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. 2016 Jun 24;5(8):716-724.
doi: 10.1016/j.molmet.2016.06.009. eCollection 2016 Aug.

The role of autonomic efferents and uncoupling protein 1 in the glucose-lowering effect of leptin therapy

Heather C Denroche  1 , Michelle M Kwon  1 , Maria M Glavas  1 , Eva Tudurí  1 , Marion Philippe  1 , Whitney L Quong  1 , Timothy J Kieffer  2
Affiliations

The role of autonomic efferents and uncoupling protein 1 in the glucose-lowering effect of leptin therapy

Heather C Denroche et al. Mol Metab. .

Abstract

Objective: Leptin reverses hyperglycemia in rodent models of type 1 diabetes (T1D). Direct application of leptin to the brain can lower blood glucose in diabetic rodents, and can activate autonomic efferents and non-shivering thermogenesis in brown adipose tissue (BAT). We investigated whether leptin reverses hyperglycemia through a mechanism that requires autonomic innervation, or uncoupling protein 1 (UCP1)-mediated thermogenesis.

Methods: To examine the role of parasympathetic and sympathetic efferents in the glucose-lowering action of leptin, mice with a subdiaphragmatic vagotomy or 6-hydroxydopamine induced chemical sympathectomy were injected with streptozotocin (STZ) to induce hyperglycemia, and subsequently leptin treated. To test whether the glucose-lowering action of leptin requires activation of UCP1-mediated thermogenesis in BAT, we administered leptin in STZ-diabetic Ucp1 knockout (Ucp1 (-/-)) mice and wildtype controls.

Results: Leptin ameliorated STZ-induced hyperglycemia in both intact and vagotomised mice. Similarly, mice with a partial chemical sympathectomy did not have an attenuated response to leptin-mediated glucose lowering relative to sham controls, and showed intact leptin-induced Ucp1 expression in BAT. Although leptin activated BAT thermogenesis in STZ-diabetic mice, the anti-diabetic effect of leptin was not blunted in Ucp1 (-/-) mice.

Conclusions: These results suggest that leptin lowers blood glucose in insulin-deficient diabetes through a manner that does not require parasympathetic or sympathetic innervation, and thus imply that leptin lowers blood glucose through an alternative CNS-mediated mechanism or redundant target tissues. Furthermore, we conclude that the glucose lowering action of leptin is independent of UCP1-dependent thermogenesis.

Keywords: 6OHDA, 6-hydroxydopamine; ANS, autonomic nervous system; BAT, brown adipose tissue; Brown adipose tissue; CCK, cholecystokinin; CNS, central nervous system; Glucose; STZ, streptozotocin; Streptozotocin; Sympathectomy; T1D, type 1 diabetes; TH, tyrosine hydroxylase; Type 1 diabetes; UCP1, uncoupling protein 1; Vagotomy; iBAT, interscapular BAT.

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Figures

Figure 1
Figure 1
Subdiaphragmatic vagotomy does not attenuate leptin action in STZ-diabetic mice. C57Bl/6J male mice received vagotomy or sham operations at 6 weeks of age, and were injected with STZ to induce diabetes at 14 weeks of age, 7 days prior to pump implantation. On day 0, osmotic pumps were implanted delivering either 10 μg/day leptin or vehicle for a calculated delivery lasting 17.7 days. Four-hour fasted parameters in sham-vehicle (open blue symbols, n = 6), sham-leptin (filled blue symbols, n = 7), and vagotomy-leptin (filled red symbols, n = 4) mice. Blood glucose (A) and body weight (B) were tracked throughout the study. Statistical analyses were performed by repeated measures two-way ANOVA with a Tukey post-hoc test. Plasma leptin (C) and insulin (D) were measured 3 weeks prior to STZ administration, and on day 13 (during leptin therapy), and day 29 (after leptin therapy had ceased). Limits of detection are shown by the broken horizontal line, and in groups where some samples were undetectable, the numbers above indicate the proportion of samples which had detectable values. Statistical analyses were performed by two-way ANOVA with a Tukey post-hoc test. (E) Empty stomach weight was measured following sacrifice on day 32 and analyzed by one-way ANOVA with a Tukey post-hoc test, n ≥ 4. (F) Food intake 1 h following cholecystokinin (CCK) or vehicle injection was measured on day −15 prior to pump implantation, n ≥ 6 per group (sham in blue bars, vagotomy in red bars), and analyzed by two-way ANOVA with a Bonferroni post-hoc test. Data are presented as mean ± SEM. *P < 0.05 vagotomy-leptin vs sham-leptin mice; †P < 0.05 sham-leptin vs sham-vehicle mice; ‡P < 0.05 vagotomy-leptin vs sham-vehicle mice.
Figure 2
Figure 2
Injection of 6OHDA to chemically sympathectomised mice does not attenuate therapeutic leptin action in STZ-diabetes. Sympathectomy was induced in male C57Bl/6J mice by injection of 6OHDA at 12 weeks of age. Sham controls received buffer only injections instead of 6OHDA. Mice were injected with STZ to induce diabetes 14 days prior to implantation of osmotic pumps delivering 20 μg/day leptin or vehicle on day 0, for a calculated delivery period of 9.2 days. Four-hour fasted parameters in sham-vehicle (open blue circles/bars, n = 5), sham-leptin (filled blue circles/bars, n = 5), and 6OHDA-leptin (filled red squares/bars, n = 5) mice. Blood glucose (A) and body weight (B) were tracked over the course of the study, and statistical analyses were performed by repeated measures two-way ANOVA with a Tukey post-hoc test. Plasma leptin (C) and insulin (D) levels were measured on indicated days relative to pump implantation. Statistical analyses for leptin were performed by two way ANOVA with a Tukey post-hoc test. Statistical analyses for insulin were performed by one way ANOVA with a Tukey post-hoc test on day −8 values only. Limits of detection are shown by the broken horizontal line, and in groups where some samples were undetectable, the numbers above indicate the proportion of samples which had detectable values. Brown adipose tissue was harvested on day 19 at the end of the study. Representative images of tyrosine hydroxylase (TH) immunofluorescence are shown in (E), scale bar is 50 μm. Quantification of TH immunoreactive area was expressed as percent total section area (F) and relative to total cell number (G). (H) BAT Ucp1 transcript was measured by RT-qPCR relative to sham-vehicle controls. Data are presented as mean ± SEM. †P < 0.05 sham-leptin vs sham-vehicle mice; ‡P < 0.05 6OHDA-leptin vs sham-vehicle mice.
Figure 3
Figure 3
Leptin-induced glucose lowering is independent of UCP1-dependent BAT thermogenesis. Interscapular brown adipose tissue (iBAT) temperature (A), and iBAT mass (B, day 6 of treatment) were measured in STZ-diabetic C57Bl/6J male mice treated with 20 μg/day leptin (STZ-leptin, red) or vehicle (STZ-vehicle, blue) and non-diabetic controls (black), n = 4–6. *P < 0.05 STZ-leptin vs STZ-vehicle. †P < 0.05 STZ-vehicle vs non-diabetic. ‡P < 0.05 STZ-leptin vs non-diabetic. Blood glucose (C) and body weight (D) were measured following a 4-hour fast on the indicated days in Ucp1−/− and Ucp1+/+ mice injected with STZ and treated with 20 μg/day leptin (Ucp1−/−leptin, filled red squares; Ucp1+/+-leptin, filled blue circles) or vehicle (Ucp1−/−-vehicle, open red squares, Ucp1+/+-vehicle, open blue circles) for 8 days, n = 5–6. *P < 0.05 Ucp1+/+-leptin vs Ucp1−/−-leptin; ‡P < 0.05 leptin vs vehicle treated Ucp1−/− mice. †P < 0.05 leptin vs vehicle treated Ucp1+/+ mice.
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