Reactive oxygen species (new actors in the hypothalamic insulin signaling involved in the control of food intake)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Insulin differently modulates hypothalamic mitochondrial energy metabolism in fed or fasted mice: Involvement in food intake regulation?

0195-6663 doi: DOI: 10.1016/j.appet.2008.04.119In hypothalamus, mitochondria have been demonstrated to be involved in nutrients sensing but little is known concerning a mitochondrial insulin effect. The aim of our studywas thus to determine, in mice, whether (i) insulin may modulate mitochondrial functions and (ii) this effect varies according to metabolic state. O2 consumption, measured by oxygraphy in fresh hypothalamus explants was similar in fed and fasted mice on glutamate whereas it was lower in fasted mice on succinate. Whatever the metabolic state of the mice, insulin (3 nM) increased O2 consumption with glutamate. By contrast, insulin increased O2 consumption with succinate only in fasted mice. This increase allows to restore O2 consumption which was observed in fed mice in basal condition thus suggesting that insulin might improve free fatty acids utilisation in fasted mice. In in vivo experiments, third ventricular insulin injection strongly inhibits food intake, in fed mice, as already described.However, this effectwas not observed with similar insulin concentration (3 nM)in fasted mice. Experiments are currently under investigation in order to assess insulin effect on mitochondrial products and to establish a link between the level of these products and food intake regulation. Altogether these data demonstrate that the effects of insulin on mitochondrial function in hypothalamus and on food intake vary according to the metabolic state. doi:10.1016/j.appet.2008.04.12

A Physiological Increase of Insulin in the Olfactory Bulb Decreases Detection of a Learned Aversive Odor and Abolishes Food Odor-Induced Sniffing Behavior in Rats

International audienceInsulin is involved in multiple regulatory mechanisms, including body weight and food intake, and plays a critical role in metabolic disorders such as obesity and diabetes. An increasing body of evidence indicates that insulin is also involved in the modulation of olfactory function. The olfactory bulb (OB) contains the highest level of insulin and insulin receptors (IRs) in the brain. However, a role for insulin in odor detection and sniffing behavior remains to be elucidated. Using a behavioral paradigm based on conditioned olfactory aversion (COA) to isoamyl-acetate odor, we demonstrated that an intracerebroventricular (ICV) injection of 14 mU insulin acutely decreased olfactory detection of fasted rats to the level observed in satiated animals. In addition, whereas fasted animals demonstrated an increase in respiratory frequency upon food odor detection, this effect was absent in fasted animals receiving a 14 mU insulin ICV injection as well as in satiated animals. In parallel, we showed that the OB and plasma insulin levels were increased in satiated rats compared to fasted rats, and that a 14 mU insulin ICV injection elevated the OB insulin level of fasted rats to that of satiated rats. We further quantified insulin receptors (IRs) distribution and showed that IRs are preferentially expressed in the caudal and lateral parts of the main OB, with the highest labeling found in the mitral cells, the main OB projection neurons. Together, these data suggest that insulin acts on the OB network to modulate olfactory processing and demonstrate that olfactory function is under the control of signals involved in energy homeostasis regulation and feeding behaviors

Insulin ICV injection (14 mU) decreases olfactory detection in fasted rats.

<p>(<i>A</i>) Validation of the COA paradigm in the two-tube experimental device. Before the conditioning, during the last day of habituation (Hab.), both tubes were filled with pure water, and during the first day of aversion acquisition (Av.), both tubes were filled with ISO 10⁻⁵. A repeated measure ANOVA revealed a significant effect of the aversive conditioning (p<0.0001). For habituation and aversion acquisition, when two identical drinks were proposed, bar graphs represent the olfactory detection index corresponding to the number of licks of the first sampled tube normalized to the total number of licks (mean ± SEM). The mean olfactory detection indexes for habituation and aversion were not statistically different (SNK, p>0.05, ns). After conditioning, rats were given the choice between pure water and ISO 10⁻⁵ for the aversion test (Aversion test) and re-test (Aversion re-test). Bar graphs represent the olfactory detection index corresponding to the number of licks of the pure-water tube normalized to the total number of licks (mean ± SEM). The mean olfactory detection indexes for the aversion test and re-test were not statistically different (SNK, p>0.05, ns). The mean olfactory detection indexes were significantly different after conditioning (SNK, compared to Hab. # p¹<0.05, compared to Av. § p¹<0.05; n = 18). The dashed line represents the chance level (50%). (<i>B</i>) Insulin injection before acquisition does not alter aversion retention. Bar graphs represent the olfactory detection index corresponding to the percentage of licks of the pure water tube normalized to the total number of licks (mean ± SEM) when rats were given the choice between pure water and ISO 10⁻⁵ 1 h after ICV NaCl injection (NaCl) and 1 h after ICV insulin injection (Insulin). The mean olfactory detection indexes were not statistically different (paired t-test, p¹>0.05, ns; n = 11). (<i>C</i>) Insulin ICV injection (14 mU) decreases olfactory detection. Bar graphs represent the olfactory detection index when rats had the choice between pure water and odorized water at ISO 10⁻⁸ and ISO 10⁻⁹ 1 h after ICV NaCl injection (NaCl) and 1 h after ICV insulin injection (Insulin). The mean olfactory detection indexes were significantly different for odor dilutions (ANOVA, p¹<0.005) and ICV treatments (p¹<0.005; n = 18). Post-hoc comparisons showed that insulin ICV injections significantly decreased the olfactory detection index for both ISO 10⁻⁹ and ISO 10⁻⁸ odor dilutions (SNK, compared to NaCl for each odor # p¹<0.05; n = 18) and that the olfactory detection index is higher for ISO 10⁻⁸ compared to ISO 10⁻⁹ for each ICV treatment (SNK, § p¹<0.05; n = 18) (<i>D</i>) Insulin ICV injection (14 mU) does not change locomotor activity. Bar graphs represent the number of side changes in the two-tube experimental device (mean ± SEM) when rats had the choice between pure water, ISO 10⁻⁸ and ISO 10⁻⁹ 1 h after ICV NaCl injection (NaCl) and 1 h after ICV insulin injection (Insulin). The mean side changes were not statistically different for odors or ICV treatments (ANOVA, p>0.05, ns; n = 18). ¹Established using transformed data.</p

IR quantification in the main OB. Bar graphs represent an arbitrary unit of the IR densitometric value obtained by quantification of the IR-Cy3 immunofluorescent signal on frozen OB sections (mean ± SEM).

<p><i>(A)</i> Specificity of the anti-IR antibody. ANOVA of densitometric values revealed significant effect of the antibody used (p<0.001). The mean densitometric value obtained with a monoclonal mouse antibody directed against the β-subunit of the IR (anti-IR), was significantly higher than the mean densitometric value obtained when using either the mouse IgG1 (isotype control) or with the omission of the primary antibody (no anti-IR) (SNK, compared to isotype control # p<0.05, compared to No anti-IR § p¹<0.05; n = 6). The mean densitometric value obtained with the omission of the primary antibody was significantly higher than the mean densitometric value obtained with the isotype control (SNK, # p<0.05). (B–E) Mean IR densitometric values of the OB sections as a function of feeding states (B), zones (C), regions (D) and layers (E). Repeated measures ANOVA revealed significant effects of zones (ANOVA, p<0.0001), regions (ANOVA, p<0.001) and layers (ANOVA, p<0.0001; n = 10 including 5 fasted rats and 5 satiated rats) but no significant effect of the feeding state (ANOVA, p>0.05). <i>(B)</i> Feeding states: The mean IR densitometric values of the OB sections of fasted (F) and satiated (S) animals are similar. <i>(C)</i> Zones: The mean IR densitometric value was significantly higher in the caudal posterior zone (PZ) compared to the rostral anterior zone (AZ) and the intermediate zone (IZ) (SNK, compared to AZ # p<0.05, compared to IZ § p<0.05). <i>(D)</i> Regions: The mean IR densitometric value was significantly lower in the dorsomedial (DM) and the ventromedial (VM) regions of the main OB compared to the dorsolateral (DL) and ventrolateral (VL) regions (SNK, compared to DL # p<0.05, compared to VL § p<0.05). <i>(E)</i> Layers: The mean IR densitometric values measured in the nerve (NL), glomerular (GL), external plexiform (EPL), mitral cell (MCL) and granular cell (GCL) layers of the main OB were all statistically different from each other (SNK, # p<0.05).</p

Acute ICV insulin injection does not alter physiological parameters such as food intake, body weight and peripheral blood glucose level.

<p>Bar graphs represent food intake <i>(A)</i> and body weight <i>(B)</i> (mean ± SEM) measured on NaCl ICV injection days (NaCl) and 14 mU insulin ICV injection days (Insulin). The means were not statistically different (paired t-test, p>0.05, ns, n = 18). <i>(C)</i> Bar graphs represent the peripheral blood glucose levels (mean ± SEM) measured from tail blood samples 1 h after ICV NaCl injection (NaCl) and 1 h after ICV insulin injection (Insulin). The means were not statistically different (paired t-test, p>0.05, ns; n = 11).</p

Comparison of the mean (± SEM) respiratory frequency in Hz between satiated NaCl (n = 10) and fasted insulin (n = 10) rats during food odor presentation.

<p>Four 10 sec periods were considered: Pre-odor (−10 sec to 0 sec); Odor presentation (start: 0 sec to 10 sec, middle: 10 sec to 20 sec, end: 20 sec to 30 sec). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051227#s3" target="_blank">Results</a> are presented for each experimental condition: in the fasted state after 14 mU insulin ICV injection; in the satiated state after NaCl ICV injection. For the four periods, the means of respiratory frequency observed in two experimental conditions were not statistically different. (Two-way repeated measures ANOVA, p>0.05, ns).</p

Overall course of the behavioral experiments.

<p><i>(A)</i> Olfactory detection experiment. Animals were tested daily in the two-tube experimental device over 16 days (D1–D16). <i>Habituation:</i> On the first 3 days (D1–D3), rats were trained to drink pure water from both tubes (W/W) of the experimental cage. <i>Aversion acquisition:</i> On the following 3 days (D4–D6), rats had access to water odorized with isoamyl-acetate (ISO) diluted at 10⁻⁵ in both tubes (10⁻⁵/10⁻⁵). ISO 10⁻⁵ consumption >0.5 mL was paired with an intraperitoneal injection of LiCl (LiCl IP) to induce a conditioned olfactory aversion (COA) to ISO. <i>Aversion test:</i> On D7, the COA efficiency was tested by giving the animals a choice between water odorized with ISO 10⁻⁵ and pure water (10⁻⁵/W). On Habituation, Aversion acquisition and Aversion test days, animals were trained to receive a daily NaCl ICV injection (NaCl ICV). <i>Olfactory detection test:</i> During the Olfactory detection test period (D8–D11), rats were offered a choice between ISO 10⁻⁹ or ISO 10⁻⁸ and pure water (10⁻⁹/W, 10⁻⁸/W). For a given odorant dilution, the animals were tested on two consecutive days: once 1 h after NaCl ICV injection (D8 and D10) and once 1 h after a 14 mU insulin ICV injection (D9 and D11). <i>Aversion re-test:</i> On D12, the COA stability was assessed by giving the rats the choice between ISO 10⁻⁵ and pure water (10⁻⁵/W). <i>Aversion retention</i>: During the Aversion retention period (D15–D16), three days after the Aversion re-test, rats were offered again the choice between ISO 10⁻⁵ and pure water (10⁻⁵/W) and the animals were tested on two consecutive days: once 1 h after a 14 mU insulin ICV injection (D15) and once after a NaCl ICV injection (D16). (<i>B</i>) Sniffing experiment. Animals were tested daily in a whole-body plethysmograph over 7 days. The rats were first allowed to familiarized with the recording chamber for 4 days (Habituation), D1–D4 without (D1, D2) or with (D3, D4) food odor stimulation. During the sniffing test period (Food odor detection test, D5–D7), the animals were tested either in the fasted state (at 10:00 a.m.), 1 h after 14 mU insulin ICV injection (D5); in the fasted state, 1 h after NaCl ICV injection (D6); in the satiated state, 1 h after NaCl ICV injection (at 4:00 p.m.) (D7).</p