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Pannexin 1 is required for full activation of insulin-stimulated glucose uptake in adipocytes.

Adamson SE, Meher AK, Chiu YH, Sandilos JK, Oberholtzer NP, Walker NN, Hargett SR, Seaman SA, Peirce-Cottler SM, Isakson BE, McNamara CA, Keller SR, Harris TE, Bayliss DA, Leitinger N - Mol Metab (2015)

Bottom Line: Finally, we measured Panx1 mRNA in human visceral adipose tissue samples by qRT-PCR and compared expression levels with glucose levels and HOMA-IR measurements in patients.Our data show that adipocytes express functional Pannexin 1 (Panx1) channels that can be activated to release ATP.We show that Panx1 channel activity regulates insulin-stimulated glucose uptake in adipocytes and thus contributes to control of metabolic homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA ; Cardiovascular Research Center, University of Virginia, Charlottesville, VA 22908, USA.

ABSTRACT

Objective: Defective glucose uptake in adipocytes leads to impaired metabolic homeostasis and insulin resistance, hallmarks of type 2 diabetes. Extracellular ATP-derived nucleotides and nucleosides are important regulators of adipocyte function, but the pathway for controlled ATP release from adipocytes is unknown. Here, we investigated whether Pannexin 1 (Panx1) channels control ATP release from adipocytes and contribute to metabolic homeostasis.

Methods: We assessed Panx1 functionality in cultured 3T3-L1 adipocytes and in adipocytes isolated from murine white adipose tissue by measuring ATP release in response to known activators of Panx1 channels. Glucose uptake in cultured 3T3-L1 adipocytes was measured in the presence of Panx1 pharmacologic inhibitors and in adipocytes isolated from white adipose tissue from wildtype (WT) or adipocyte-specific Panx1 knockout (AdipPanx1 KO) mice generated in our laboratory. We performed in vivo glucose uptake studies in chow fed WT and AdipPanx1 KO mice and assessed insulin resistance in WT and AdipPanx1 KO mice fed a high fat diet for 12 weeks. Panx1 channel function was assessed in response to insulin by performing electrophysiologic recordings in a heterologous expression system. Finally, we measured Panx1 mRNA in human visceral adipose tissue samples by qRT-PCR and compared expression levels with glucose levels and HOMA-IR measurements in patients.

Results: Our data show that adipocytes express functional Pannexin 1 (Panx1) channels that can be activated to release ATP. Pharmacologic inhibition or selective genetic deletion of Panx1 from adipocytes decreased insulin-induced glucose uptake in vitro and in vivo and exacerbated diet-induced insulin resistance in mice. Further, we identify insulin as a novel activator of Panx1 channels. In obese humans Panx1 expression in adipose tissue is increased and correlates with the degree of insulin resistance.

Conclusions: We show that Panx1 channel activity regulates insulin-stimulated glucose uptake in adipocytes and thus contributes to control of metabolic homeostasis.

No MeSH data available.


Related in: MedlinePlus

Insulin induces Panx1 channel activation and ATP release. (A) Adipocytes isolated from perigonadal adipose tissue of WT mice release ATP upon insulin stimulation, which can be blocked by treatment with the Panx1 inhibitor probenecid. Data are expressed as mean ± s.e.m. *p < 0.05 by Student's t-test. (B) Whole cell patch clamp of HEK cells transfected with human insulin receptor and mouse Panx1 reveals a Panx1 dependent current upon treatment with insulin that is abolished by addition of the Panx1 inhibitor carbenoxolone. Current–voltage relationship curve is shown in middle. Insulin treatment significantly increases CBX-sensitive current density (n = 8 cells). (C) Whole cell patch clamp of HEK cells transfected with human insulin receptor and human Panx1 in which the C-terminal caspase cleavage site has been replaced with a TEV protease cleavage site reveals a Panx1 dependent and CBX-sensitive current upon treatment with insulin. Current–voltage relationship curve is shown in middle. Insulin treatment significantly increases CBX-sensitive current density (n = 8 cells).
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fig3: Insulin induces Panx1 channel activation and ATP release. (A) Adipocytes isolated from perigonadal adipose tissue of WT mice release ATP upon insulin stimulation, which can be blocked by treatment with the Panx1 inhibitor probenecid. Data are expressed as mean ± s.e.m. *p < 0.05 by Student's t-test. (B) Whole cell patch clamp of HEK cells transfected with human insulin receptor and mouse Panx1 reveals a Panx1 dependent current upon treatment with insulin that is abolished by addition of the Panx1 inhibitor carbenoxolone. Current–voltage relationship curve is shown in middle. Insulin treatment significantly increases CBX-sensitive current density (n = 8 cells). (C) Whole cell patch clamp of HEK cells transfected with human insulin receptor and human Panx1 in which the C-terminal caspase cleavage site has been replaced with a TEV protease cleavage site reveals a Panx1 dependent and CBX-sensitive current upon treatment with insulin. Current–voltage relationship curve is shown in middle. Insulin treatment significantly increases CBX-sensitive current density (n = 8 cells).

Mentions: To examine whether insulin activates Panx1 channels, we first assessed Panx1-dependent ATP release from primary adipocytes isolated from WT mice. Treatment with insulin for 30 min caused a significant increase in extracellular ATP, which was blocked by addition of the Panx1 inhibitor probenecid (Prob) (Figure 3A). Next, we tested the ability of insulin to activate Panx1 channels using HEK293 cells co-transfected with expression plasmids for the human insulin receptor (hIR) and murine Panx1. Insulin treatment evoked a current with Panx1-like voltage-dependent properties that was blocked by the Panx1 inhibitor carbenoxolone (CBX) (Figure 3B). Insulin-stimulated Panx1 currents were also seen in HEK293 cells co-transfected with expression plasmids for the human insulin receptor (hIR) and human Panx1 with a mutated caspase cleavage site [32] (Figure 3C). These data identify insulin as a novel mediator of Panx1 channel activation and show that insulin-induced activation of Panx1 is independent of caspase-mediated cleavage.


Pannexin 1 is required for full activation of insulin-stimulated glucose uptake in adipocytes.

Adamson SE, Meher AK, Chiu YH, Sandilos JK, Oberholtzer NP, Walker NN, Hargett SR, Seaman SA, Peirce-Cottler SM, Isakson BE, McNamara CA, Keller SR, Harris TE, Bayliss DA, Leitinger N - Mol Metab (2015)

Insulin induces Panx1 channel activation and ATP release. (A) Adipocytes isolated from perigonadal adipose tissue of WT mice release ATP upon insulin stimulation, which can be blocked by treatment with the Panx1 inhibitor probenecid. Data are expressed as mean ± s.e.m. *p < 0.05 by Student's t-test. (B) Whole cell patch clamp of HEK cells transfected with human insulin receptor and mouse Panx1 reveals a Panx1 dependent current upon treatment with insulin that is abolished by addition of the Panx1 inhibitor carbenoxolone. Current–voltage relationship curve is shown in middle. Insulin treatment significantly increases CBX-sensitive current density (n = 8 cells). (C) Whole cell patch clamp of HEK cells transfected with human insulin receptor and human Panx1 in which the C-terminal caspase cleavage site has been replaced with a TEV protease cleavage site reveals a Panx1 dependent and CBX-sensitive current upon treatment with insulin. Current–voltage relationship curve is shown in middle. Insulin treatment significantly increases CBX-sensitive current density (n = 8 cells).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4563021&req=5

fig3: Insulin induces Panx1 channel activation and ATP release. (A) Adipocytes isolated from perigonadal adipose tissue of WT mice release ATP upon insulin stimulation, which can be blocked by treatment with the Panx1 inhibitor probenecid. Data are expressed as mean ± s.e.m. *p < 0.05 by Student's t-test. (B) Whole cell patch clamp of HEK cells transfected with human insulin receptor and mouse Panx1 reveals a Panx1 dependent current upon treatment with insulin that is abolished by addition of the Panx1 inhibitor carbenoxolone. Current–voltage relationship curve is shown in middle. Insulin treatment significantly increases CBX-sensitive current density (n = 8 cells). (C) Whole cell patch clamp of HEK cells transfected with human insulin receptor and human Panx1 in which the C-terminal caspase cleavage site has been replaced with a TEV protease cleavage site reveals a Panx1 dependent and CBX-sensitive current upon treatment with insulin. Current–voltage relationship curve is shown in middle. Insulin treatment significantly increases CBX-sensitive current density (n = 8 cells).
Mentions: To examine whether insulin activates Panx1 channels, we first assessed Panx1-dependent ATP release from primary adipocytes isolated from WT mice. Treatment with insulin for 30 min caused a significant increase in extracellular ATP, which was blocked by addition of the Panx1 inhibitor probenecid (Prob) (Figure 3A). Next, we tested the ability of insulin to activate Panx1 channels using HEK293 cells co-transfected with expression plasmids for the human insulin receptor (hIR) and murine Panx1. Insulin treatment evoked a current with Panx1-like voltage-dependent properties that was blocked by the Panx1 inhibitor carbenoxolone (CBX) (Figure 3B). Insulin-stimulated Panx1 currents were also seen in HEK293 cells co-transfected with expression plasmids for the human insulin receptor (hIR) and human Panx1 with a mutated caspase cleavage site [32] (Figure 3C). These data identify insulin as a novel mediator of Panx1 channel activation and show that insulin-induced activation of Panx1 is independent of caspase-mediated cleavage.

Bottom Line: Finally, we measured Panx1 mRNA in human visceral adipose tissue samples by qRT-PCR and compared expression levels with glucose levels and HOMA-IR measurements in patients.Our data show that adipocytes express functional Pannexin 1 (Panx1) channels that can be activated to release ATP.We show that Panx1 channel activity regulates insulin-stimulated glucose uptake in adipocytes and thus contributes to control of metabolic homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA ; Cardiovascular Research Center, University of Virginia, Charlottesville, VA 22908, USA.

ABSTRACT

Objective: Defective glucose uptake in adipocytes leads to impaired metabolic homeostasis and insulin resistance, hallmarks of type 2 diabetes. Extracellular ATP-derived nucleotides and nucleosides are important regulators of adipocyte function, but the pathway for controlled ATP release from adipocytes is unknown. Here, we investigated whether Pannexin 1 (Panx1) channels control ATP release from adipocytes and contribute to metabolic homeostasis.

Methods: We assessed Panx1 functionality in cultured 3T3-L1 adipocytes and in adipocytes isolated from murine white adipose tissue by measuring ATP release in response to known activators of Panx1 channels. Glucose uptake in cultured 3T3-L1 adipocytes was measured in the presence of Panx1 pharmacologic inhibitors and in adipocytes isolated from white adipose tissue from wildtype (WT) or adipocyte-specific Panx1 knockout (AdipPanx1 KO) mice generated in our laboratory. We performed in vivo glucose uptake studies in chow fed WT and AdipPanx1 KO mice and assessed insulin resistance in WT and AdipPanx1 KO mice fed a high fat diet for 12 weeks. Panx1 channel function was assessed in response to insulin by performing electrophysiologic recordings in a heterologous expression system. Finally, we measured Panx1 mRNA in human visceral adipose tissue samples by qRT-PCR and compared expression levels with glucose levels and HOMA-IR measurements in patients.

Results: Our data show that adipocytes express functional Pannexin 1 (Panx1) channels that can be activated to release ATP. Pharmacologic inhibition or selective genetic deletion of Panx1 from adipocytes decreased insulin-induced glucose uptake in vitro and in vivo and exacerbated diet-induced insulin resistance in mice. Further, we identify insulin as a novel activator of Panx1 channels. In obese humans Panx1 expression in adipose tissue is increased and correlates with the degree of insulin resistance.

Conclusions: We show that Panx1 channel activity regulates insulin-stimulated glucose uptake in adipocytes and thus contributes to control of metabolic homeostasis.

No MeSH data available.


Related in: MedlinePlus