Leptin resistance is a secondary consequence of the obesity in ciliopathy mutant mice

Abstract

Although primary cilia are well established as important sensory and signaling structures, their function in most tissues remains unknown. Obesity is a feature associated with some syndromes of cilia dysfunction, such as Bardet-Biedl syndrome (BBS) and Alström syndrome, as well as in several cilia mutant mouse models. Recent data indicate that obesity in BBS mutant mice is due to defects in leptin receptor trafficking and leptin resistance. Furthermore, induction of cilia loss in leptin-responsive proopiomelanocortin neurons results in obesity, implicating cilia on hypothalamic neurons in regulating feeding behavior. Here, we directly test the importance of the cilium as a mediator of the leptin response. In contrast to the current dogma, a longitudinal study of conditional Ift88 cilia mutant mice under different states of adiposity indicates that leptin resistance is present only when mutants are obese. Our studies show that caloric restriction leads to an altered anticipatory feeding behavior that temporarily abrogates the anorectic actions of leptin despite normalized circulating leptin levels. Interestingly, preobese Bbs4 mutant mice responded to the anorectic effects of leptin and did not display other phenotypes associated with defective leptin signaling. Furthermore, thermoregulation and activity measurements in cilia mutant mice are inconsistent with phenotypes previously observed in leptin deficient ob/ob mice. Collectively, these data indicate that cilia are not directly involved in leptin responses and that a defect in the leptin signaling axis is not the initiating event leading to hyperphagia and obesity associated with cilia dysfunction.

Fig. 1.

Paradigm of leptin sensitivity testing, body composition, and serum leptin in Ift88 conditional mutant mice. (A) Longitudinal paradigm showing body weights of Ift88^Δ/Δ and Ift88^flox/flox mice after Cre induction (C), lean time point I (I), obese time point II (II), and lean time point III (III). (B) Body weight, fat mass, and lean mass at each time point. (C) Serum leptin levels at each time point. All points and bars are means ± SEMs. *P < 0.05, **P < 0.01; Student t test. Numbers within parentheses or bars indicate number of animals.

Fig. 2.

Feeding behavior and arcuate nucleus pSTAT3 and cFos staining in Ift88 conditional mice after leptin injection. (A–F) Feeding data and cumulative food intake (CFI) after i.p. injection of leptin in Ift88^flox/flox (A–C and Insets) and Ift88^Δ/Δ (D–F and Insets) mice at time points I, II, and III. Insets show total CFI. (G–I) Representative images showing pSTAT3 staining in the arcuate nucleus after i.p. leptin or vehicle injection at time points I, II, and III. (Scale bar, 90 µm.) Dotted lines indicate approximate border of the arcuate nucleus. (J and K) Quantification of pSTAT3 and cFos staining in the arcuate nucleus after leptin or vehicle injection at time point I. *P < 0.05, **P < 0.01; Student t test. All points and bars in graphs represent means ± SEMs. Numbers within parentheses or bars indicate number of animals.

Fig. 3.

Food anticipatory activity (FAA) feeding behavior in ad libitum–fed Ift88 mutant mice that were calorie restricted. (A) Graphic output of real-time food intake. Days 1–3 show Ift88^Δ/Δ FAA during calorie restriction, blue line (calorie restricted). Steep initial slope indicates that Ift88^Δ/Δ consumed the majority of their food immediately. Ad libitum feeding began on day 4 (AL). FAA deteriorated after 9 d (ad libitum food but FAA behavior) and normal feeding resumed (FAA lost). Vehicle and leptin injection during FAA are indicated (VE and LE). Time point III vehicle and leptin injection nights are indicated (V3 and L3). Gray shading indicates dark cycle (night). (B) Percentage of food consumed between 5:00 and 8:00 PM (corresponding to the period just after food addition during the caloric restricted paradigm), indicating the persistence of FAA. Ift88^Δ/Δ mice ate the majority of their ad libitum food during the first 3 h of night, compared with Ift88^flox/flox mice that gradually ate approximately a quarter of their total intake during this same period. *P < 0.05, Student t test. (C and Inset) Feeding data and CFI after leptin injection in Ift88^Δ/Δ during FAA. P = 0.07, P = 0.54; Student t test. All points and bars in graphs represent means ± SEMs. (D) pSTAT3 staining in the arcuate nucleus after IP leptin or vehicle injection during FAA. (Scale bar, 90 µm.) Dotted lines indicate approximate border of arcuate nucleus. Numbers within parentheses or bars indicate number of animals.

Fig. 4.

Thermoregulation and locomotor activity between Ift88 and ob/ob mice. (A) Body temperature of Ift88^flox/flox, Ift88^Δ/Δ, and ob/ob mice measured for baseline, after 30, 120, and 240 min of exposure to 4 °C, and at 270, 390, and 480 min during room temperature recovery. The ob/ob mice were pulled from the experiment due to an inability to thermoregulate. *P < 0.05, one-way ANOVA followed by post hoc Tukey's HSD test. (B) Locomotor activity at time point I comparing Ift88^flox/flox, Ift88^Δ/Δ, and ob/ob mice. *P < 0.05, **P < 0.01; one-way ANOVA followed by post hoc Tukey's HSD test. Numbers within parentheses or bars indicate number of animals.

Fig. 5.

Serum leptin, thermoregulation and leptin sensitivity analysis in Bbs4^−/− mice. (A) Serum leptin levels of Bbs4^+/+ and preobese Bbs4^−/− mice. (B) Quantification of pSTAT3 staining in the arcuate nucleus of Bbs4^+/+ and Bbs4^−/− mice after IP leptin and vehicle. *P < 0.05, Student t test. (C) pSTAT3 staining in the arcuate nucleus after i.p. injection of leptin or vehicle in Bbs4^+/+ and Bbs4^−/− preobese mice. (Scale bar, 90 µm.) Dotted lines indicate approximate border of arcuate nucleus. (D) Feeding data after leptin and vehicle injection in Bbs4^−/− mutant mice. (Inset) Cumulative food intake (CFI) for Bbs4^+/+ and Bbs4^−/− mice after leptin or vehicle i.p. injection. *P < 0.05, Student t test. (E) Body temperature of Bbs4^+/+ and Bbs4^−/− mice measured for baseline, and then at 30, 120, and 240 min of exposure to 4 °C, and at 270, 390, and 480 min during room temperature recovery. All points and bars represent means ± SEMs. Numbers within parentheses or bars indicate number of animals.