Research ArticlePHYSIOLOGY

Beneficial metabolic role of β-arrestin-1 expressed by AgRP neurons

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Science Advances  03 Jun 2020:
Vol. 6, no. 23, eaaz1341
DOI: 10.1126/sciadv.aaz1341
  • Fig. 1 HFD AgRP-barr1-KO mice show impairments in glucose homeostasis.

    (A) Representative immunofluorescence images showing Cre activity in AgRP neurons of AgRP-Cre-Z/EG-barr1 f/f mice. In the left panel, only Cre-expressing neurons display GFP fluorescence. In the center panel, AgRP neurons were identified with an anti-AgRP antibody. (B) Body weights of AgRP-barr1-KO and control mice maintained on an HFD (HFD feeding was initiated when mice were 6 weeks old). (C) Fat and lean mass of HFD AgRP-barr1-KO and control mice (age, 20 weeks; 14 weeks on HFD). (D) Food intake (cumulative over 3 days) of HFD AgRP-barr1-KO and control mice (age, 20 weeks; 13 weeks on HFD). (E) Glucose tolerance test (GTT; 1 g glucose/kg i.p.) carried out with HFD AgRP-barr1-KO and control mice (age, 14 weeks; 8 weeks of HFD). (F) Insulin tolerance test (ITT; 0.75 U/kg i.p.) performed with HFD AgRP-barr1-KO and control mice (age, 15 weeks; 9 weeks on HFD). (G and H) Fasting and fed blood glucose (G) and plasma insulin (H) levels (age, 14 to 16 weeks; 8 to 10 weeks on HFD). (I and J) Plasma FFA (I) and resistin (J) levels (age, 14 to 16 weeks; 8 to 10 weeks on HFD). Mice had free access to food. Male mice were used for all studies. Data are given as means ± SEM (n = 5 to 9 per group). *P < 0.05; **P < 0.01 [two-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test (E and F) and two-tailed Student’s t test (G to J)].

  • Fig. 2 HFD AgRP-barr1-KO mice exhibit increased HGP and impaired hepatic insulin signaling.

    All studies were carried out with male HFD AgRP-barr1-KO and control littermates. (A to D) Euglycemic-hyperinsulinemic clamp studies. (A) Blood glucose levels. (B) GIR. AUC, area under the curve. (C and D) HGP under basal (C) and steady-state conditions (D). (E to I) Tissue glucose uptake by skeletal muscle and adipose tissues after intraperitoneal insulin (0.75 U/kg) injection. (J) Liver weights (HFD for 12 weeks; age, 20 weeks). (K) Liver triglyceride levels (HFD for 12 weeks; age, 20 weeks). (L) Histological staining of liver sections (HFD for 12 weeks; age, 20 weeks). (M) Hepatic expression levels of genes involved in gluconeogenesis and inflammatory processes (HFD for 12 weeks; age, 20 weeks). (N) Hepatic insulin signaling studied by Western blotting analysis after intravenous insulin (5 U per mouse) (HFD for 12 weeks; age, 20 weeks). (O) Quantification of the Western blotting data shown in (N). The data shown in (K) to (O) were obtained with mice that had been fasted for 4 hours. Data are given as means ± SEM (n = 5 to 9 male mice per group). *P < 0.05; **P < 0.01; ***P < 0.001 (Student’s t test). AU, arbitrary units.

  • Fig. 3 Disruption of hepatic vagal innervation prevents the metabolic deficits caused by HFD feeding of AgRP-barr1-KO mice.

    All experiments were carried out with male mice that had been maintained on an HFD for at least 8 weeks. Mice in which the vagal innervation of the liver had been disrupted are referred to as AgRP-barr1-KO-Vgtm and Control-Vgtm mice, respectively. Mice that had been subjected to sham surgery are referred to as AgRP-barr1-KO-Sham and Control-Sham mice, respectively. (A) Body weights of AgRP-barr1-KO-Sham and Control-Sham mice (age, 18 weeks; 8 weeks on HFD). (B) Body weights of AgRP-barr1-KO-Vgtm and Control-Vgtm mice (age, 18 weeks; 8 weeks on HFD). (C and D) Intraperitoneal glucose tolerance test (IGTT; 1 g glucose/kg i.p.) carried out with mice subjected to sham surgery (C) or vagotomy (D) (age, 18 weeks; 8 weeks on HFD). (E and F) Insulin tolerance test (1 U/kg i.p.) performed with mice subjected to sham surgery (E) or vagotomy (F) (age, 19 weeks; 9 weeks on HFD). (G and H) Pyruvate tolerance test (PTT; 2g/kg i.p.) carried out with mice subjected to sham surgery (G) or vagotomy (H) (age, 20 weeks; 10 weeks on HFD). (I and J) Fasting and fed blood glucose levels of sham-operated (I) or vagotomized (J) mice (age, 18 to 19 weeks; 8 to 9 weeks on HFD). (K and L) Fasting and fed plasma insulin levels of sham-operated (K) or vagotomized (L) mice (age, 18 to 19 weeks; 8 to 9 weeks on HFD). Data are given as means ± SEM (n = 6 to 9 male mice per group). *P < 0.05; **P < 0.01 [two-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test (C, E, and G) and two-tailed Student’s t test (I and K)].

  • Fig. 4 Barr1 deficiency impairs insulin action on AgRP neurons.

    (A to M) Electrophysiological studies with AgRP neurons from 8- to 12-week-old male mice maintained on standard chow. (A to D) Bright-field (A) or fluorescein isothiocyanate (GFP) illumination (B) of an AgRP neuron from AgRP-Z/EG reporter mice. (C) Complete dialysis of Alexa Fluor 350 from the intracellular pipette. (D) Merged image of a targeted AgRP neuron (arrow). Scale bar, 50 μm. (E to H) Current-clamp recording depicting insulin-induced hyperpolarization and reduced action potential (AP) firing frequency of control AgRP neurons (E and F) and lack of these responses in barr1-deficient AgRP neurons (G and H). ACSF, artificial cerebrospinal fluid. (I and J) The PI3 kinase activator 740Y-P elicits insulin-like responses in barr1-deficient AgRP neurons. (K and L) Reexpression of barr1 in AgRP neurons of adult AgRP-barr1-KO mice restores normal insulin responses. (M) Summary of the effects of insulin and 740Y-P on the membrane potential of AgRP neurons. (N) Central administration of insulin (200 μU into the lateral ventricle) fails to inhibit pyruvate (2 g/kg i.p.)–induced hyperglycemia in AgRP-barr1-KO mice (HFD for 12 weeks; 20-week-old males; n = 4 or 5). Data are given as means ± SEM. *P ≤ 0.05; **P ≤ 0.01 [two-tailed Student’s t test (E to L) and one-tailed Student’s t test (N)]. ns, no significant difference.

  • Fig. 5 Overexpression of barr1 in AgRP neurons improves whole-body glucose tolerance and insulin sensitivity in lean and obese mice.

    Experiments were carried out with male mice maintained on regular chow (RC) (A to I) or with male mice that had consumed an HFD for at least 7 weeks (F to M). (A) Representative immunofluorescence images showing the expression of mCerulean (left), a marker for barr1 overexpression, and mCherry (control mice; right) in the arcuate nucleus of AgRP-barr1-OE and AgRP-mCherry mice, respectively. 3rd v, third ventricle. (B) Glucose tolerance test (1 g glucose/kg i.p.) carried out with regular chow AgRP-barr1-OE and control mice (age, 12 weeks). (C) Insulin tolerance test (0.75 U/kg i.p.) (11-week-old regular chow mice). (D) Fasting and fed blood glucose levels (regular chow mice, 11 to 12 weeks old). (E) Food intake of regular chow AgRP-barr1-OE and control mice after a 24-hour fast (refeeding; mouse age, 13 weeks). (F) Body weight gain of AgRP-barr1-OE and control mice consuming an HFD (mouse age, 13 to 17 weeks). (G) Fat and lean mass composition of HFD AgRP-barr1-OE and control mice (mouse age, 22 weeks; 9 weeks on HFD). (H) Glucose tolerance test (1 g glucose/kg i.p.) carried out with HFD AgRP-barr1-OE and control mice (mouse age, 21 weeks; 8 weeks on HFD). (I) Insulin tolerance test (0.75 U/kg i.p.) (mouse age, 20 weeks; 7 weeks on HFD). (J to M) Fasting and fed blood glucose (J) and plasma insulin (K), FFA (L), and leptin (M) levels (mouse age, 20 to 22 weeks; 7 to 9 weeks on HFD). Data are given as means ± SEM (n = 6 to 9 mice per group). *P < 0.05; **P < 0.01 [two-way ANOVA followed by Bonferroni’s post hoc test (B, C, H, and I) and two-tailed Student’s t test (D, G, and J to M)].

  • Fig. 6 Overexpression of barr1 in AgRP neurons protects against diet-induced liver steatosis.

    AgRP-barr1-OE and control mice were maintained on an HFD for at least 9 weeks. (A and B) Liver weights (A) and liver triglyceride levels (B) of HFD AgRP-barr1-OE and control mice. (C) Histological staining of liver sections from HFD AgRP-barr1-OE and control mice. (D) Hepatic expression levels of genes involved in gluconeogenesis and proinflammatory genes in HFD AgRP-barr1-OE and control mice. (E) Liver glycogen levels of HFD AgRP-barr1-OE and control mice. (F) Western blotting analysis of hepatic insulin signaling after intravenous insulin treatment (5 U per mouse) of HFD AgRP-barr1-OE and control mice. (G) Quantification of the Western blotting data shown in (F). Data are given as means ± SEM (n = 4 to 6 male mice per group; mouse age, 24 to 25 weeks, 11 to 12 weeks on HFD). *P < 0.05 (two-tailed Student’s t test).

Supplementary Materials

  • Supplementary Materials

    Beneficial metabolic role of β-arrestin-1 expressed by AgRP neurons

    Sai P. Pydi, Zhenzhong Cui, Zhenyan He, Luiz F. Barella, Jonathan Pham, Yinghong Cui, Douglas J. Oberlin, Hale Ergin Egritag, Nikhil Urs, Oksana Gavrilova, Gary J. Schwartz, Christoph Buettner, Kevin W. Williams, Jürgen Wess

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