Acute leptin deficiency, leptin resistance, and the physiologic response to leptin withdrawal

Abstract

Food restriction and weight loss result in reduced plasma leptin, which is associated with a pleiotropic biologic response. However, because weight loss itself is also associated with changes in numerous other humoral and metabolic signals, it can be difficult to determine the precise features of the biologic response to acute leptin deficiency. To study this response in the absence of changes in nutritional state, we have developed a protocol that allows such analysis in normal, non-food-restricted animals. Wild-type mice are treated with high-dose leptin until fat mass is depleted and, as a consequence, endogenous leptin production is reduced. At this point, exogenous leptin is abruptly withdrawn, thus inducing a state of leptin deficiency in otherwise normal mice. Leptin deficiency is sustained by feeding the animals only as much as they consumed voluntarily before leptin withdrawal. The biologic response to leptin deficiency induced in this manner includes altered neuropeptide levels, decreased energy expenditure, and impaired reproductive and immune function. Replacement of leptin at physiological concentrations after withdrawal of high-dosage leptin blunts, but does not completely block, the hyperphagia and weight regain caused by acute leptin deficiency, nor does it correct the resulting reproductive and immune dysfunction. This suggests that high-dosage leptin treatment induces a state of partial leptin resistance. In aggregate, these studies establish the role of acute hypoleptinemia in regulating energy balance, the immune system, and reproductive function, and further suggest that high-dosage leptin treatment can induce a state of acquired leptin resistance.

Fig. 1.

Food intake and body weight in response to leptin treatment and subsequent withdrawal. (A) Daily food intake in leptin-FF mice (•), leptin-NC mice (▴), and PBS control mice (▪). (B) Body mass of leptin-FF mice, leptin-NC mice, and PBS control mice. Leptin treatment begins on day 0 and withdrawal begins on day 8. [n = 50 per group and decreases by 6–10 per day (days 1–4 and 7), beginning on day 8 as animals were killed for analysis.]

Fig. 2.

Effect of leptin withdrawal on 24-h energy expenditure. Energy expenditure and locomotor activity was monitored on days 6–8 in leptin-treated mice (Lep), days 1–7 after leptin withdrawal in leptin-FF (FF1–7) and leptin-NC (NC) mice, days 1–3 after leptin withdrawal in PBS controls, and for 72 consecutive hours in ob/ob mice (n = 4 per group for all groups). Because the PBS control mice, ob/ob mice, and leptin-NC mice showed little day-to-day variability in energy expenditure and activity, all data were averaged and reported as a single value. The data for leptin-FF mice vary over time and are thus reported at 24-h intervals. (A) Energy expenditure. (B) Locomotor activity (*, P < 0.01 vs. PBS; †, P < 0.05 vs. FF at every time point).

Fig. 3.

Quantitative real-time PCR analysis of neuropeptide expression in the hypothalamus. Neuropeptide RNA expression levels in PBS control mice, leptin-FF mice, and leptin-NC mice were analyzed at the end of the dark cycle on day 8 of leptin treatment (L8) and days 1, 2, 3, 4, and 7 after leptin withdrawal (W1–W7, n = 6 per group). Data for TaqMan analysis are reported in relative fluorometric units (×10) normalized to cyclophilin expression. *, P < 0.5 vs. PBS, FF; **, P < 0.005 vs. PBS.

Fig. 4.

Effect of leptin withdrawal on the immune system. (A) Numbers of thymocytes as measured in PBS and leptin-NC mice on day 7 after leptin withdrawal, leptin-FF mice on day 2 after leptin withdrawal, and age-matched ob/ob mice. *, P < 0.05 vs. PBS; **, P < 0.01 vs. PBS; †, P < 0.05 vs. FF; ‡, P < 0.01 vs. FF; #, P < 0.05 vs. ob/ob. (B) Analysis of thymic apoptosis. *, P < 0.05 vs. PBS; **, P < 0.001 vs. PBS; †, P < 0.001 vs. FF; #, P < 0.001 vs. ob/ob. (n = 6 per group for both experiments.)

Fig. 5.

Leptin replacement in adipose-depleted mice. (A) After 8 days of leptin treatment, high-dosage leptin was withdrawn and replaced with PBS (0 ng/h) or increasing amounts of leptin as indicated (ng/h). Mice were then allowed to feed freely for 7 days (R1–R7) (n = 6 per group). (B) Daily body weight of mice receiving leptin replacement.