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. 2013 Jun 14;288(24):17675-88.
doi: 10.1074/jbc.M113.475343. Epub 2013 May 2.

Obesity induces hypothalamic endoplasmic reticulum stress and impairs proopiomelanocortin (POMC) post-translational processing

Isin Cakir  1 , Nicole E Cyr, Mario Perello, Bogdan Patedakis Litvinov, Amparo Romero, Ronald C Stuart, Eduardo A Nillni
Affiliations

Obesity induces hypothalamic endoplasmic reticulum stress and impairs proopiomelanocortin (POMC) post-translational processing

Isin Cakir et al. J Biol Chem. .

Abstract

It was shown previously that abnormal prohormone processing or inactive proconverting enzymes that are responsible for this processing cause profound obesity. Our laboratory demonstrated earlier that in the diet-induced obesity (DIO) state, the appetite-suppressing neuropeptide α-melanocyte-stimulating hormone (α-MSH) is reduced, yet the mRNA of its precursor protein proopiomelanocortin (POMC) remained unaltered. It was also shown that the DIO condition promotes the development of endoplasmic reticulum (ER) stress and leptin resistance. In the current study, using an in vivo model combined with in vitro experiments, we demonstrate that obesity-induced ER stress obstructs the post-translational processing of POMC by decreasing proconverting enzyme 2, which catalyzes the conversion of adrenocorticotropin to α-MSH, thereby decreasing α-MSH peptide production. This novel mechanism of ER stress affecting POMC processing in DIO highlights the importance of ER stress in regulating central energy balance in obesity.

Keywords: Endoplasmic Reticulum Stress; Energy Metabolism; Hypothalamus; Neuropeptide; Protein Processing.

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Figures

FIGURE 1.
FIGURE 1.
α-MSH peptide is lower in DIO rodents, whereas POMC mRNA remains unchanged. Feeding rats a high fat diet for 12 weeks generated DIO (gray bars), whereas lean controls (black bars) were kept on regular chow. A, POMC mRNA in lean and DIO ARC was measured by RT-PCR. B, Western blot analysis depicted the accumulation of POMC protein in the DIO ARC. C, RIA of ACTH in lean and DIO rats. D, RIA of α-MSH in lean and DIO rats. E, ObRb levels in lean and DIO rat ARC as measured by Western blot. F, Western blot of pSTAT3 over total STAT3 in the ARC of lean and DIO rats. G, SOCS3 protein levels in lean and DIO ARC were measured by Western blot. H, PTP1B protein levels in lean and DIO ARC measured by Western blot. n = 12 lean and 8 DIO. Data are mean ± S.E. (error bars). *, p < 0.05; **, p < 0.01 versus lean control.
FIGURE 2.
FIGURE 2.
Leptin action on POMC, ACTH, and α-MSH in DIO rats. For leptin experiments, fasted animals were without food for 48 h, and leptin was infused icv (3.5 μg/rat). Black bars, lean; gray bars, DIO. For C and D, AG490 (54 nmol/rat; gray bars) or vehicle control (black bars) was infused icv. A, RT-PCR of POMC mRNA levels. B, ACTH RIA analyses in lean and DIO ARC. C, α-MSH RIA analyses in lean and DIO ARC. D, ACTH response to AG490 in lean and DIO ARC. E, α-MSH response to AG490 in lean and DIO ARC. n = 12 lean and 8 DIO (A–D). Data are mean ± S.E. (error bars). *, p < 0.05; **, p < 0.01 versus lean fed vehicle (A–C) or lean vehicle (D and E); #, p < 0.05; ##, p < 0.01 versus fasted within the same group (lean or DIO).
FIGURE 3.
FIGURE 3.
POMC processing is altered in DIO. A, the POMC processing cascade is modeled here and has been described previously. Black bars, lean; gray bars, DIO. B, Western blot of PC1 protein levels in lean and DIO ARC. C, RT-PCR of PC1 mRNA levels in lean and DIO ARC. D, Western blot of PC2 protein levels in lean and DIO ARC. E, RT-PCR of PC2 mRNA levels in lean and DIO ARC. F, Western blot of CPE protein levels in lean and DIO ARC. G, RT-PCR of CPE mRNA levels in lean and DIO ARC. Depicted analyses were performed on n = 8 lean and 8 DIO (A–D) (B, D, and F were repeated n = 8 with the same results. Data are mean ± S.E. (error bars). *, p < 0.05 versus lean control.
FIGURE 4.
FIGURE 4.
DIO and pharmacologically induced ER stress increases p-PERK and SOCS3 and PTP1B expression. Black bars, lean; gray bars, DIO. Protein levels were analyzed by Western blot. A, p-PERK protein levels in lean and DIO ARC. B, p-eIF2α protein levels in lean and DIO ARC. For C–F, ER stress inducers thapsigargin (thaps; 10 ng/rat; dark gray bars) or tunicamycin (tunic; 10 μg/rat; light gray bars) were icv infused in lean rats and compared with vehicle controls (black bars). C, p-PERK protein levels in the ARC of lean rats treated with tunic or vehicle control. D, protein levels of p-eIF2α in the ARC of lean rats treated with tunicamycin, thapsigargin, or vehicle control. E, analysis of SOCS3 expression by end point PCR in the ARC of lean rats treated with tunicamycin, thapsigargin, or vehicle control. F, SOCS3 and PTP1B protein levels in the ARC of lean rats treated with tunicamycin, thapsigargin, or vehicle control. n = 12 lean and 8 DIO (A–D and F) n = 3 (E). Data are mean ± S.E. (error bars). *, p < 0.05; **, p < 0.01 versus lean control (A and B) or vehicle control (C–F).
FIGURE 5.
FIGURE 5.
Pharmacologically induced ER stress alters ACTH, α-MSH, and PC2. A–D, ER stress inducers thapsigargin (thaps; 10 ng/rat; dark gray bars) or tunicamycin (tunic; 10 μg/rat; light gray bars) were icv infused in lean rats and compared with vehicle controls (black bars). A–D, n = 8/group; E and F, n = 6/group. A, RIA of ACTH peptide content in the ARC of lean rats treated with tunicamycin, thapsigargin, or vehicle control. B, RIA of α-MSH peptide content in the ARC of lean rats treated with tunicamycin, thapsigargin, or vehicle control. C, RT-PCR of PC2 mRNA levels in the ARC of lean rats treated with tunicamycin, thapsigargin, or vehicle control. D, Western blot of PC2 protein levels in the ARC of lean rats treated with tunicamycin, thapsigargin, or vehicle control. E, fasting glucose levels in rats treated with thapsigargin or vehicle control. F, oxygen consumption following thapsigargin or vehicle control infusions. Data are mean ± S.E. (error bars). *, p < 0.05 versus vehicle control.
FIGURE 6.
FIGURE 6.
Changes in ACTH and α-MSH in DIO are reversed with a chemical chaperone. Lean and DIO rats were treated with (gray bars) and without (black bars) the chemical chaperone TUDCA (200 mg/kg for 2 days). LV, lean vehicle (n = 4); LT, lean TUDCA (n = 4); DV, DIO vehicle (n = 5); DT, DIO TUDCA (n = 5). A, TUDCA reduced p-eIF2α in the ARC of DIO rats. B, RIA of ACTH peptide content in the ARC of lean and DIO rats treated with and without TUDCA. C, RIA of α-MSH peptide content in the ARC of lean and DIO rats treated with and without TUDCA. D, body weight of lean and DIO rats treated with and without TUDCA. Animals were weighed immediately prior to the first TUDCA infusion and a second time immediately prior to sacrifice. Weight was measured as the percentage weight gain (g) from the first to the second measurement. E, TUDCA reduced food intake in DIO rats. F, oxygen consumption in DIO rats following TUDCA or vehicle control infusions. G, fasting glucose levels in rats treated with TUDCA or vehicle control. Data are mean ± S.E. (error bars). *, p < 0.05 versus vehicle control.
FIGURE 7.
FIGURE 7.
There were no changes detected in leptin signaling, prolylcarboxypeptidase (PRCP), or NPY due to TUDCA. Lean and DIO rats were treated with (gray bars) and without (black bars) the chemical chaperone TUDCA (200 mg/kg for 2 days). LV, lean vehicle (n = 4); LT, lean TUDCA (n = 4); DV, DIO vehicle (n = 5); DT, DIO TUDCA (n = 5). A, Western blot of pSTAT3 protein levels in the ARC of lean and DIO rats treated with and without TUDCA. B, Western blot of SOCS3 protein levels in the ARC of lean and DIO rats treated with and without TUDCA. C, Western blot of ObRb protein levels in the ARC of lean and DIO rats treated with and without TUDCA. D, Western blot of PRCP protein levels in the ARC of lean and DIO rats treated with and without TUDCA. E, RIA of NPY content in the ARC of lean and DIO rats treated with and without TUDCA. Data are mean ± S.E. (error bars). *, p < 0.05 versus vehicle control.
FIGURE 8.
FIGURE 8.
ER stress attenuates PC2 protein (but not PC2 promoter activity or processing) specifically in POMC-positive N43/5 neuronal cells, and salubrinal-enhanced p-eIF2α increases PC2 and appears to protect against the ER stress decrease in PC2. A, PC2 and p-eIF2α protein in POMC-positive N43/5 cells treated with tunicamycin (tunic; 0.1 μg/ml; gray bars) and with and without PBA (20 mm, 14 h) pretreatment. n = 6; results were replicated in three independent experiments. B, dose-response curve for PC2 protein in N43/5 cells treated with vehicle, 7.5 μg of salubrinal, and 30 μg of salubrinal. p-eIF2α is also shown. n = 4/group. C, PC2 protein in N43/5 cells pretreated with vehicle (VV), pretreated with vehicle then thapsigargin (thaps; for 6 h) (VT), or pretreated with 7.5 μg of salubrinal (14 h) and then thapsigargin (6 h) (ST). p-eIF2α is also shown. n = 6/group. D, PC2 promoter activity with and without tunicamycin (1.5 h) treatment as analyzed by luciferase assay. n = 8. E, Western blot analysis of 7B2 in control (white bars) and tunicamycin-treated (0.1 μg/ml, 2 h; gray bars) N43/5 cells. F, Western blot analysis of 7B2 in the ARC of lean (white bars) and DIO (gray bars) animals. E and F, n = 6–9. Data are mean ± S.E. (error bars). *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus vehicle control at time 0 (A), vehicle (B and D), vehicle/vehicle (C), or lean controls (F).
FIGURE 9.
FIGURE 9.
PC2 protein and α-MSH release decreases with induction of ER stress in POMC-positive AtT20 cells. PC2 protein (A) and α-MSH release into the medium (B) in AtT20 cells pretreated with vehicle (VV), pretreated with vehicle and then thapsigargin (thaps for 6 h) (VT), or pretreated with 20 mm PBA (14 h) and then thapsigargin (6 h) (PT). PC2 protein (C) and α-MSH release into the medium (D) in AtT20 cells transfected with 2 μg of PC2 cDNA and treated with vehicle, transfected with 2 μg of PC2 cDNA and treated with thapsigargin, or transfected with 4 μg PC2 cDNA and treated with thapsigargin (all n = 5/group). *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus vehicle/vehicle (A and B) or PC2 (2 μg)/vehicle (C and D). #, p < 0.05 versus vehicle/thapsigargin (A and B) or PC2 (2 μg)/thapsigargin (C and D). Error bars, S.E.
FIGURE 10.
FIGURE 10.
Model depicting the changes in POMC, its processing enzyme PC2, and α-MSH under conditions of fasting (i.e. low leptin; first panel), fed (i.e. basal leptin; second panel), and DIO (i.e. high leptin; third panel). Compared with the fed (basal leptin) condition, fasting causes diminished α-MSH by leptin-mediated reduction in POMC mRNA along with reduced POMC processing due to lower PC1 and PC2 levels. In the DIO state, ER stress triggers an unfolded response causing an accumulation of POMC in the ER partly caused by an unfolded response and a possible degradation of PC2 through the ER-associated degradation system, conditions that ultimately cause less α-MSH production.
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