Research ArticleNEUROPHYSIOLOGY

Insulin signaling in AgRP neurons regulates meal size to limit glucose excursions and insulin resistance

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Science Advances  26 Feb 2021:
Vol. 7, no. 9, eabf4100
DOI: 10.1126/sciadv.abf4100
  • Fig. 1 TCPTP in AgRP but not POMC neurons regulates feeding behavior.

    Ten-week-old Ptpn2fl/fl and AgRP-TC male mice were fed a chow diet, and (A) body weights, (B and C) food intake, and (D to H) feeding behavior were determined. Ten-week-old Ptpn2fl/fl and POMC-TC male mice were fed a chow diet, and (I) food intake and (J) feeding behavior were determined. Representative and quantified results are shown (means ± SEM) for the indicated number of mice; significance was determined using (E, G, and H) Student’s t test. *P < 0.05, **P < 0.01.

  • Fig. 2 IR heterozygosity corrects the altered feeding behavior in AgRP-TC mice.

    Ten-week-old Ptpn2fl/fl, AgRP-TC, and AgRP-TC-IRfl/+ male mice were fed a chow diet, and (A) body weights, (B and C) food intake, and (D to H) feeding behavior were determined. Representative and quantified results are shown (means ± SEM) for the indicated number of mice; significance was determined using one-way ANOVA. *P < 0.05, **P < 0.01.

  • Fig. 3 IR signaling in AgRP neurons regulates feeding behavior.

    Ten-week-old C57BL/6 mice fed a chow diet were administered either 0.5 or 0.75 mU/g of insulin immediately before the beginning of the dark cycle, and (A and B) food intake and (C to G) feeding behavior were determined. Ten-week-old Ptpn2fl/fl, AgRP-TC, and AgRP-IRfl/fl male mice were fed a chow diet, and (H) body weights, (I and J) food intake, and (K and L) feeding behavior were determined. Representative and quantified results are shown (means ± SEM) for the indicated number of mice; significance was determined using one-way ANOVA; *P < 0.05, **P < 0.01, ***P < 0.001. In (L), #P < 0.05 represents Ptpn2fl/fl versus AgRP-IRfl/fl using Student’s t test.

  • Fig. 4 Leptin signaling regulates caloric intake but not feeding behavior.

    Eight- to 10-week-old Ptpn1fl/fl, AgRP-1B, and AgRP-TC male mice were administered vehicle or insulin, and brains were processed for immunohistochemistry monitoring for (A) insulin-induced p-AKT. Ten-week-old Ptpn1fl/fl and AgRP-1B male mice were fed a chow diet, and (B) body weights, (C) body composition (EchoMRI), (D and E) food intake, and (F to J) feeding behavior were determined. Nine-week-old Stat3fl/fl male mice were bilaterally injected with AAV-Cre-GFP into the ARC. Fourteen days after rAVV injection, Stat3fl/fl mice versus 10-week-old Ptpn1fl/fl and AgRP-1B male mice were administered vehicle or leptin, and brains were processed for immunohistochemistry monitoring for (K) leptin-induced p-STAT3. Nine-week-old Stat3fl/fl male mice were bilaterally injected with AAV-GFP or AAV-Cre-GFP into the ARC. Fourteen days after rAVV injection, Stat3fl/fl mice were fed a chow diet, and (L) body weights, (M) body composition (EchoMRI), (N and O) food intake, and (P to T) feeding behavior were determined. Representative and quantified results are shown (means ± SEM) for the indicated number of mice; significance was determined using (A and K) two-way ANOVA and (E, G, H, O, Q, and R) Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar, 100 μm.

  • Fig. 5 IR signaling in AgRP neurons does not regulate stereotypic feeding behaviors.

    (A) Ten- to 14-week-old male Ptpn2fl/fl, AgRP-TC, and AgRP-TC-IRfl/+ male mice were subjected to elevated plus maze test to assess anxiety-like behavior in both fed and fasted states, and (B) the number of entries and (C) the time spent in different parts of the maze were determined. To assess effects on anxiety-related food-seeking behaviors, (D) 10- to 14-week-old male Ptpn2fl/fl, AgRP-TC, and AgRP-TC-IRfl/+ male mice were subjected to baited open-field tests in both fed and fasted states, and (E) time spent in the different parts of the open field, (F) the number of entries, and (G) food intake were determined. To measure repetitive behaviors, (H) 10-week-old male Ptpn2fl/fl, AgRP-TC, and AgRP-TC-IRfl/+ mice were placed in a cage containing 24 marbles uniformly dispatched, and the (I) percentage of marbles buried within 30 min was determined. Representative and quantified results are shown (means ± SEM) for the indicated number of mice. PB, peanut butter.

  • Fig. 6 IR signaling in AgRP neurons does not regulate reward behaviors.

    To measure reward behaviors, mice were subjected to (A to C) a saccharin preference test, (D and E) a conditioned place preference test, or (F to H) a nose-poke operant conditioning paradigm. (A to C) Twelve-week-old male Ptpn2fl/fl, AgRP-TC, and AgRP-TC-IRfl/+ mice were subjected to saccharin preference tests, and fluid intake in either (B) fed or (C) fasted state was determined. (D and E) Ten- to 14-week-old male Ptpn2fl/fl, AgRP-TC, and AgRP-TC-IRfl/+ mice were associated with the side of the conditioning apparatus with either chow or peanut butter chip for 10 days. On day 10, food was removed, and mice were placed into the neutral region (white box). (D) Schematic of the conditioned place preference test, and (E) time spent in each quadrant of the apparatus was determined. (F to H) Twelve-week-old male Ptpn2fl/fl and AgRP-TC mice were subjected to a PR nose-poke operant conditioning paradigm. Mice were fed a chow diet during the FR and the first PR session and then were fed a high-fat diet (HFD) for 5 days before the second PR session. (F) Schematic of operant conditioning test and the motivation breakpoint (the ratio at which mice stop nose poking to receive a sucrose reward) in mice fed with (G) chow or (H) high-fat diet. Representative and quantified results are shown (means ± SEM) for the indicated number of mice.

  • Fig. 7 TCPTP deletion in AgRP neurons attenuates the consumption of high-fat diet.

    (A) Schematic of high-fat diet preference paradigm. Ten-week-old chow-fed Ptpn2fl/fl or AgRP-TC mice were exposed to both chow and high-fat diet for 5 days, and (B) feeding behavior and (C to E) the composition of food intake were determined. The dashed blue lines represent the opening of the food hopper gates to allow access to high-fat diet. Representative and quantified results are shown (means ± SEM) for the indicated number of mice using (D and E) two-way ANOVA. ***P < 0.001.

  • Fig. 8 Insulin signaling in AgRP neurons attenuates the acute development of insulin resistance.

    Eight-week-old chow-fed Ptpn2fl/fl, AgRP-TC, and AgRP-TC-IRfl/+ male mice were fed a chow diet, and (A) blood glucose and (B) plasma insulin levels were determined at 6:00 and 11:00 p.m. (C) Schematic of high-fat diet preference paradigm. Ten-week-old chow-fed Ptpn2fl/fl or AgRP-TC mice were given access to both chow and high-fat diet for 5 days, and (D) blood glucose and (E) plasma insulin levels were measured (at 2:00 p.m.), and (F) HOMA-IRs were calculated on days −2 (chow only) and 4 (5 days of exposure to chow and high-fat diet). Representative and quantified results are shown (means ± SEM) for the indicated number of mice; significance was determined using (A, B, and D to F) two-way ANOVA; *P < 0.05, **P < 0.01, and ***P < 0.001. In (A) and (B), #P < 0.05, ##P < 0.01, and ###P < 0.001 represent significance determined using Student’s t test.

  • Fig. 9 Insulin signaling in AgRP neurons attenuates the consumption of sucrose- but not saccharin-sweetened water.

    (A) Schematic of sucrose preference test. Eight-week-old chow-fed Ptpn2fl/fl or AgRP-TC mice were given access to both water and 10% sucrose solution, and (B) fluid intake and (C) drinking behavior were determined. (D) Schematic of saccharin preference test. Eight-week-old chow-fed Ptpn2fl/fl or AgRP-TC mice were given access to both water and 0.1% saccharin solution, and (E) fluid intake and (F) drinking behavior were determined. Representative and quantified results are shown (means ± SEM) for the indicated number of mice; significance was determined using (C) Student’s t test. *P < 0.05.

  • Fig. 10 IR signaling in AgRP neurons overrides compensatory feeding responses to restricted feeding and limits glucose excursions.

    Ten-week-old Ptpn2fl/fl, AgRP-TC, and AgRP-IRfl/+ male mice were subjected to a restricted feeding paradigm, whereby mice were granted access to food during only the first 4 hours of the diurnal feeding cycle (7:00 p.m. until 11:00 a.m.). (A) Schematic of restricted feeding paradigm. (B and D) Food intake and (E) feeding behavior were determined on days 1 and 3 of restricted feeding. (C) Blood glucose levels were determined at 6:00 and 11:00 p.m. on day 2 of restricted feeding. (F) Daily body weights of Ptpn2fl/fl and AgRP-TC male mice throughout the 4 days of restricted feeding. (G) Body composition (EchoMRI) following 4 days of restricted feeding. Representative and quantified results are shown (means ± SEM) for the indicated number of mice; significance was determined using (B and C) two-way ANOVA, (F) two-way ANOVA with repeated measures, and (G) one-way ANOVA; *P < 0.05, **P < 0.01, and ***P < 0.001. In (F) #P < 0.05 represents significance determined using Student’s t test.

Supplementary Materials

  • Supplementary Materials

    Insulin signaling in AgRP neurons regulates meal size to limit glucose excursions and insulin resistance

    Garron T. Dodd, Seung Jae Kim, Mathieu Méquinion, Chrysovalantou E. Xirouchaki, Jens C. Brüning, Zane B. Andrews, Tony Tiganis

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