Obesity-associated gene TMEM18 has a role in the central control of appetite and body weight regulation

Abstract

An intergenic region of human chromosome 2 (2p25.3) harbors genetic variants which are among those most strongly and reproducibly associated with obesity. The gene closest to these variants is TMEM18, although the molecular mechanisms mediating these effects remain entirely unknown. Tmem18 expression in the murine hypothalamic paraventricular nucleus (PVN) was altered by changes in nutritional state. Germline loss of Tmem18 in mice resulted in increased body weight, which was exacerbated by high fat diet and driven by increased food intake. Selective overexpression of Tmem18 in the PVN of wild-type mice reduced food intake and also increased energy expenditure. We provide evidence that TMEM18 has four, not three, transmembrane domains and that it physically interacts with key components of the nuclear pore complex. Our data support the hypothesis that TMEM18 itself, acting within the central nervous system, is a plausible mediator of the impact of adjacent genetic variation on human adiposity.

Keywords: GWAS; TMEM18; hypothalamus; obesity.

Fig. 1.

Tmem18 expression within the hypothalamic paraventricular nucleus is nutritionally regulated. qRT-PCR analysis showing changes in Tmem18 gene expression in the PVN of wild-type mice that have been fed, fasted for 48 h, or fasted for 48 h with leptin administration. Data are expressed as mean ± SEM; *P < 0.05, **P < 0.01 vs. fasted mice.

Fig. 2.

Germline loss of Tmem18 results in increased body weight in male mice. Body weights of Tmem18 ^wt/wt (WT) and Tmem18 ^tm1a/tm1a (HOM) male mice on normal chow from weaning (WT, n = 23; HOM, n = 23) (A) or HFD (B) from 8 wk of age (WT, n = 15; HOM, n = 20). Body composition analyses showing total, lean, or fat mass of 14-wk old WT and HOM male mice on normal chow (WT, n = 16; HOM, n = 16) (C) or HFD from 8 wk of age (WT, n = 18; HOM, n = 20) (D). Data are expressed as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 vs. WT mice.

Fig. 3.

Effect of loss of expression of Tmem18 on energy homeostasis, activity levels, and food intake in male mice fed a HFD. Average 24-h energy intake of WT and homozygous male mice both measured in their home cage (A) and measured in the indirect calorimetry cage (B). (C) ANCOVA analysis of 24-h energy expenditure (EE) with body weight at time of analysis. (D) Average bodyweight of individual animals during 72-h analysis of energy expenditure. For all graphs, WT, n = 13; HOM, n = 18. Data are expressed as mean ± SEM; *P < 0.05, **P < 0.01 vs. WT mice.

Fig. 4.

Overexpression of Tmem18 within the PVN. (A) Change in Tmem18 gene expression within the PVN of mice 7 wk after bilateral injection of an adeno-associated vector (AAV-T18) (n = 10–11). Change in body weight of mice, measured weekly for 6 wk after bilateral PVN injections with either an AAV-T18 cDNA or AAV-GFP (n = 15 each group), expressed as grams (B) and as percentage of starting body weight (C). (D) Average 24-h energy intake of mice, measured in their home cage, 2 wk after surgery (n = 15 each group). (E) Average 24-h energy intake of mice, in the indirect calorimetry cage, 6 wk after surgery (n = 13 each group). (F) ANCOVA analysis of total EE, assessed over a 24-h period 6 wk after injection, with covariates evaluated at body weight = 29.41 g (n = 13 each group). Data are expressed as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 vs. GFP-injected mice.

Fig. 5.

Topology of the TMEM18 protein. Shown is the overexpression of N-terminal FLAG-tagged TMEM18 in COS cells treated with either TX-100 (permeabilizes both plasma and nuclear membrane) or digitonin (permeabilizes plasma membrane only). TMEM18 expression was detected with either a FLAG antibody (red, A and B) or an antibody to the C terminus of TMEM18 (green, C and D).

Fig. 6.

BiFC and co-IP confirmation of TMEM18 interaction with NDC1 and AAAS. (A–G) Physical interaction between TMEM18 and both NDC1 and AAAS was confirmed by BiFC. The N terminus of YFP was fused to FLAG-tagged NDC1 (YN-F-NDC1; C) or AAAS (YN-F-AAAS; E) or NUP35 (negative control, YN-F-NUP35; G), while the C terminus of YFP was fused to TMEM18 (TMEM18-Yc). FLAG expression was detected by using a FLAG antibody (red) while YFP signal is depicted in green. YN-F-NDC1 and AAAS-YC was used as a positive BiFC control (B). (H and I) Coimmunoprecipitation experiments using FLAG-tagged TMEM18 and GFP-tagged NDC1 (H) or AAAS (I) overexpressed in HEK cells. Dashed lines in the blot represents two different lanes in the same blot put together.