Preclinical Assessment of Leptin Transport into the Cerebrospinal Fluid in Diet-Induced Obese Minipigs

Abstract

Objective: A minipig model was employed to explore the changes in endogenous leptin transport into the central nervous system and in hypothalamic sensitivity to exogenous leptin when individuals are placed on high-fat diet (HFD) compared with standard diet.

Methods: Serum and cerebrospinal fluid (CSF) leptin concentrations during 10 weeks of HFD versus standard diet and exogenous leptin-induced STAT3 phosphorylation in the hypothalamus of minipigs were assessed, and the hypothalamic leptin-sensitive cells were characterized by immunofluorescence.

Results: The efficiency of the passage of endogenous blood-borne leptin into the CSF (measured as the log [CSF:serum leptin ratio]) decreased over time in minipigs fed a HFD (β = -0.04 ± 0.005 per kilogram of weight gain in HFD; P < 0.0001), while it remained stable in minipigs fed a standard diet. However, the ability of peripherally administered leptin to activate its receptor in hypothalamic neurons was preserved in obese minipigs at 10 weeks of HFD.

Conclusions: Together, these data are consistent with the existence of an early-onset tranport deficiency for endogenous circulating leptin into the brain in individuals developing obesity, preceding the acquisition of hypothalamic leptin resistance. Although additional studies are required to identify the underlying mechanisms, our study paves the way for the development of new preclinical pharmacological models targeting the restoration of the shuttling of peripheral leptin into the central nervous system to manage obesity.

Figure 1

Diet‐induced obesity in minipigs. (A) Body weight curves in minipigs fed normal‐fat diet (NFD) and high‐fat diet (HFD), revealing diet‐induced obesity in minipigs. (B) Adiposity measurement by MRI in minipigs. Graphs represent the volume of each fat compartment. Values shown are means ± SEM. **P < 0.001, ***P < 0.0001, HFD vs. NFD. VAT, visceral adipose tissue; SAT, subcutaneous adipose tissue.

Figure 2

Longitudinal relationships of serum leptin, cerebrospinal fluid (CSF) leptin, CSF:serum leptin ratio, and body weight in minipigs fed normal‐fat diet (NFD) and high‐fat diet (HFD). (A‐C) Evolution of serum and CSF leptin and CSF:serum leptin ratios with time. Values are means and 95% CI. Bonferroni post hoc analysis, ***P < 0.0001, HFD vs. NFD. Evolution of (D) serum leptin, (E) CSF leptin, and (F) CSF:serum leptin ratio according to body weight in HFD minipigs. Values are means ± SEM at each week.

Figure 3

Peripheral leptin activates leptin receptor signaling in hypothalamic neurons of minipigs even after 10 weeks of high‐fat diet (HFD). (A,B) Distribution of pSTAT3 immunoreactivity (arrows) in coronal sections of the ARH in minipigs after subcutaneous administration of leptin (right images in panels A and B) or vehicle (left image in panel A) (n = 3 per group). Insets show higher magnification of the area delineated by a white rectangle in the main image. In panel B, the white (left panel) and green (right panel) signals show HuC/D‐ and β‐endorphin‐immunoreactive (IR) neurons, respectively, some of which express pSTAT3 (arrows). Empty arrows point to single‐labeled pSTAT3‐immunoreactive cells. Scale bars: 300 μm (100 μm in insets). Number of pSTAT3‐immunoreactive neurons in the (C) ARH, (D) VMH, and (E) DMH in minipigs fed normal‐fat diet (NFD) (n = 6) and HFD (n = 6) for 10 weeks and subjected to vehicle (n = 3 per group) or leptin treatment (n = 3 per group). *P < 0.05, ***P < 0.0001, vehicle vs. leptin. 3V, third ventricle; ME, median eminence; ARH, arcuate nucleus of the hypothalamus; VMH, ventromedial nucleus of the hypothalamus; DMH, dorsomedial nucleus of the hypothalamus. [Colour figure can be viewed at wileyonlinelibrary.com]