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. 2015 Apr;156(4):1283-91.
doi: 10.1210/en.2014-1909. Epub 2015 Jan 21.

Nedd4 haploinsufficient mice display moderate insulin resistance, enhanced lipolysis, and protection against high-fat diet-induced obesity

Jing Jing Li  1 , Robert J Ferry Jr, Shiyong Diao, Bingzhong Xue, Suleiman W Bahouth, Francesca-Fang Liao
Affiliations

Nedd4 haploinsufficient mice display moderate insulin resistance, enhanced lipolysis, and protection against high-fat diet-induced obesity

Jing Jing Li et al. Endocrinology. 2015 Apr.

Abstract

Neural precursor cell expressed developmentally down-regulated protein 4 (Nedd4) is the prototypical protein in the Nedd4 ubiquitin ligase (E3) family, which governs ubiquitin-dependent endocytosis and/or degradation of plasma membrane proteins. Loss of Nedd4 results in embryonic or neonatal lethality in mice and reduced insulin/IGF-1 signaling in embryonic fibroblasts. To delineate the roles of Nedd4 in vivo, we examined the phenotypes of heterozygous knockout mice using a high-fat diet-induced obesity (HFDIO) model. We observed that Nedd4+/- mice are moderately insulin resistant but paradoxically protected against HFDIO. After high-fat diet feeding, Nedd4+/- mice showed less body weight gain, less fat mass, and smaller adipocytes vs the wild type. Despite ameliorated HFDIO, Nedd4+/- mice did not manifest improvement in glucose tolerance vs the wild type in both genders. Nedd4+/- male, but not female, mice displayed significantly lower fasting blood glucose levels and serum insulin levels. Under obesogenic conditions, Nedd4+/- mice displayed elevated stimulated lipolytic activity, primarily through a β2-adrenergic receptor. Combined, these data support novel complex roles for Nedd4 in metabolic regulation involving altered insulin and β-adrenergic signaling pathways.

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Figures

Figure 1.
Figure 1.. Nedd4+/− mice partially resist HFDIO.
A and B, Body weight curves of male (A) and female (B) littermates fed ND or HFD ad libitum over a 30-week period starting at age 6 weeks (n = 8–11). Body weights were monitored weekly. C, Percentages of epididymal (Epi), inguinal sc (SubQ), or brown adipose tissue (BAT) depot weights to total body weight in wild-type (WT) or Nedd4+/− (Het) male mice at age 22 weeks (n = 5–7). D, Percentages of liver, gastrocnemius (Gas), or quadriceps (Qcp) weight to total body weight in WT or Het male mice at age 22 weeks (n = 5–7). E, Leptin mRNA levels in epididymal fat were measured by quantitative real-time PCR and normalized to gapdh mRNA levels (ND, n = 3; HFD, n = 5–6 with both genders). Data represent mean ± SEM. *, P < .05, **; P < .01; ***, P < .001.
Figure 2.
Figure 2.. Nedd4+/− mice on ND are insulin resistant but glucose tolerant.
A and B, GTT was carried out in wild-type (WT) and Nedd4+/− (Het) mice on ND. Mice fasted overnight were ip injected with glucose at 1.5 g/kg body weight. Blood glucose levels were measured at time 0 and then 30, 60, 90, and 120 minutes after glucose injection. Glucose clearance ability was similar between genotypes for males (A) or females (B) (n = 7–11). C and D, After a 4-hour fast, mice received ip insulin 0.75 U/kg body weight, and then blood glucose was measured at 0, 30, 60, and 90 minutes relative to insulin injection. Het mice were less responsive to insulin (vs WT littermate controls), based on glucose clearance rate in males (A) and females (B) (n = 8–11). E, Serum insulin levels at time 0, 30, and 60 minutes after glucose injection during GTT (n = 8; 4 males and 4 females). Data represent mean ± SEM. *, P < .05; **, P < .01; and ***, P < .001.
Figure 3.
Figure 3.. Nedd4+/− mice on ND have reduced insulin signaling in WAT, skeletal muscle, and liver.
A–C, ND-fed wild-type (WT) and Nedd4+/− (Het) male mice were fasted overnight and ip injected with saline or insulin 5 U/kg body weight. Insulin-stimulated phosphorylation of IRβ and AKT in WAT (A), skeletal muscle (B), and liver (C) were measured by Western blot analysis (n = 7). Representative Western blots and quantification data were shown. Data represent mean ± SEM. *, P < .05; **, P < .01.
Figure 4.
Figure 4.. Nedd4+/− male mice on HFD exhibit reduced fasting glucose and insulin levels.
A and B, GTT was carried out in wild-type (WT) and Nedd4+/− (Het) mice on HFD. After an overnight fast, mice received ip glucose 1.5 g/kg body weight, and then blood glucose levels were measured at time 0, 30, 60, 90, 120, and 180 minutes relative to glucose injection. Glucose clearance ability was similar between genotypes in males (A) or females (B) (n = 6–7). C and D, ITT was carried out in WT and Het male (A) and female (B) mice (n = 5–7). After a 4-hour fast, mice received ip insulin 0.75 U/kg body weight, and then blood glucose levels were measured at time 0, 30, 60, and 90 minutes relative to insulin injection. E and F, Serum insulin levels at time 0, 30, and 60 minutes after glucose injection during GTT in males (E) and females (F) (n = 6). G and H, After an overnight fast, blood glucose levels in male (G) and female (H) mice (n = 10–13). Data represent mean ± SEM. *, P < .05; ***, P < .001.
Figure 5.
Figure 5.. Nedd4+/− mice are protected against HFD-induced adipocyte hypertrophy.
A, Representative H&E staining of adipocytes. Scale bar, 100 μm. B, Average adipocyte size in epididymal WAT of littermate wild-type (WT) and Nedd4+/− (Het) mice on ND or HFD (ND, n = 3, two males and one female; HFD, n = 4, two males and two females). Area of adipocytes was measured within 300–400 cells per mouse using ImageJ software (National Institutes of Health). Data represent mean ± SEM. *, P < .05.
Figure 6.
Figure 6.. HFD-fed Nedd4+/− mice display enhanced isoproterenol-stimulated lipolysis in vivo and ex vivo.
A, Serum-free glycerol levels were measured in a cohort of 22-week-old wild-type (WT) and Nedd4+/− (Het) mice on ND or HFD at indicated time after ip isoproterenol, 10 mg/kg body weight (n = 5; three males and two females). B, Primary adipocytes purified from epididymal fat of WT and Het mice were incubated with or without 1 μM isoproterenol, xamoterol, or salmeterol as described in Materials and Methods. The β-antagonists ICI-118551 and CGP-20712A were preincubated for 10 minutes, and then glycerol levels in conditioned media were measured (n = 4; two males and two females). Data represent mean ± SEM. *, P < .05; **, P < .01; ***, P < .001.
Figure 7.
Figure 7.. Primary adipocytes from HFD-fed Nedd4+/− mice are associated with increased β2-AR protein concentrations.
A, Representative Western blots for β2-AR expression in total tissue lysates from epididymal WAT in wild-type (WT) and Nedd4+/− (Het) mice after 16 weeks of HFD feeding (n = 4; two males and two females). Quantification is shown in the right panel. B, Representative Western blots for β2-AR expression in crude membrane fraction from primary adipocytes purified from WT and Het male mice after 16 weeks of HFD feeding (n = 3). Data represent mean ± SEM. *, P < .05.
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