Adipose overexpression of desnutrin promotes fatty acid use and attenuates diet-induced obesity

Abstract

Objective: To investigate the role of desnutrin in adipose tissue triacylglycerol (TAG) and fatty acid metabolism.

Research design and methods: We generated transgenic mice overexpressing desnutrin (also called adipose triglyceride lipase [ATGL]) in adipocytes (aP2-desnutrin) and also performed adenoviral-mediated overexpression of desnutrin in 3T3-L1CARDelta1 adipocytes.

Results: aP2-desnutrin mice were leaner with decreased adipose tissue TAG content and smaller adipocyte size. Overexpression of desnutrin increased lipolysis but did not result in increased serum nonesterified fatty acid levels or ectopic TAG storage. We found increased cycling between diacylglycerol (DAG) and TAG and increased fatty acid oxidation in adipocytes from these mice, as well as improved insulin sensitivity.

Conclusions: We show that by increasing lipolysis, desnutrin overexpression causes reduced adipocyte TAG content and attenuation of diet-induced obesity. Desnutrin-mediated lipolysis promotes fatty acid oxidation and re-esterification within adipocytes.

FIG. 1.

Desnutrin overexpression in adipose tissue of mice. A: Transgene expression was verified by RT-PCR in tissues from aP2-desnutrin mice. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. B: Desnutrin mRNA level in gonadal WAT and BAT as determined by RT-qPCR (n = 5–6). □, wild type; ■, aP2-desnutrin. C: CGI-58 mRNA level in gonadal WAT as determined by RT-qPCR (n = 6–7). D: Immunoblot and quantification of desnutrin-HA fusion protein in gonadal WAT and BAT. □, endogenous; ■, endogenous + transgene. E: TAG lipase activity in WAT homogenates from wild-type and aP2-desnutrin mice. *P < 0.05, **P < 0.01, ***P < 0.001. All data are from female mice. WT, wild type. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 2.

Weight gain, fat pad weight, and adipocyte size in aP2-desnutrin mice. A: Gross appearance of female wild-type and aP2-desnutrin littermates (upper panel). Food intakes of female mice at 8 and 20 weeks (Wk) (lower panel). B: Time course of body weights over 20 weeks in mice fed an HFD. Inset: Average body weights at 20 weeks in mice fed a chow diet or an HFD (n = 9–11). C and D: Organ and fat pad weights in female mice fed a chow diet (C) or an HFD (D) (n = 9–11). Gon, gonadal fat pad; Ing, inguinal fat pad; Ren, renal fat pad; SC, subcutaneous fat pad. E: RT-qPCR for C/EBPα, Pref-1, PPARγ, and aP2/aFABP from WAT of female wild-type and aP2-desutrin mice fed an HFD (n = 4–6). F: Representative images of hematoxylin and eosin–stained sections of gonadal WAT and frequency distribution of adipocyte cell size in gonadal WAT. *P < 0.05, **P < 0.01, ***P < 0.001. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 3.

Overexpression of desnutrin in adipocytes decreases TAG content. A: TAG content of gonadal fat pads (n = 6–9). WT, wild type. B: Immunoblot of endogenous desnutrin and desnutrin-GFP in differentiated 3T3-L1CARΔ1 adipocytes. *Nonspecific band. DES, desnutrin. C: Confocal microscopy images showing localization of desnutrin-GFP to TAG-rich lipid droplets in contrast to the diffuse localization of GFP alone. D: Endogenous TAG content from 3T3-L1CARΔ1 adipocytes. Three days after infection with GFP or desnutrin-GFP, medium was replaced with serum-free medium and cells were maintained for an additional 6 h either with dibutyryl cAMP (stimulated) or without (basal) before analysis of TAG content. E: Time course of TAG breakdown in 3T3-L1CARΔ1 adipocytes labeled with [¹⁴C]palmitate before infection with GFP or desnutrin-GFP and chase with cold medium. *P < 0.05, **P < 0.01, ***P < 0.001. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 4.

In vivo measures of TAG metabolism in WAT. A: Fractional in vivo synthesis of TAG-glycerol in gonadal WAT (n = 6). B: In vivo lipolysis in gonadal WAT (n = 3–6). C: Fractional in vivo DNL in gonadal WAT (n = 6). D: Ratio of fractional in vivo DNL/fractional in vivo synthesis of TAG-glycerol in gonadal WAT (n = 6). *P < 0.05, †P = 0.07.

FIG. 5.

Desnutrin overexpression increases lipolysis. A: Glycerol and FA release from isolated adipocytes of fed female wild-type or aP2-desnutrin mice under basal conditions or stimulated with adenosine deaminase (ADA) or isoproterenol (iso). FA (B) and glycerol (C) release from 50 mg explants of fresh gonadal WAT incubated under basal conditions or stimulated with 0.5 mmol/l dibutyryl cAMP (n = 9–12). D: Radiolabeled-FA release from 3T3-L1CARΔ1 adipocytes. Cells were labeled with [¹⁴C]palmitate and then infected with adenoviral desnutrin-GFP or GFP (control) and chased with cold medium. [¹⁴C]FA in the medium were quantified 30 h after infection. E: Glycerol release from 3T3-L1CARΔ1 adipocytes after infection with GFP or desnutrin-GFP for 3 days. Six hours before assessment of glycerol concentrations, medium was replaced with serum-free medium either with dibutyryl cAMP (stimulated) or without (basal). □, control; ■, desnutrin. *P < 0.05, **P < 0.01, ***P < 0.001. WT, wild type.

FIG. 6.

Improved insulin sensitivity in aP2-desnutrin mice. A and B: Plasma glucose (A) and glucose infusion (GINF) (B) rates (n = 7–8) C: Whole-body glucose uptake (n = 7–8). D: Skeletal muscle (gastrocnemius) glucose uptake (n = 7–8). E: Epididymal WAT glucose uptake (n = 7–8). F: Hepatic glucose production during hyperinsulinemic-euglycemic clamps (n = 7–8). G: Cryosections of frozen livers stained with Oil red O. H: Tissue TAG content in indicated organs (n = 3–9). *P < 0.05. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 7.

Increased thermogenesis and energy expenditure in aP2-desnutrin mice. A: Body temperatures of 20-week-old male mice. Temperatures were measured beginning at 10 am following a 17-h overnight fast and before resumption of feeding and were monitored for 24 h (n = 4–6). Inset: Average body temperature (°C) over the day. B: Oxygen consumption rate (Vo₂) measured through indirect calorimetry and the average Vo₂ over 24 h (inset) (n = 4). *P < 0.05. FFM, fat-free mass.

FIG. 8.

Gene expression in WAT, BAT, liver, and skeletal muscle and increased FA oxidation in adipocytes. Relative mRNA levels were measured by RT-qPCR for the indicated genes from WAT (A), BAT (B), liver (C), and skeletal muscle (D) (n = 5–6). E: Oxidation of [¹⁴C]palmitate to ¹⁴CO₂ by adipocytes isolated from aP2-desnutrin mice or wild-type (WT) controls. WT, light gray bars; aP2-desnutrin, black bars. *P < 0.05, **P < 0.01, ***P < 0.001. AOx, α-oxidation.