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TrpC5 Mediates Acute Leptin and Serotonin Effects via Pomc Neurons - PubMed

  • ️Sun Jan 01 2017

TrpC5 Mediates Acute Leptin and Serotonin Effects via Pomc Neurons

Yong Gao et al. Cell Rep. 2017.

Abstract

The molecular mechanisms underlying acute leptin and serotonin 2C receptor-induced hypophagia remain unclear. Here, we show that neuronal and pro-opiomelanocortin (Pomc)-specific loss of transient receptor potential cation 5 (TrpC5) subunits is sufficient to decrease energy expenditure and increase food intake resulting in elevated body weight. Deficiency of Trpc5 subunits in Pomc neurons is also sufficient to block the anorexigenic effects of leptin and serotonin 2C receptor (Ht2Cr) agonists. The loss of acute anorexigenic effects of these receptors is concomitant with a blunted electrophysiological response to both leptin and Ht2Cr agonists in arcuate Pomc neurons. We also demonstrate that the Ht2Cr agonist lorcaserin-induced improvements in glucose and insulin tolerance are blocked by TrpC5 deficiency in Pomc neurons. Together, our results link TrpC5 subunits in the brain with leptin- and serotonin 2C receptor-dependent changes in neuronal activity, as well as energy balance, feeding behavior, and glucose metabolism.

Keywords: diabetes; electrophysiology; glycemia; leptin; lorcaserin; melanocortin; obesity; patch-clamp; serotonin; thermogenesis; transient receptor potential cation channels.

Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1
Figure 1

Body weight and metabolic assessment of male WT and Pomc-creERT2∷TrpC5lox/Y mice on chow diet. (A) Schematic of genomic DNA region around exon 5 of TrpC5 in mice carrying a targeted TrpC5 allele (TrpC5lox/Y). After Cre-mediated excision of exon 5, a PCR amplification product identifying the deleted TrpC5 allele (TrpC5lox/Y) becomes detectable. (B) PCR amplification products from genomic DNA using primers detect wild type, floxed, and deleted TrpC5. Tissues were dissected from a ZP3-cre∷TrpC5lox/Y mouse. From left to right; lane 1 Arcuate nucleus (positive for recombination); lane 2 forebrain (positive for recombination); lane 3 midbrain (positive for recombination); lane 4 hindbrain (positive for recombination); lane 5 and 6 wildtype hypothalamus (negative for recombination); lane 6 DNA ladder; and lane 7 hypothalamus from floxed mouse negative for ZP3-cre. Body weight curve of (C) male CamkIIα-cre∷TrpC5lox/Y and (D) male Pomc-cre∷TrpC5lox/Y mice (*p<0.05). (E-I) Depicts (E) unchanged oxygen consumption - VO2, (F) unchanged carbon dioxide production - VCO2, (G) increased respiratory exchange ratio - RER (H) decreased heat production, and (I) unchanged ambulatory activity in male Pomc-cre∷TrpC5lox/Y mice. (J) Male Pomc-cre∷TrpC5lox/Y mice exhibited increased food intake in the light cycle which resulted in hyperphagia over 24h. For (A)–(I), n = 8-15 per group; *p < 0.05. Error bars indicate SEM. Note: mice used in (E-I) were age-matched male littermates (8 weeks of age), and had comparable body weight and lean mass.

Figure 2
Figure 2

Leptin-, mCPP-, and lorcaserin-induced hypophagia is blunted in mice deficient for TrpC5 subunits. (A) and (B) Food consumption was measured 1 hour after leptin administration (5mg/kg, i.p.) and compared with food consumption in each animal following saline administration. (C) and (D) Cumulative food intake measured at 1, 4 and/or 6 hours after administration of Ht2Cr agonists mCPP (3mg/kg) or lorcaserin (1, 3, or 6mg/kg). (E) and (F) Food intake measured in response to lorcaserin (3mg/kg, i.p.). For (A)–(F), n = 9-14 per group; *p < 0.05.

Figure 3
Figure 3

Trpc5 subunits are required for the acute leptin-induced depolarization of arcuate Pomc neurons. (A) Brightfield illumination of Pomc-hrGFP∷Lepr-cre∷tdtomato neuron from PLT mice. (B) and (C) The same neuron under FITC (hrGFP) and Alexafluor 594 (tdtomato) illumination. (D) Complete dialysis of Alexa Fluor 350 from the intracellular pipette. (E) Merge image illustrates colocalization of hr-GFP, tdtomato, and Alexa Fluor 350 indicative of a Pomc neuron which expresses Leprs. (F) Electrophysiological study demonstrates a Pomc-hrGFP∷Lepr-cre∷tdtomato (green/red) neuron that is depolarized in response to leptin (100nM). (G) Traces showing decreased voltage deflection and increased action potential frequency after leptin application. (H) Current versus voltage (I-V) plot from same WT neuron illustrating a characteristic decrease in input resistance subsequent to leptin application. Shown are responses before (control) and during leptin application. (I) demonstrates a current clamp recording of a Pomc-hrGFP∷Lepr-cre∷tdtomato∷TrpC5KO (green/red) neuron in which leptin fails to induce a depolarization. (J) Histogram summarizing the acute effect of leptin on the membrane potential of Pomc neurons which express leptin receptors as well as express or do not express Trpc5 subunits (n= 12-14 per group). (K) Rostro-caudal and medio-lateral distribution of electrophysiological responses to leptin from Pomc neurons which express leptin receptors as well as express or do not express Trpc5 subunits.

Figure 4
Figure 4

Trpc5 subunits are required for the acute mCPP-induced depolarization of arcuate Pomc neurons. (A) Brightfield illumination of Pomc-hrGFP neuron from PLT mice. (B) and (C) The same neuron under FITC (hrGFP) and Alexafluor 594 (tdtomato) illumination. (D) Complete dialysis of Alexa Fluor 350 from the intracellular pipette. (E) Merge image illustrates colocalization of hr-GFP and Alexa Fluor 350 indicative of a Pomc neuron which does not express Leprs. (F) Electrophysiological study demonstrates a Pomc-hrGFP (green) neuron from PLT mice that depolarized in response to mCPP (4μM). Traces showing decreased voltage deflection and increased action potential frequency after mCPP application. (H) Current versus voltage (I-V) plot from same WT neuron illustrating a characteristic decrease in input resistance subsequent to mCPP application. Shown are responses before (control) and during mCPP application. (I) demonstrates a current clamp recording of a Pomc-hrGFP∷Trcp5 -/Y (green) neuron in which mCPP fails to induce a depolarization. (J) Histogram summarizing the acute effect of mCPP on the membrane potential of Pomc neurons which do not express leptin receptors as well as express or do not express Trpc5 subunits (n= 13-19 per group). (K) Rostro-caudal and medio-lateral distribution of electrophysiological responses to mCPP from Pomc neurons which do not express leptin receptors as well as express or do not express Trpc5 subunits.

Figure 5
Figure 5

Trpc5 subunits are required for the acute lorcaserin-induced depolarization of arcuate Pomc neurons. (A) Electrophysiological study demonstrates a Pomc-hrGFP (green) neuron from PLT mice that depolarized in response to lorcaserin (4μM). (B) Traces showing decreased voltage deflection and increased action potential frequency after lorcaserin application. (C) Current versus voltage (I-V) plot from same WT neuron illustrating a characteristic decrease in input resistance subsequent to lorcaserin application. Shown are responses before (control) and during lorcaserin application. (D) Demonstrates a current clamp recording of a Pomc-hrGFP∷Trcp5lox/Y (green) neuron from PLT mice in which lorcaserin fails to induce a depolarization. (E) Histogram summarizing the acute effect of lorcaserin on the membrane potential of Pomc neurons which do not express leptin receptors as well as express or do not express TrpC5 subunits (n= 9-12 per group). (F) Rostro-caudal and medio-lateral distribution of electrophysiological responses to lorcaserin from Pomc neurons which do not express leptin receptors as well as express or do not express TrpC5 subunits.

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