 7.0 ± 0.1 6.8 ± 0.1 6.6 ± 0.2 
    IL 7.0 ± 0.1 6.8 ± 0.3 6.9 ± 0.2 7.0 ± 0,2 6.5 ± 0.2 
Insuline (mmol/l)      
    NaCl 59 ± 06 74 ± 23 68 ± 05 72 ± 07 74 ± 10 
    IL 60 ± 10 51 ± 07 57 ± 16 58 ± 06 61 ± 07 
Leptine (mmol/l)      
    NaCl 5.7 ± 0.7 ND 3.8 ± 0.3 5.4 ± 0.9 4.7 ± 1.0 
    IL 5.4 ± 1.1 ND 3.0 ± 0.3 4.6 ± 0.9 3.8 ± 0.8 

Data are means ± SE (n = 6).

*

Significant difference vs. controls. IL, intralipid. ND, not determined.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This work was supported by grants from Agence Nationale de la Recherche number ANR-05-PNRA-004. B.A. is a recipient of a Centre National de la Recherche Scientifique fellowship.

We wish to thank Jean-Marc Lerme and Christine Fourreau for their special care for animals and Valentin Barquissau, Anne-Marie Bessac, Pascale Guillou, Maryse Nibbelink, and Geraldine Offer for their technical assistance.

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