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. 2018 May:11:1-17.
doi: 10.1016/j.molmet.2018.02.013. Epub 2018 Mar 2.

Adipocyte Xbp1s overexpression drives uridine production and reduces obesity

Yingfeng Deng  1 , Zhao V Wang  2 , Ruth Gordillo  1 , Yi Zhu  1 , Aktar Ali  1 , Chen Zhang  1 , Xiaoding Wang  3 , Mengle Shao  1 , Zhuzhen Zhang  1 , Puneeth Iyengar  4 , Rana K Gupta  1 , Jay D Horton  5 , Joseph A Hill  6 , Philipp E Scherer  7
Affiliations

Adipocyte Xbp1s overexpression drives uridine production and reduces obesity

Yingfeng Deng et al. Mol Metab. 2018 May.

Abstract

Objective: The spliced transcription factor Xbp1 (Xbp1s), a transducer of the unfolded protein response (UPR), regulates lipolysis. Lipolysis is stimulated by fasting when uridine synthesis is also activated in adipocytes.

Methods: Here we have examined the regulatory role Xbp1s in stimulation of uridine biosynthesis in adipocytes and triglyceride mobilization using inducible mouse models.

Results: Xbp1s is a key molecule involved in adipocyte uridine biosynthesis and release by activation of carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, dihydroorotase (CAD), the rate-limiting enzyme for UMP biosynthesis. Adipocyte Xbp1s overexpression drives energy mobilization and protects mice from obesity through activation of the pyrimidine biosynthesis pathway.

Conclusion: These observations reveal that Xbp1s is a potent stimulator of uridine production in adipocytes, enhancing lipolysis and invoking a potential anti-obesity strategy through the induction of a futile biosynthetic cycle.

Keywords: CAD; ER stress; Obesity; Pyrimidine; UPR; Uridine; Xbp1.

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Figures

Figure 1
Figure 1
Xbp1s induction in adipocyte associates with lipolysis. (A) Xbp1s mRNA levels in fat pads were elevated after an overnight fast compared to fed group (n = 4). (B) Xbp1s mRNA levels in fat pads were elevated upon β3 adrenergic receptor stimulation with CL 316,243 compared to vehicle injected group (n = 4). (C) Xbp1s mRNA levels in fat pads were elevated in mice undergoing LLC tumor-induced cachexia compared to control group (n = 5). (D) Xbp1s mRNA levels in fat pads were elevated in ob/ob mice compared to lean littermates (n = 5). eWAT, epididymal white adipose tissue. sWAT, subcutaneous white adipose tissue. BAT, interscapular brown adipose tissue. Data were analyzed with two-tailed Student t test. *, p < 0.05; **, p < 0.01, ***, p < 0.001. Error bars denote SEM.
Figure 2
Figure 2
Adipocyte Xbp1s is sufficient and necessary for plasma uridine elevation triggered by lipolysis. (A) Strategy for the generation of mouse with adipocyte-specific fat tissue inducible Xbp1s overexpression (FIXs). (B) Verification of Xbp1s expression by western blotting after 7 days of Dox chow feeding. Lamin was used as a loading control. * denotes a non-specific cross-reacting band. (C) Adipocyte Xbp1s overexpression increased plasma uridine levels (n = 5–6). Plasma uridine concentration from each mouse was normalized to basal level prior to Dox. Statistical analysis was performed for the two genotypes at time points indicated. (D) Adipocyte Xbp1s overexpression increased uridine concentration in fat depots (n = 6). (E) Strategy for the generation of mouse with adipocyte-specific fat tissue inducible Xbp1 knockout (FIX KO). (F) Adipocyte Xbp1 deficiency prevented plasma uridine elevation in fasted mice (n = 3). Statistical analysis was performed for each genotype using the fed state of that group as base line. (G) Serum NEFA was increased by CL316, 243 in both WT and FIX KO mice (n = 3). Data in (C), (D), and (G) were analyzed with two-tailed Student t test, and data in (F) was analyzed with paired t test. *, p < 0.05; **, p < 0.01, ***, p < 0.001. Error bars denote SEM.
Figure 3
Figure 3
Xbp1s and ER stress regulates pyrimidine synthesis in adipocyte. (A) Adipocyte Xbp1s overexpression increased Cad mRNA in fat depots (n = 8). (B) Adipocyte Xbp1s overexpression did not change Dhodh mRNA in fat depots (n = 8). (C) Adipocyte Xbp1s overexpression did not change Umps mRNA in fat depots (n = 8). (D) Adipocyte Xbp1s overexpression induced elevation of Cad protein as assessed by immunoblotting. GAPDH was used as loading control. (E) Cad expression was increased by Xbp1s overexpression in vitro (24 h incubation with Dox). Adipocytes were differentiated from SVF and Dox were supplemented at day 6 post differentiation (n = 6). (F) Fetal Bovine Serum (FBS) but not DMEM contained uridine (n = 3) (G) Concentrations of uridine in cell culture medium were elevated by Xbp1s overexpression at 48 h post Dox addition (n = 3) and serum starvation (n = 3) (H) Concentrations of uridine in tissue culture medium were elevated by ER stress inducer treatment (n = 3). (I) Tunicamycin (TM) induced elevation of uridine in culture medium were suppressed by PALA in WT adipocyte but not Xbp1-knockout adipocytes (FIX KO). PALA was added at 0.1 μM, 10 μM, and 1 mM. (n = 3). (J) Xbp1s overexpression increased Cad mRNA in liver and heart (n = 3–6). LIXs, hepatocyte-specific liver inducible Xbp1s overexpression mouse as described . HIXs, cardiomyocyte-specific heart inducible Xbp1s overexpression mouse as described . Data were analyzed with two-tailed Student t test. *, p < 0.05; **, p < 0.01, ***, p < 0.001. Error bars denote SEM.
Figure 3
Figure 3
Xbp1s and ER stress regulates pyrimidine synthesis in adipocyte. (A) Adipocyte Xbp1s overexpression increased Cad mRNA in fat depots (n = 8). (B) Adipocyte Xbp1s overexpression did not change Dhodh mRNA in fat depots (n = 8). (C) Adipocyte Xbp1s overexpression did not change Umps mRNA in fat depots (n = 8). (D) Adipocyte Xbp1s overexpression induced elevation of Cad protein as assessed by immunoblotting. GAPDH was used as loading control. (E) Cad expression was increased by Xbp1s overexpression in vitro (24 h incubation with Dox). Adipocytes were differentiated from SVF and Dox were supplemented at day 6 post differentiation (n = 6). (F) Fetal Bovine Serum (FBS) but not DMEM contained uridine (n = 3) (G) Concentrations of uridine in cell culture medium were elevated by Xbp1s overexpression at 48 h post Dox addition (n = 3) and serum starvation (n = 3) (H) Concentrations of uridine in tissue culture medium were elevated by ER stress inducer treatment (n = 3). (I) Tunicamycin (TM) induced elevation of uridine in culture medium were suppressed by PALA in WT adipocyte but not Xbp1-knockout adipocytes (FIX KO). PALA was added at 0.1 μM, 10 μM, and 1 mM. (n = 3). (J) Xbp1s overexpression increased Cad mRNA in liver and heart (n = 3–6). LIXs, hepatocyte-specific liver inducible Xbp1s overexpression mouse as described . HIXs, cardiomyocyte-specific heart inducible Xbp1s overexpression mouse as described . Data were analyzed with two-tailed Student t test. *, p < 0.05; **, p < 0.01, ***, p < 0.001. Error bars denote SEM.
Figure 4
Figure 4
Adipocyte Xbp1s overexpression reduces fat mass but not lifespan. (A) Adipocyte Xbp1s overexpression led to reduction of bodyweight (n = 4). (B) Overexpression of Xbp1s in adipocytes reduced fat mass (n = 6). (C) Overexpression of Xbp1s led to reduction of fat depot mass (n = 9–10). (D) Representative H&E staining image of WATs. Scale bar, 200 μM. (E) Overexpression of Xbp1s in adipocytes caused lower levels of free fatty acids and ketone bodies under fasted condition (n = 6). (F) Overexpression of Xbp1s in adipocytes did not change insulin levels under fasted condition (n = 6). (G) Lifespan of male mice on Dox chow (n = 34 for WT, n = 24 for FIXs). (H) Representative H&E staining image of WATs from 40 weeks old male mice that were maintained on Dox chow from 7 weeks of age. Scale bar 100 μM, Data were analyzed with two-tailed Student t test. *, p < 0.05; **, p < 0.01, ***, p < 0.001. Error bars denote SEM.
Figure 4
Figure 4
Adipocyte Xbp1s overexpression reduces fat mass but not lifespan. (A) Adipocyte Xbp1s overexpression led to reduction of bodyweight (n = 4). (B) Overexpression of Xbp1s in adipocytes reduced fat mass (n = 6). (C) Overexpression of Xbp1s led to reduction of fat depot mass (n = 9–10). (D) Representative H&E staining image of WATs. Scale bar, 200 μM. (E) Overexpression of Xbp1s in adipocytes caused lower levels of free fatty acids and ketone bodies under fasted condition (n = 6). (F) Overexpression of Xbp1s in adipocytes did not change insulin levels under fasted condition (n = 6). (G) Lifespan of male mice on Dox chow (n = 34 for WT, n = 24 for FIXs). (H) Representative H&E staining image of WATs from 40 weeks old male mice that were maintained on Dox chow from 7 weeks of age. Scale bar 100 μM, Data were analyzed with two-tailed Student t test. *, p < 0.05; **, p < 0.01, ***, p < 0.001. Error bars denote SEM.
Figure 5
Figure 5
Adipocyte Xbp1s overexpression protects mice from obesity. (A) Mice were maintained on regular chow until 27–32 weeks old to start Dox HFD feeding. Bodyweight of FIXs mice was reduced by Dox HFD and increased by regular HFD, which was not observed in WT mice (n = 7–11). The Chow group were age-matched WT mice that were maintained on regular chow during the course of study (n = 15). (B) Fat mass and lean mass of mice were determined by MRI after 100 days on HFD Dox (n = 6). (C)–(E) Mice on ob/ob background were maintained on regular chow until 11 weeks old to start Dox Chow feeding. Bodyweight mice were monitored for 166 days on Dox. Fat volume and muscle volume of mice were measured by CT scan at indicated time after switch to Dox chow (n = 3). (F) Mice were maintained on regular chow until 27–32 weeks old to start Dox HFD feeding. Bodyweight of mice was monitored after switch to Dox HFD, back to regular HFD and then Dox HFD again (n = 6). The Chow group were age-matched WT mice shared in the study of Figure 5A. (G) Mice were maintained on regular chow until 69 weeks old to start Dox HFD. Fat mass and lean mass of mice were determined by MRI 4 weeks after switch to HFD Dox (n = 8). Data were analyzed with two-tailed Student t test. *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001. Error bars denote SEM.
Figure 5
Figure 5
Adipocyte Xbp1s overexpression protects mice from obesity. (A) Mice were maintained on regular chow until 27–32 weeks old to start Dox HFD feeding. Bodyweight of FIXs mice was reduced by Dox HFD and increased by regular HFD, which was not observed in WT mice (n = 7–11). The Chow group were age-matched WT mice that were maintained on regular chow during the course of study (n = 15). (B) Fat mass and lean mass of mice were determined by MRI after 100 days on HFD Dox (n = 6). (C)–(E) Mice on ob/ob background were maintained on regular chow until 11 weeks old to start Dox Chow feeding. Bodyweight mice were monitored for 166 days on Dox. Fat volume and muscle volume of mice were measured by CT scan at indicated time after switch to Dox chow (n = 3). (F) Mice were maintained on regular chow until 27–32 weeks old to start Dox HFD feeding. Bodyweight of mice was monitored after switch to Dox HFD, back to regular HFD and then Dox HFD again (n = 6). The Chow group were age-matched WT mice shared in the study of Figure 5A. (G) Mice were maintained on regular chow until 69 weeks old to start Dox HFD. Fat mass and lean mass of mice were determined by MRI 4 weeks after switch to HFD Dox (n = 8). Data were analyzed with two-tailed Student t test. *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001. Error bars denote SEM.
Figure 6
Figure 6
Adipocyte Xbp1s overexpression increases fatty acid oxidation and heat production. (A) Xbp1s overexpression in adipocytes for 6 days increased fatty acid oxidation without affecting lipid uptake (n = 6). (B) Xbp1s induction in adipocytes for 90 days increased fatty acid oxidation in multiple fat depots without affecting lipid uptake (n = 6). (C) Body temperature was monitored after mice were switched to Dox chow (n = 4). (D) Xbp1s overexpression in adipocytes for 6 days reduced expression of most thermogenic genes in BAT (n = 8). (E)–(I) Metabolic cage studies were conducted to measure food intake, physical activities, O2 consumption, CO2 release, and heat production in mice. A 24 h fast was performed from 12 AM to 12 AM during the study (n = 5). Data were analyzed with two-tailed Student t test. *, p < 0.05, **, p < 0.01, ***, p < 0.001. ns, not significant. Error bars denote SEM.
Figure 6
Figure 6
Adipocyte Xbp1s overexpression increases fatty acid oxidation and heat production. (A) Xbp1s overexpression in adipocytes for 6 days increased fatty acid oxidation without affecting lipid uptake (n = 6). (B) Xbp1s induction in adipocytes for 90 days increased fatty acid oxidation in multiple fat depots without affecting lipid uptake (n = 6). (C) Body temperature was monitored after mice were switched to Dox chow (n = 4). (D) Xbp1s overexpression in adipocytes for 6 days reduced expression of most thermogenic genes in BAT (n = 8). (E)–(I) Metabolic cage studies were conducted to measure food intake, physical activities, O2 consumption, CO2 release, and heat production in mice. A 24 h fast was performed from 12 AM to 12 AM during the study (n = 5). Data were analyzed with two-tailed Student t test. *, p < 0.05, **, p < 0.01, ***, p < 0.001. ns, not significant. Error bars denote SEM.
Figure 7
Figure 7
Adipocyte Cad knockout prevents pyrimidine synthesis and weight loss triggered by Xbp1s overexpression. (A) Strategy for the generation of mouse model for inducible adipocyte Xbp1s overexpression and Cad knockout (FIXs, Cad KO). (B) Representative gel image of PCR products of CAD from BAT derived cDNA. Exon 3 of CAD, which contains 130 nucleotides, is flanked by two loxP sites. The removal of exon 3 in CAD gene produces a CAD mutant transcript which can generate a 104 bp PCR product amplified from the cDNA with specifically designed primer set (Supplemental Table 1). (C) Representative western blot image of full-length CAD protein in adipocytes isolated from sWAT. GAPDH was blotted as loading control. (D) Bodyweight of mice prior to and post switch to Dox chow (WT, n = 9; FIXs, n = 7; FIXs, Cad KO, n = 6; Cad KO, n = 5). (E) Representative H&E staining image of adipose tissues from mice. Scale bar, 200 μM. (F) Gene expression of Xbp1s in fat depots (n = 3–4 each group). (G) Uridine concentrations in fat depots and liver (n = 3–4). Statistical analysis was performed for each tissue type using WT mice as base line if not specified. (H) Uridine concentrations in liver and fat depots from WT and ob/ob mice that were 19–23 weeks old on chow (n = 5). The ob/ob mice lack visible BAT at the time of harvest. (I) Uridine concentrations in liver and fat depots from WT and db/db mice that were 28–31 weeks old on chow (n = 5). (J) Uridine concentrations in liver and fat depots from WT male mice that were 19–21 weeks old and on HFD for 8 weeks (n = 4). (K) Uridine concentrations in liver and fat depots from WT male mice that were 26–31 weeks old and on Dox chow from 8 weeks of age (n = 5–6). Data were analyzed with two-tailed Student t test. *, p < 0.05, **, p < 0.01. ns, not significant. Error bars denote SEM.
Figure 7
Figure 7
Adipocyte Cad knockout prevents pyrimidine synthesis and weight loss triggered by Xbp1s overexpression. (A) Strategy for the generation of mouse model for inducible adipocyte Xbp1s overexpression and Cad knockout (FIXs, Cad KO). (B) Representative gel image of PCR products of CAD from BAT derived cDNA. Exon 3 of CAD, which contains 130 nucleotides, is flanked by two loxP sites. The removal of exon 3 in CAD gene produces a CAD mutant transcript which can generate a 104 bp PCR product amplified from the cDNA with specifically designed primer set (Supplemental Table 1). (C) Representative western blot image of full-length CAD protein in adipocytes isolated from sWAT. GAPDH was blotted as loading control. (D) Bodyweight of mice prior to and post switch to Dox chow (WT, n = 9; FIXs, n = 7; FIXs, Cad KO, n = 6; Cad KO, n = 5). (E) Representative H&E staining image of adipose tissues from mice. Scale bar, 200 μM. (F) Gene expression of Xbp1s in fat depots (n = 3–4 each group). (G) Uridine concentrations in fat depots and liver (n = 3–4). Statistical analysis was performed for each tissue type using WT mice as base line if not specified. (H) Uridine concentrations in liver and fat depots from WT and ob/ob mice that were 19–23 weeks old on chow (n = 5). The ob/ob mice lack visible BAT at the time of harvest. (I) Uridine concentrations in liver and fat depots from WT and db/db mice that were 28–31 weeks old on chow (n = 5). (J) Uridine concentrations in liver and fat depots from WT male mice that were 19–21 weeks old and on HFD for 8 weeks (n = 4). (K) Uridine concentrations in liver and fat depots from WT male mice that were 26–31 weeks old and on Dox chow from 8 weeks of age (n = 5–6). Data were analyzed with two-tailed Student t test. *, p < 0.05, **, p < 0.01. ns, not significant. Error bars denote SEM.
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