Skip to main content
NCBI home page
American Journal of Physiology - Regulatory, Integrative and Comparative Physiology logoLink to American Journal of Physiology - Regulatory, Integrative and Comparative Physiology
. 2016 Apr 20;310(11):R1322–R1331. doi: 10.1152/ajpregu.00549.2015

DEPTOR in POMC neurons affects liver metabolism but is dispensable for the regulation of energy balance

Alexandre Caron 1,2, Sébastien M Labbé 1,2, Mathilde Mouchiroud 1,2, Renaud Huard 2, Damien Lanfray 1,2, Denis Richard 1,2, Mathieu Laplante 1,2,✉
PMCID: PMC4935503  PMID: 27097662

Abstract

We have recently demonstrated that specific overexpression of DEP-domain containing mTOR-interacting protein (DEPTOR) in the mediobasal hypothalamus (MBH) protects mice against high-fat diet-induced obesity, revealing DEPTOR as a significant contributor to energy balance regulation. On the basis of evidence that DEPTOR is expressed in the proopiomelanocortin (POMC) neurons of the MBH, the present study aimed to investigate whether these neurons mediate the metabolic effects of DEPTOR. Here, we report that specific DEPTOR overexpression in POMC neurons does not recapitulate any of the phenotypes observed when the protein was overexpressed in the MBH. Unlike the previous model, mice overexpressing DEPTOR only in POMC neurons 1) did not show differences in feeding behavior, 2) did not exhibit changes in locomotion activity and oxygen consumption, 3) did not show an improvement in systemic glucose metabolism, and 4) were not resistant to high-fat diet-induced obesity. These results support the idea that other neuronal populations are responsible for these phenotypes. Nonetheless, we observed a mild elevation in fasting blood glucose, insulin resistance, and alterations in liver glucose and lipid homeostasis in mice overexpressing DEPTOR in POMC neurons. Taken together, these results show that DEPTOR overexpression in POMC neurons does not affect energy balance regulation but could modulate metabolism through a brain-liver connection.

Keywords: DEPTOR, mTOR, POMC, energy balance, glucose metabolism

the control of food intake and energy expenditure is ensured by neurons of several brain regions, including the mediobasal hypothalamus (MBH), which has emerged as a major center of integration for nutrient and hormonal cues (26, 33). MBH hosts several neuronal populations, including the proopiomelanocortin (POMC) and neuropeptide Y (NPY)/agouti-related protein (AgRP)/GABA-producing neurons of the arcuate nucleus, as well as steroidogenic factor 1 (SF1) neurons of the ventromedial hypothalamus. Over the years, these neurons were shown to play critical roles in the regulation of systemic metabolic homeostasis (17, 21). Intensive efforts are currently being made to better understand the molecular mechanisms by which MBH neurons control energy balance and systemic metabolism (11, 15).

The mechanistic target of rapamycin (mTOR) plays an important role in the hypothalamic regulation of energy balance and glucose homeostasis (3, 8, 9). mTOR is a serine/threonine kinase that nucleates two protein complexes named mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (20). These complexes are part of a well-conserved anabolic pathway that modulates growth and metabolism in response to nutrients and growth factors. They include several proteins, including DEP-domain containing mTOR-interacting protein (DEPTOR), a component of both mTORC1 and mTORC2 (31). Recently, we have characterized the expression of Deptor in the rat and mouse brain and have shown that this gene is highly expressed in several structures involved in energy balance regulation, including the MBH (5, 6). Using transgenic mice, we have shown that MBH-specific overexpression of DEPTOR protects mice against high-fat diet-induced obesity and improves systemic glucose homeostasis (6). These phenotypes were associated with a reduction in food intake and feed efficiency and with an elevation in oxygen consumption. Although these findings established hypothalamic DEPTOR as an important player in the control of body weight and systemic metabolism, the identity of the neurons that mediated these effects was not determined.

Previous studies have shown that DEPTOR is expressed in POMC neurons of the MBH (5). Here, to specifically test the physiological importance of DEPTOR in POMC neurons, we have generated a transgenic mouse model, allowing the specific overexpression of DEPTOR in these cells. Contrary to mice overexpressing DEPTOR in the whole MBH, specific overexpression of DEPTOR in POMC neurons had no beneficial effect on energy balance and glucose homeostasis. When challenged with a high-fat diet, these mice were not protected against the development of obesity and metabolic disturbances. Conversely, we noted a mild hyperglycemia, an elevation in the capacity for hepatic glucose production, and the development of hepatosteatosis in mice overexpressing DEPTOR in POMC neurons. These results clearly demonstrate that the modulation in energy balance and the improvement in glucose homeostasis brought by the overexpression of DEPTOR in MBH are not reproduced when DEPTOR is specifically overexpressed in POMC neurons. Overall, these findings indicate that overexpression of DEPTOR in other neuronal population of the MBH probably plays a more prominent role in regulating energy balance and systemic glucose metabolism. These results also suggest that DEPTOR expression in POMC neurons could modulate metabolism through a brain-liver connection.

MATERIALS AND METHODS

Animals.

Animal care and handling were performed in accordance with the Canadian Guide for the Care and Use of Laboratory Animals, and all experimental procedures received prior approval from the Laval University Animal Care Committee (CPAUL). Male mice were maintained on a 12:12-h light-dark cycle (lights on 0600–1800), while individually housed in ventilated cages at an ambient temperature of 23 ± 1°C. They were fed ad libitum chow (Harlan Teklad, 2918) or a high-fat diet (Research Diets, D12492, 60 kcal% fat).

Generation of POMC-DeptorO/E mice.

Pomc-Cre mice were obtained from The Jackson Laboratory (stock no: 0005965) and were backcrossed with C57BL/6J mice for at least 10 generations. They were then mated with our previously described pure C57BL/6J conditional overexpressor transgenic model carrying a CAAGSLox-stop-Lox Deptor allele (6). POMC-DeptorO/E mice were compared with littermate controls carrying only the CAAGSLox-stop-Lox Deptor allele.

PCR validation.

Total DNA was extracted from the mediobasal hypothalamus (MBH), cerebral cortex, white adipose tissue, brown adipose tissue, and liver. Briefly, tissues were incubated for 30 min at 95°C in 50 mM NaOH, and Tris·HCl 1 M at pH 6.8 was added to neutralize the solution. The CAAGSLox-stop-Lox Deptor allele was amplified using the following primers: forward, 5′-TTC GGC TTC TGG CGT GTG-3′ and reverse, 5′-ACT TCA GCC ATG CGT TCC-3′. In the presence of the Cre recombinase, the Lox-stop-Lox cassette upstream of the Deptor transgene recombines, and the Lox-stop-Lox cassette can be measured by the production of a ΔLox-stop-Lox using the following primers: forward, 5′-GCC TCT TTT ACC CTT TTC CTC TTC C-3′ and reverse, 5′-TTT GTC GTC TCT GTC AAT GGC C-3′.

Food intake and body composition.

Body weight and food intake were measured weekly. Body composition was measured by dual-energy X-ray absorptiometry using the PIXIMUS mouse densitometry apparatus (Lunar, Madison, WI) under isoflurane anesthesia.

Indirect calorimetry and locomotor activity.

Oxygen consumption (V̇o2) and respiratory quotient (RQ) were evaluated over 24 h in an open-circuit system with an O2 (S-3A1; Applied Electrochemistry, Naperville, IL) and a CO2 analyzer (CD-3A; Applied Electrochemistry). Locomotor activity was measured with the AccuScan Digiscan Activity Monitor (AccuScan Instruments, Columbus, OH) using the VersaMax software (version 1.30; AccuScan Instruments). Locomotion was determined by breaks in photo beams. Mice were housed individually in acrylic chambers for a 72-h adaptation period, and V̇o2, RQ, and movement were measured for 24 h.

Glucose, insulin, and pyruvate tolerance tests.

For the glucose tolerance test (GTT), mice were fasted for 12 h and were injected with 1 g/kg ip of d-glucose (Sigma Aldrich, St. Louis, MO). For the insulin tolerance test (ITT), animals were fasted for 6 h and were injected with 0.75 U/kg ip of insulin (Humulin, Eli Lilly, Toronto, ON, Canada). For the pyruvate tolerance test (PTT), mice were fasted for 16 h and then were injected with 1.5 g/kg ip of sodium pyruvate (Sigma Aldrich). Blood samples were collected from the tail vein, and glucose was measured using a glucometer (OneTouch).

Plasma and hepatic lipid determinations.

Blood was collected by puncture of the tail vein in a tube containing EDTA. Plasma was stored at −80°C for further biochemical analyses. Plasma insulin was measured using high-sensitivity ELISA kit (ALPCO) Triglycerides in plasma and in liver lipid extracts were measured using colorimetric assays, according to the manufacturers' instructions (RANDOX). The homeostatic model assessment of insulin resistance (HOMA-IR) was calculated according to the formula: fasting insulin (μU/l) × fasting glucose (mmol/l)/22.5.

RNA isolation, cDNA preparation, and quantitative PCR.

Total mRNA was isolated from MBH and liver, as described previously (6). The MBH dissection extended from the optic chiasm to the mammillary bodies, bilaterally to the optic tract. mRNA extraction and cDNA synthesis were performed following the manufacturer's instructions. cDNA was diluted in DNase-free water (1:25) before the quantification by real-time PCR. mRNA transcript levels were measured in duplicate samples using a CFX96 touchTM real-time PCR (Bio-Rad, Mississauga, ON, Canada). Chemical detection of the PCR products was achieved with SYBR Green (Bio-Rad; 172–5271). At the end of each run, melt curve analyses were performed, and representative samples of each experimental group were run on agarose gel to ensure the specificity of the amplification, as previously described (6). Fold differences in target mRNA expression were measured using the 2Δ-cycle threshold method by comparison with the housekeeping gene large P0 ribosomal protein (36b4) and expressed as a fold change vs. controls. Primers sequences are presented in Table 1.

Table 1.

Quantitative PCR primers sequences

Gene Accession Number Forward Primer Reverse Primer
36B4 NM_007475.5 5′-AGAAACTGCTGCCTCACATC-3′ 5′-CATCACTCAGAATTTCAATGG-3′
Acadm NM_007382.5 5′-GAAGGTTGAACTCGCTAGGC-3′ 5′-GCTAGCTGATTGGCAATGTC-3′
Acox1 NM_015729.3 5′-GCCTGAGCTTCATGCCCTCA-3′ 5′-ACCAGAGTTGGCCAGACTGC-3′
Acs NM_001302163.1 5′-GCTGATCCAGAAGGGGTTCA-3′ 5′-CCACCCCACACTTCTTGCCT-3′
Ap2 NM_024406.2 5′-GACGACAGGAAGGTGAAGAG-3′ 5′-ACATTCCACCACCAGCTTGT-3′
Cd36 NM_001159558.1 5′-GTCCTGGCTGTGTTTGGAGG-3′ 5′-GCTGCTACAGCCAGATTCAG-3′
Cpt1a NM_013495.2 5′-TGCCTCTATGTGGTGTCCAA-3′ 5′-CATGGCTTGTCTCAAGTGCT-3′
Cytc NM_007808.4 5′-TGGACCAAATCTCCACGGTCTGTT-3′ 5′-TAGGTCTGCCCTTTCTCCCTTCTT-3′
Deptor NM_145470.3 5′-AGCAGAGAGAGCTGGAACGC-3′ 5′-CAGAGGCCTCCTTATGTTCA-3′
Dgat1 NM_010046.2 5′-GGCCTGCCCCATGCGTGATTAT-3′ 5′-CCCCACTGACCTTCTTCCCTGTAGA-3′
Dgat2 NM_026384.3 5′-GAAGCTGCCCGCAGCGAAAA-3′ 5′-TCTTGGGCGTGTTCCAGTCAA-3′
Fabp1 NM_017399.4 5′-GCCCGAGGACCTCATCCAGAAA-3′ 5′-CTCTCTTGTAGACAATGTCGCCCA-3′
Fas NM_007988.3 5′-CTGGCCCCGGAGTCGCTTGAGTATA-3′ 5′-GGAGCCTCCGAAGCCAAATGA-3′
Fatp1 NM_011977.3 5′-CCAGACGGACGTGGCTGTGTAT-3′ 5′-GTCCCTGCTTCAGGTCTAGAAAGA-3′
Gyk NM_001294138.1 5′-AATCCGTTACTCCACATGGA-3′ 5′-ACCCGATCTTAACTGTCAAT-3′
Pepck NM_011044.2 5′-GAGTGGAGACCGCAGGAC-3′ 5′-CAGGTATTTGCCGAAGTTGTAG-3′
Pgc1a NM_008904.2 5′-AAGATCAAGGTCCCCAGGCAGTAG-3′ 5′-TGTCCGCGTTGTGTCAGGTC-3′
Ppara NM_011144.6 5′-AGAGCCCCATCTGTCCTCTC-3′ 5′-ACTGGTAGTCTGAAAACCAAA-3′
Ppard NM_011145.3 5′-GGACAATCCGCATGAAGCTC-3′ 5′-GGATGACAAAGGGTGCGTTG-3′
Scd1 NM_009127.4 5′-TCACTGCACCTCCCTCCGGAAA-3′ 5′-TGTGGCTCCAGAGGCGATGA-3′
Open in a new tab

Brain in situ hybridization histochemistry.

Brains were kept in paraformaldehyde (4%) for 7 days, transferred to a solution containing paraformaldehyde (4%) and sucrose (10%), and cut 24 h later using a sliding microtome (Histoslide 2000; Reichert-Jung, Heidelberg, Germany). Brain sections (25 μm) were taken from the olfactory bulb to the brain stem and stored at −20°C in a cryoprotectant solution before performing in situ hybridization histochemistry, as previously described (5). The neuropeptides cRNA riboprobes were prepared as follows: Agrp, from a 389-bp fragment of the 5′-region cDNA of Agrp subcloned into a pCRII plasmid (from M. Graham) and linearized with T7-HindIII (Pharmacia Biotech, Baie-D'Urfé, QC, Canada); Npy, from a 189-bp fragment of the 5′-region cDNA of Npy subcloned into a pGem2 plasmid (from D. Larhammar) and linearized with T7-EcoR1; and Pomc, from a 852-bp fragment of the 5-region cDNA of Pomc subcloned into a pBluescript II plasmid (from B. T. Bloomquisf) and linearized with T3-Xho1. The specificity of these probes was confirmed in our previous studies (12, 18).

Statistical analysis.

Results are expressed as means ± SE. Statistical analyses were performed using GraphPad Prism Software version 6.0 for Mac (San Diego, CA). The two-tailed Student's t-test for nonpaired values was used for two-group comparisons. Comparisons between several groups were achieved using ANOVA followed by Bonferroni post hoc test. A P value <0.05 was considered statistically significant.

RESULTS

Generation of POMC-specific DEPTOR overexpresser mice.

We have recently reported the generation of a new transgenic mouse model for conditional overexpression of DEPTOR (6). Briefly, we have inserted in the ColA1 locus a single copy of a transgene composed of a CAGGS promoter, a LoxP-stop-LoxP cassette, and the Deptor coding sequence (Fig. 1A). In the presence of a Cre recombinase, the stop codon surrounded by LoxP site is eliminated, which allows a significant overexpression of the DEPTOR in tissues (6). To test the role of DEPTOR in POMC neurons, we crossed our transgenic model with Pomc-Cre mice, a mouse that selectively expresses the Cre recombinase in these cells (1). The Pomc-Cre mouse was widely used over the last decade to study the specific contribution of several proteins to the function of POMC neurons (1, 15, 35). From now on, we will refer to the POMC-specific DEPTOR overexpresser as the POMC-DeptorO/E mouse (Fig. 1B). As shown in Fig. 1C, PCR analyses confirmed that this strategy led to recombination of the LoxP-stop-LoxP allele only in the MBH, where the POMC neurons reside and not in any other tissue tested. In addition, POMC-DeptorO/E mice exhibited a 20% increase in Deptor mRNA expression in the MBH (Fig. 1D). Although this increase looks modest, one has to consider that it was measured in whole MBH homogenates where only 3,000 POMC neurons reside (24).

Fig. 1.

Fig. 1.

Open in a new tab

Development and validation of the proopiomelancortin (POMC)-DeptorO/E mouse model. A: schematic view of the transgenic alleles allowing the conditional overexpression of DEP-domain containing mTOR-interacting protein (DEPTOR) in mouse tissues. The CAAGSLox-stop-Lox Deptor allele was inserted in the mouse ColA1 locus. The presence of the Lox-stop-Lox cassette upstream of the Deptor transgene prevents the expression of Deptor. When exposed to a Cre recombinase, the stop codon is eliminated, which allows the expression of Deptor downstream of the CAAGS promoter. B: schematic presentation of the breeding strategy used to generate POMC-DeptorO/E mice. C: validation of the POMC-DeptorO/E mouse by genotyping. PCR analyses show the presence of the CAAGSLox-stop-Lox Deptor allele in all of the tissues tested. When the Cre is expressed under the control of the Pomc promoter, the Lox-stop-Lox cassette upstream of the Deptor transgene recombines in the POMC neurons of the hypothalamus. The specific recombination of the Lox-stop-Lox cassette can be measured by PCR by the production of a ΔLox-stop-Lox allele in the hypothalamus. Importantly, the ΔLox-stop-Lox allele is not detected in any other tissue tested such as the cortex, brown adipose tissue (BAT), white adipose tissue (WAT), and the liver. Representative images are presented. D: expression of Deptor mRNA in the mediobasal hypothalamus (MBH), as assessed by quantitative RT-PCR (n = 17–19/group). Data represent means ± SE.

POMC-DeptorO/E mice fed a chow diet have normal body composition and feeding behavior.

POMC-DeptorO/E mice were born at the expected Mendelian ratio and did not show apparent physiological difference. To determine the physiological impact linked to DEPTOR overexpression in POMC neurons, we first measured body weight and food consumption in control and POMC-DeptorO/E male mice fed a standard laboratory chow diet. As depicted in Fig. 2, A and B, there was no difference in body weight and food intake between the groups. No significant difference in lean and fat mass was observed between control and POMC-DeptorO/E mice (Fig. 2, C and D). Supporting the absence of phenotype, overexpression of DEPTOR in POMC neurons did not affect the expression of Pomc, Agrp, and Npy (Fig. 3).

Fig. 2.

Fig. 2.

Open in a new tab

POMC-DeptorO/E mice fed a chow diet have a normal body composition and feeding behavior. Body weight (A), daily food intake (B), fat mass (C), and lean mass (D) of control and POMC-DeptorO/E mice fed a laboratory chow diet for 14 wk (n = 11–13/group). Diet starts at 8–12 wk old. Fat mass and lean mass were evaluated by dual-energy X-ray absorptiometry (DEXA) scanning on 12-wk-old animals. Data represent means ± SE. Control represents littermates.

Fig. 3.

Fig. 3.

Open in a new tab

DEPTOR overexpression in POMC neurons does not impair the expression of Agrp, Npy, and Pomc. In situ hybridization histochemistry was performed on brain slices using riboprobes specific for Agrp, Npy, and Pomc mRNA. Male control and POMC-DeptorO/E mice fed a laboratory chow diet and aged 8–10 wk old were used (n = 5 or 6/group). Data represent means ± SE. Control represents littermates.

DEPTOR overexpression in POMC neurons does not affect locomotor activity and energy expenditure in mice fed a standard chow diet.

MBH-specific overexpression of DEPTOR increases physical activity in mice (6). To test whether the expression of DEPTOR in POMC neurons is involved in these effects, we placed POMC-DeptorO/E male mice and control littermates in metabolic chambers. Unlike MBH-specific DEPTOR-expressing mice, we did not detect any difference in locomotor activity between control and POMC-DeptorO/E mice (Fig. 4, A and B). Additionally, we did not observe variation in oxygen consumption and the respiratory quotient between the groups (Fig. 4, C–F). Together, these results indicate that DEPTOR overexpression in POMC neurons has no impact on energy balance regulation when mice are fed a normal diet. These observations also indicate that DEPTOR found in POMC neurons does not contribute to the elevation in physical activity reported in mice overexpressing DEPTOR in the whole MBH (6).

Fig. 4.

Fig. 4.

Open in a new tab

DEPTOR overexpression in POMC neurons does not affect locomotor activity and energy expenditure in mice fed a standard chow diet. Control and POMC-DeptorO/E mice fed a laboratory chow diet (n = 8/group) were placed in metabolic chambers at 14 wk old, where activity (A and B) and oxygen consumption (C and D) were measured. E and F: respiratory quotient was calculated from these experiments. Data represent means ± SE. Control represents littermates.

POMC-specific DEPTOR overexpression does not improve but worsens glucose metabolism in chow-fed mice.

We have shown before that overexpression of DEPTOR in the MBH improves glucose metabolism even in young, lean, and healthy mice (6). To determine whether this phenotype is recapitulated when DEPTOR overexpression is restricted to POMC neurons, GTT and ITT were performed in POMC-DeptorO/E male mice. As shown in Fig. 5, A–D, we did not measure any improvement in glucose homeostasis in response to DEPTOR overexpression. In contrast, we found that the maximal capacity for hepatic glucose production inferred from PTT was increased in POMC-DeptorO/E mice (Fig. 5, E–F). This was associated with a tendency for higher fasting blood glucose in POMC-DeptorO/E mice (Fig. 5G). Circulating insulin levels were not affected between the groups (Fig. 5H). Interestingly, calculation of HOMA-IR indicated systemic insulin resistance in POMC-DeptorO/E mice (Fig. 5I). Circulating triglycerides levels were not affected between the groups (Fig. 5J). These results show that DEPTOR expression in POMC neurons does not improve but appears to worsen glucose homeostasis in mice. These observations also support the idea that POMC neurons are not responsible for the improvement in glucose metabolism observed in response to MBH-specific DEPTOR overexpression.

Fig. 5.

Fig. 5.

Open in a new tab

POMC-specific DEPTOR overexpression does not improve glucose metabolism in mice fed a standard chow diet. A: glucose tolerance test (GTT) was performed in control and POMC-DeptorO/E mice aged 12–14 wk old and fed a laboratory chow diet (n = 9–10/group). B: area under the curve for the GTT was calculated from the experiment shown in A. C: insulin tolerance test (ITT) was performed in control and POMC-DeptorO/E mice fed the same diet (n = 9 or 10/group). D: area under the curve for the ITT was calculated from the experiment shown in C. E: pyruvate tolerance test (PTT) was performed in control and POMC-DeptorO/E mice fed the same diet (n = 9 or 10/group). F: area under the curve for the PTT was calculated from the experiment shown in E. Plasma glucose (G), insulin (H), homeostatic model assessment of insulin resistance (HOMA-IR) (I), and triglyceride (J) levels measured in control and POMC-DeptorO/E fasted for 16 h (n = 5 or 6/group). *P < 0.05 and ***P < 0.001 compared with fasted animals. The data represent means ± SE. Control represents littermates.

POMC-DeptorO/E mice fed a high-fat diet are not resistant to obesity.

One striking observation that we made using the MBH-specific overexpressing mice is that these animals were completely protected against the development of obesity when fed a high-fat diet (6). This effect was associated with a reduction in food intake and feed efficiency, with an elevation in physical activity and with an increase in oxygen consumption (6). To test whether these phenotypes could be recapitulated in POMC-DeptorO/E male mice, we challenged the latter with a high-fat diet for a period of 6 wk. As shown in Fig. 6, A and B, control and POMC-DeptorO/E mice gained a similar amount of weight when fed the obesogenic diet. No difference in food intake and body composition were detected between the groups (Fig. 6, C–E). Control and POMC-DeptorO/E mice fed a high-fat diet were then placed in metabolic chambers to measure locomotor activity and oxygen consumption. Exactly as observed in chow-fed animals, we did not detect any effect of DEPTOR overexpression in POMC neurons in a context of obesity (Fig. 7, A–F). Altogether, these results reinforce the idea that DEPTOR expression in POMC neurons has no impact on the control of food intake and energy expenditure in mice.

Fig. 6.

Fig. 6.

Open in a new tab

POMC-DeptorO/E mice fed a high-fat diet are not resistant to obesity. Body weight (A), body weight gain (B), and cumulative food intake (C) of control and POMC-DeptorO/E mice fed a high-fat diet for 6 wk (n = 11–13/group). Diet starts at 14 wk old. Fat mass (D) and lean mass (E) were evaluated by DEXA scanning. The data represent means ± SE. Control represents littermates.

Fig. 7.

Fig. 7.

Open in a new tab

DEPTOR overexpression in POMC neurons does not affect locomotor activity and energy expenditure in mice fed a high-fat diet. Control and POMC-DeptorO/E mice fed a high-fat diet aged 18–20 wk old (n = 8/group) were placed in metabolic chambers where activity (A and B) and oxygen consumption (C and D) was measured. E and F: respiratory quotient was calculated from these experiments. Data represent means ± SE. Control represents littermates.

POMC-specific DEPTOR overexpression causes hepatosteatosis in obese mice.

We next sought to evaluate the impact of POMC-specific overexpression of DEPTOR on glucose homeostasis in high-fat diet-fed mice. GTT and ITT were performed in control and POMC-DeptorO/E male mice following 6 wk of high-fat feeding. As shown in Fig. 8, A and B, we did not observe any improvement in glucose homeostasis in response to DEPTOR overexpression, as reported in mice expressing DEPTOR in the MBH (6). On the contrary, we observed a mild deterioration in glucose tolerance in POMC-DeptorO/E mice as observed in chow-fed mice. Supporting the idea that POMC overexpression of DEPTOR impairs glucose homeostasis, we noticed that POMC-DeptorO/E mice accumulated more hepatic triglycerides when exposed to the high-fat diet (Fig. 9A). This effect, which likely contributes to the elevation in glycemia, was associated with a significant increase in the expression of several lipogenic genes in the liver (Fig. 9B). Interestingly, several genes involved in lipid oxidation were drastically increased in POMC-DeptorO/E mice following a high-fat diet, suggesting an overall increase in fatty acid turnover that is oriented toward lipogenesis. Altogether, these results indicate that overexpression of DEPTOR in POMC neurons is deleterious rather than beneficial for glucose tolerance and liver metabolism.

Fig. 8.

Fig. 8.

Open in a new tab

POMC-specific DEPTOR overexpression does not improve glucose metabolism in mice fed a high-fat chow diet. A: glucose tolerance test (GTT) was performed in control and POMC-DeptorO/E mice aged 18 wk old and fed a high-fat diet (n = 9–11/group). B: area under the curve for the GTT was calculated from the experiment shown in A. C: insulin tolerance test (ITT) was performed in control and POMC-DeptorO/E mice fed the same diet (n = 12/group). D: area under the curve for the ITT was calculated from the experiment shown in C. *P < 0.05 compared with control animals. Data represent means ± SE. Control represents littermates.

Fig. 9.

Fig. 9.

Open in a new tab

DEPTOR overexpression in POMC neurons induces hepatosteatosis in obese mice. A: liver triglycerides (TG) content in POMC-DeptorO/E mice and control mice fed a high-fat diet for 6 wk (n = 10–12). B: liver quantitative PCR of genes involved in lipid metabolism in POMC-DeptorO/E mice and control mice fed a high-fat diet (n = 10–12). *P < 0.05 and **P < 0.01 compared with control animals. Data represent means ± SE. Control represents littermates. Primers sequences are presented in Table 1.

DISCUSSION

Over the last years, several groups have shown that the mTOR pathway, a signaling node that is highly sensitive to nutritional cues, plays key roles in the regulation of energy balance and glucose metabolism (3, 7, 9, 16, 28, 29, 35). In two recent studies (5, 6), we have demonstrated that DEPTOR, an endogenous inhibitor of mTOR, is widely expressed in the brain, with high levels being found in the MBH. Functional studies using transgenic animals revealed that targeted overexpression of DEPTOR in the MBH protects mice against the development of obesity and metabolic disturbances (6). These effects were associated with a reduction in food intake and feed efficiency and with an elevation in oxygen consumption. Forced expression of DEPTOR in the MBH was also associated with the improvement in systemic glucose homeostasis (6). Although these findings established hypothalamic DEPTOR as an important player in the control of body weight and systemic metabolism, the identity of the neurons that mediated these effects was not determined.

Within the brain, POMC neurons are principally expressed in the arcuate nucleus of the hypothalamus, where they have been shown to regulate several physiological processes, including energy balance, as well as glucose and lipid metabolism, through the melanocortin receptors (24, 37). Their critical function in energy homeostasis is supported by the striking obesity phenotype, resulting from loss-of-function mutations of the POMC gene (4, 40). We have shown in a previous report that DEPTOR colocalizes with POMC neurons in the arcuate nucleus, suggesting that DEPTOR may be important in regulating the function of these neurons (5). To determine whether POMC neurons mediate the effects of hypothalamic DEPTOR overexpression on the protection against obesity and the improvement in glucose metabolism, we have produced a transgenic mouse model allowing the specific overexpression of DEPTOR in these neurons. Here, we report that DEPTOR overexpression in POMC neurons did not recapitulate any of the phenotypes observed when the protein was overexpressed in the MBH. Overall, POMC-DeptorO/E mice 1) did not show difference in feeding behavior, 2) did not exhibit changes in locomotion activity, 3) did not show an improvement in systemic glucose metabolism, and 4) were not resistant to high-fat diet-induced obesity. Taken together, these results clearly show that POMC neurons are not mediating the metabolic effects linked to DEPTOR overexpression in the MBH. Importantly, these observations also indicate that other MBH neuronal populations, such as AgRP or SF1 neurons, which are the other dominant neuronal populations of the MBH (32), could be the neurons mediating the beneficial metabolic effects of DEPTOR. Assessing the impact of DEPTOR overexpression in AgRP and SF1 neurons using Agrp-Cre (36) and Sf1-Cre (10) mice will certainly help to dissect the contribution of each neuronal population to the phenotype originally described when DEPTOR is overexpressed in the whole MBH.

We have previously observed that overexpression of DEPTOR in the MBH improves glucose homeostasis and reduces hepatic steatosis (6). Surprisingly, we report in the present study a mild deterioration of glucose tolerance, an elevation in the capacity for hepatic glucose production, and the development of hepatosteatosis in mice expressing DEPTOR in POMC-DeptorO/E mice. The reason underlying the opposite phenotype of these models is unknown. It is likely that DEPTOR overexpression in the whole MBH simultaneously modulates the action of several neuronal populations and that some effects resulting from this intervention can dominate more than others and affect the phenotype of the mice. Here, by expressing DEPTOR only in POMC neurons, we clearly showed that these cells play a role in modulating brain-liver connection. Interestingly, several groups have reported that POMC neurons are important in the modulation of liver metabolism (2, 14, 34, 38, 39). A recent study showed that POMC-specific ablation of p70 S6 kinase 1 (S6K1), a downstream substrate of mTORC1, affects hepatic glucose production and peripheral lipid metabolism without affecting energy balance (35). It was reported that reducing S6K1 activity, an effect observed in response to DEPTOR overexpression (6, 19, 31), increases hepatic glucose output (35). Supporting these results, we observed that hepatic glucose production was significantly increased in POMC-DeptorO/E mice. Further investigations are needed to determine whether the activation of S6K1 in POMC neurons plays an important role in mediating the effect of DEPTOR on liver metabolism. Over the years, it was shown several times that DEPTOR promotes insulin signaling by dampening the feedback inhibition from mTORC1 to phosphoinositide-3-kinase (PI3K), which leads to an activation of protein kinase B (Akt/PKB) (6, 19, 22, 23, 31). Defining whether changes in insulin signaling contribute to modulate the brain-liver connection that we report here represents another key question that will require additional experiments.

Although the results described here strongly suggest that POMC neurons do not mediate the beneficial effects of overexpressing DEPTOR in the MBH (6), one should consider differences between the model described in the present study and the one that we have used before (6). First, in our previous study, MBH-specific overexpression of DEPTOR was achieved through stereotaxic injections of adeno-associated virus type 2 (AAV2) encoding the Cre recombinase in adult mice carrying the CAAGSLox-stop-Lox Deptor allele (6). Here, POMC-specific overexpression of DEPTOR was achieved by crossing Pomc-Cre mice with mice carrying the CAAGSLox-stop-Lox Deptor allele. Contrary to the AAV-based approach, the use of the Pomc-Cre mouse allows the overexpression of DEPTOR during both the developmental and adult stages. It is possible that prenatal overexpression of DEPTOR could have led to compensatory mechanisms that may have covered effects observed when the protein is overexpressed only in adults (30). However, we previously observed that constitutive (prenatal) whole body overexpression of DEPTOR using a Cmv-Cre mouse improved energy and glucose metabolism, which argues against such compensatory mechanisms. Again, the mild deterioration in glucose tolerance and perturbations in liver metabolism described in the present study demonstrate that targeting DEPTOR in POMC neurons has effects similar to those of specific ablation of S6K1 in POMC neurons (35), reinforcing the idea that the improvements observed in the MBH model were mediated through another neuronal population. Future studies with models allowing the expression of the Cre-recombinase only in adult POMC neurons will be required to fully address the impact of postnatal overexpression of DEPTOR on metabolism. It is key to point out that, in addition to mediating recombination of floxed alleles in POMC neurons of the hypothalamus, Pomc-Cre lines also mediate recombination in extra-hypothalamic POMC-neurons and in some non-POMC neurons in adults (27). This limitation is inherent to most developmental Cre lines presently used by most research groups (13, 25, 30). Thus, it is possible that the phenotype reported here could be related to unspecific recombination of the CAAGSLox-stop-LoxDeptor allele. Nevertheless, the absence of a metabolic improvement in POMC-DeptorO/E mice strongly suggests that this neuronal population, or any other possibly targeted by the Pomc-Cre, are not responsible for the phenotype previously reported in mice overexpressing DEPTOR in the MBH.

In summary, the present study demonstrates that overexpressing DEPTOR in POMC neurons did not affect energy homeostasis and did not protect mice from high-fat diet-induced obesity. These results indicate that POMC neurons are not mediating the improvement in energy and glucose metabolism linked to DEPTOR overexpression in the MBH. Importantly, a mild glucose intolerance, an elevation in the capacity for hepatic glucose production, and the development of hepatosteatosis were observed in mice expressing DEPTOR in POMC neurons. Taken together, these results show that DEPTOR overexpression in POMC neurons does not affect energy balance regulation but could modulate metabolism through a brain-liver connection.

GRANTS

This work was supported by grants from the Canadian Institutes of Health Research (CIHR), the Natural Sciences and Engineering Research Council of Canada (NSERC), Les Fonds de la Recherche Santé Québec (FRQS), the Canadian Liver Foundation, Le Réseau de Recherche en Santé Cardiométabolique, Diabète et Obésité (CMDO), Le Réseau de Bio-Imagerie du Québec (RBIQ), Diabète Québec, and La Fondation de l'Institut Universitaire de Cardiologie et de Pneumologie de Québec (IUCPQ) to M. Laplante. A. Caron held a fellowship from the CIHR Training Program in Obesity/Healthy Body Weight Research and now holds a Canadian Diabetes Association postdoctoral fellowship. S. M. Labbé. holds a CIHR postdoctoral fellowship.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

A.C., D.R., and M.L. conception and design of research; A.C., S.M.L., M.M., and R.H. performed experiments; A.C. and S.M.L. analyzed data; A.C., D.R., and M.L. interpreted results of experiments; A.C. and M.L. prepared figures; A.C. drafted manuscript; A.C., D.R., and M.L. edited and revised manuscript; A.C., S.M.L., M.M., R.H., D.R., and M.L. approved final version of manuscript.

ACKNOWLEDGMENTS

The authors are grateful to Marie-Claude Roy, Julie Plamondon, and Yves Gélinas for their helpful technical assistance.

REFERENCES

  • 1.Balthasar N, Coppari R, McMinn J, Liu SM, Lee CE, Tang V, Kenny CD, McGovern RA, Chua SC Jr, Elmquist JK, Lowell BB. Leptin receptor signaling in POMC neurons is required for normal body weight homeostasis. Neuron 42: 983–991, 2004. [DOI] [PubMed] [Google Scholar]
  • 2.Berglund ED, Vianna CR, Donato J Jr, Kim MH, Chuang JC, Lee CE, Lauzon DA, Lin P, Brule LJ, Scott MM, Coppari R, Elmquist JK. Direct leptin action on POMC neurons regulates glucose homeostasis and hepatic insulin sensitivity in mice. J Clin Invest 122: 1000–1009, 2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Blouet C, Ono H, Schwartz GJ. Mediobasal hypothalamic p70 S6 kinase 1 modulates the control of energy homeostasis. Cell Metab 8: 459–467, 2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Butler AA. The melanocortin system and energy balance. Peptides 27: 281–290, 2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Caron A, Baraboi ED, Laplante M, Richard D. DEP domain-containing mTOR-interacting protein in the rat brain: Distribution of expression and potential implication. J Comp Neurol 523: 93–107, 2015. [DOI] [PubMed] [Google Scholar]
  • 6.Caron A, Labbe SM, Lanfray D, Blanchard PG, Villot R, Roy C, Sabatini DM, Richard D, Laplante M. Mediobasal hypothalamic overexpression of DEPTOR protects against high-fat diet-induced obesity. Mol Metab 5: 102–112, 2016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Cota D. Mammalian target of rapamycin complex 1 (mTORC1) signaling in energy balance and obesity. Physiol Behav 97: 520–524, 2009. [DOI] [PubMed] [Google Scholar]
  • 8.Cota D, Matter EK, Woods SC, Seeley RJ. The role of hypothalamic mammalian target of rapamycin complex 1 signaling in diet-induced obesity. J Neurosci 28: 7202–7208, 2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Cota D, Proulx K, Smith KA, Kozma SC, Thomas G, Woods SC, Seeley RJ. Hypothalamic mTOR signaling regulates food intake. Science 312: 927–930, 2006. [DOI] [PubMed] [Google Scholar]
  • 10.Dhillon H, Zigman JM, Ye C, Lee CE, McGovern RA, Tang V, Kenny CD, Christiansen LM, White RD, Edelstein EA, Coppari R, Balthasar N, Cowley MA, Chua S Jr, Elmquist JK, Lowell BB. Leptin directly activates SF1 neurons in the VMH, and this action by leptin is required for normal body-weight homeostasis. Neuron 49: 191–203, 2006. [DOI] [PubMed] [Google Scholar]
  • 11.Elmquist JK, Coppari R, Balthasar N, Ichinose M, Lowell BB. Identifying hypothalamic pathways controlling food intake, body weight, and glucose homeostasis. J Comp Neurol 493: 63–71, 2005. [DOI] [PubMed] [Google Scholar]
  • 12.Guesdon B, Denis RG, Richard D. Additive effects of olanzapine and melanin-concentrating hormone agonism on energy balance. Behav Brain Res 207: 14–20, 2010. [DOI] [PubMed] [Google Scholar]
  • 13.Harno E, Cottrell EC, White A. Metabolic pitfalls of CNS Cre-based technology. Cell Metab 18: 21–28, 2013. [DOI] [PubMed] [Google Scholar]
  • 14.Hill JW, Elias CF, Fukuda M, Williams KW, Berglund ED, Holland WL, Cho YR, Chuang JC, Xu Y, Choi M, Lauzon D, Lee CE, Coppari R, Richardson JA, Zigman JM, Chua S, Scherer PE, Lowell BB, Bruning JC, Elmquist JK. Direct insulin and leptin action on pro-opiomelanocortin neurons is required for normal glucose homeostasis and fertility. Cell Metab 11: 286–297, 2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Hill JW, Xu Y, Preitner F, Fukuda M, Cho YR, Luo J, Balthasar N, Coppari R, Cantley LC, Kahn BB, Zhao JJ, Elmquist JK. Phosphatidyl inositol 3-kinase signaling in hypothalamic proopiomelanocortin neurons contributes to the regulation of glucose homeostasis. Endocrinology 150: 4874–4882, 2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Kocalis HE, Hagan SL, George L, Turney MK, Siuta MA, Laryea GN, Morris LC, Muglia LJ, Printz RL, Stanwood GD, Niswender KD. Rictor/mTORC2 facilitates central regulation of energy and glucose homeostasis. Mol Metab 3: 394–407, 2014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Koch L, Wunderlich FT, Seibler J, Konner AC, Hampel B, Irlenbusch S, Brabant G, Kahn CR, Schwenk F, Bruning JC. Central insulin action regulates peripheral glucose and fat metabolism in mice. J Clin Invest 118: 2132–2147, 2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Lanfray D, Caron A, Roy MC, Laplante M, Morin F, Leprince J, Tonon MC, Richard D. Involvement of the Acyl-CoA binding domain containing 7 in the control of food intake and energy expenditure in mice. eLife 5: pii: e11742, 2016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Laplante M, Horvat S, Festuccia WT, Birsoy K, Prevorsek Z, Efeyan A, Sabatini DM. DEPTOR cell-autonomously promotes adipogenesis, and its expression is associated with obesity. Cell Metab 16: 202–212, 2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell 149: 274–293, 2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Lenard NR, Berthoud HR. Central and peripheral regulation of food intake and physical activity: pathways and genes. Obesity (Silver Spring) 16 Suppl 3: S11–S22, 2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Meng ZX, Li S, Wang L, Ko HJ, Lee Y, Jung DY, Okutsu M, Yan Z, Kim JK, Lin JD. Baf60c drives glycolytic metabolism in the muscle and improves systemic glucose homeostasis through DEPTOR-mediated Akt activation. Nature Med 19: 640–645, 2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Meng ZX, Wang L, Xiao Y, Lin JD. The Baf60c/DEPTOR pathway links skeletal muscle inflammation to glucose homeostasis in obesity. Diabetes 63: 1533–1545, 2014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Mercer AJ, Hentges ST, Meshul CK, Low MJ. Unraveling the central proopiomelanocortin neural circuits. Front Neurosci 7: 19, 2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Morrison CD, Munzberg H. Capricious Cre: the devil is in the details. Endocrinology 153: 1005–1007, 2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Morton GJ, Cummings DE, Baskin DG, Barsh GS, Schwartz MW. Central nervous system control of food intake and body weight. Nature 443: 289–295, 2006. [DOI] [PubMed] [Google Scholar]
  • 27.Mountjoy KG. Functions for pro-opiomelanocortin-derived peptides in obesity and diabetes. Biochem J 428: 305–324, 2010. [DOI] [PubMed] [Google Scholar]
  • 28.Muta K, Morgan DA, Rahmouni K. The role of hypothalamic mTORC1 signaling in insulin regulation of food intake, body weight and sympathetic nerve activity in male mice. Endocrinology: en20141660, 2015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Ono H, Pocai A, Wang Y, Sakoda H, Asano T, Backer JM, Schwartz GJ, Rossetti L. Activation of hypothalamic S6 kinase mediates diet-induced hepatic insulin resistance in rats. J Clin Invest 118: 2959–2968, 2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Padilla SL, Carmody JS, Zeltser LM. Pomc-expressing progenitors give rise to antagonistic neuronal populations in hypothalamic feeding circuits. Nature Med 16: 403–405, 2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Peterson TR, Laplante M, Thoreen CC, Sancak Y, Kang SA, Kuehl WM, Gray NS, Sabatini DM. DEPTOR is an mTOR inhibitor frequently overexpressed in multiple myeloma cells and required for their survival. Cell 137: 873–886, 2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Richard D. The cognitive and autonomic determinants of energy homeostasis behind obesity. Nat Rev Endocrinol 11: 489–501, 2015. [DOI] [PubMed] [Google Scholar]
  • 33.Richard D. Cognitive and autonomic determinants of energy homeostasis in obesity. Nat Rev Endocrinol 11: 489–501, 2015. [DOI] [PubMed] [Google Scholar]
  • 34.Shi X, Zhou F, Li X, Chang B, Li D, Wang Y, Tong Q, Xu Y, Fukuda M, Zhao JJ, Li D, Burrin DG, Chan L, Guan X. Central GLP-2 enhances hepatic insulin sensitivity via activating PI3K signaling in POMC neurons. Cell Metab 18: 86–98, 2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Smith MA, Katsouri L, Irvine EE, Hankir MK, Pedroni SM, Voshol PJ, Gordon MW, Choudhury AI, Woods A, Vidal-Puig A, Carling D, Withers DJ. Ribosomal S6K1 in POMC and AgRP neurons regulates glucose homeostasis but not feeding behavior in mice. Cell Rep 11: 335–343, 2015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Tong Q, Ye CP, Jones JE, Elmquist JK, Lowell BB. Synaptic release of GABA by AgRP neurons is required for normal regulation of energy balance. Nat Neurosci 11: 998–1000, 2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Varela L, Horvath TL. Leptin and insulin pathways in POMC and AgRP neurons that modulate energy balance and glucose homeostasis. EMBO Rep 13: 1079–1086, 2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Williams KW, Liu T, Kong X, Fukuda M, Deng Y, Berglund ED, Deng Z, Gao Y, Liu T, Sohn JW, Jia L, Fujikawa T, Kohno D, Scott MM, Lee S, Lee CE, Sun K, Chang Y, Scherer PE, Elmquist JK. Xbp1s in Pomc neurons connects ER stress with energy balance and glucose homeostasis. Cell Metab 20: 471–482, 2014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Xu Y, Berglund ED, Sohn JW, Holland WL, Chuang JC, Fukuda M, Rossi J, Williams KW, Jones JE, Zigman JM, Lowell BB, Scherer PE, Elmquist JK. 5-HT2CRs expressed by pro-opiomelanocortin neurons regulate insulin sensitivity in liver. Nat Neurosci 13: 1457–1459, 2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Yaswen L, Diehl N, Brennan MB, Hochgeschwender U. Obesity in the mouse model of pro-opiomelanocortin deficiency responds to peripheral melanocortin. Nat Med 5: 1066–1070, 1999. [DOI] [PubMed] [Google Scholar]

Articles from American Journal of Physiology - Regulatory, Integrative and Comparative Physiology are provided here courtesy of American Physiological Society

RESOURCES