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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

Full activation of insulin-stimulated glucose uptake in adipocytes requires ATP release by Panx1 channels. (A) Blockade of Pannexin-1 channels with carbenoxolone (100 μM, CBX) or probenecid (1 mM, Prob) significantly decreases insulin-stimulated 3H-glucose uptake in 3T3L1-adipocytes. Data are expressed as mean ± s.e.m. *p < 0.001 by 2 way ANOVA with Tukey's multiple comparisons test. (B) Insulin-stimulated glucose uptake is significantly decreased in adipocytes isolated from perigonadal adipose tissue of adipocyte-specific Pannexin-1  mice. Addition of exogenous ATP (50 μM) restores insulin-stimulated 14C-glucose uptake in adipocytes isolated from Panx1  mice. Data are expressed as mean ± s.e.m. *p < 0.003 by 1 way ANOVA with Tukey's multiple comparisons test. (C) In vivo [3H] 2-deoxy-d-glucose uptake was assessed in perigonadal white adipose tissue (WAT) and gastrocnemius muscle (MUS) in age-matched, male, chow fed WT and AdipPanxKO littermates (n = 6). Data are presented as mean ± s.e.m. *p < 0.05 by paired t-test. Dotted line indicates the 20% decrease in glucose uptake in WAT in AdipPanxKO mice compared to WT.
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fig2: Full activation of insulin-stimulated glucose uptake in adipocytes requires ATP release by Panx1 channels. (A) Blockade of Pannexin-1 channels with carbenoxolone (100 μM, CBX) or probenecid (1 mM, Prob) significantly decreases insulin-stimulated 3H-glucose uptake in 3T3L1-adipocytes. Data are expressed as mean ± s.e.m. *p < 0.001 by 2 way ANOVA with Tukey's multiple comparisons test. (B) Insulin-stimulated glucose uptake is significantly decreased in adipocytes isolated from perigonadal adipose tissue of adipocyte-specific Pannexin-1 mice. Addition of exogenous ATP (50 μM) restores insulin-stimulated 14C-glucose uptake in adipocytes isolated from Panx1 mice. Data are expressed as mean ± s.e.m. *p < 0.003 by 1 way ANOVA with Tukey's multiple comparisons test. (C) In vivo [3H] 2-deoxy-d-glucose uptake was assessed in perigonadal white adipose tissue (WAT) and gastrocnemius muscle (MUS) in age-matched, male, chow fed WT and AdipPanxKO littermates (n = 6). Data are presented as mean ± s.e.m. *p < 0.05 by paired t-test. Dotted line indicates the 20% decrease in glucose uptake in WAT in AdipPanxKO mice compared to WT.

Mentions: To examine a role for Panx1 in glucose uptake in adipocytes, we treated cultured or primary adipocytes with insulin for 15 min, and basal and insulin-stimulated glucose uptake was measured in the presence and absence of two distinct pharmacological Panx1 inhibitors. Glucose uptake into cultured 3T3-L1 adipocytes was significantly increased after treatment with insulin; notably, pretreatment with carbenoxolone (CBX) or probenecid (Prob) resulted in significantly blunted insulin-stimulated glucose uptake (Figure 2A). In addition, insulin-stimulated glucose uptake was significantly impaired in adipocytes isolated from AdipPanx1KO mice compared to adipocytes isolated from WT mice (Figure 2B). Since pharmacological as well as genetic inhibition of Panx1 in adipocytes resulted in blunted insulin-stimulated glucose uptake, we hypothesized that Panx1-mediated ATP release was responsible for the effect. Indeed, the addition of exogenous ATP rescued the compromised insulin-stimulated glucose uptake in Panx1-deficient adipocytes (Figure 2B).


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)

Full activation of insulin-stimulated glucose uptake in adipocytes requires ATP release by Panx1 channels. (A) Blockade of Pannexin-1 channels with carbenoxolone (100 μM, CBX) or probenecid (1 mM, Prob) significantly decreases insulin-stimulated 3H-glucose uptake in 3T3L1-adipocytes. Data are expressed as mean ± s.e.m. *p < 0.001 by 2 way ANOVA with Tukey's multiple comparisons test. (B) Insulin-stimulated glucose uptake is significantly decreased in adipocytes isolated from perigonadal adipose tissue of adipocyte-specific Pannexin-1  mice. Addition of exogenous ATP (50 μM) restores insulin-stimulated 14C-glucose uptake in adipocytes isolated from Panx1  mice. Data are expressed as mean ± s.e.m. *p < 0.003 by 1 way ANOVA with Tukey's multiple comparisons test. (C) In vivo [3H] 2-deoxy-d-glucose uptake was assessed in perigonadal white adipose tissue (WAT) and gastrocnemius muscle (MUS) in age-matched, male, chow fed WT and AdipPanxKO littermates (n = 6). Data are presented as mean ± s.e.m. *p < 0.05 by paired t-test. Dotted line indicates the 20% decrease in glucose uptake in WAT in AdipPanxKO mice compared to WT.
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fig2: Full activation of insulin-stimulated glucose uptake in adipocytes requires ATP release by Panx1 channels. (A) Blockade of Pannexin-1 channels with carbenoxolone (100 μM, CBX) or probenecid (1 mM, Prob) significantly decreases insulin-stimulated 3H-glucose uptake in 3T3L1-adipocytes. Data are expressed as mean ± s.e.m. *p < 0.001 by 2 way ANOVA with Tukey's multiple comparisons test. (B) Insulin-stimulated glucose uptake is significantly decreased in adipocytes isolated from perigonadal adipose tissue of adipocyte-specific Pannexin-1 mice. Addition of exogenous ATP (50 μM) restores insulin-stimulated 14C-glucose uptake in adipocytes isolated from Panx1 mice. Data are expressed as mean ± s.e.m. *p < 0.003 by 1 way ANOVA with Tukey's multiple comparisons test. (C) In vivo [3H] 2-deoxy-d-glucose uptake was assessed in perigonadal white adipose tissue (WAT) and gastrocnemius muscle (MUS) in age-matched, male, chow fed WT and AdipPanxKO littermates (n = 6). Data are presented as mean ± s.e.m. *p < 0.05 by paired t-test. Dotted line indicates the 20% decrease in glucose uptake in WAT in AdipPanxKO mice compared to WT.
Mentions: To examine a role for Panx1 in glucose uptake in adipocytes, we treated cultured or primary adipocytes with insulin for 15 min, and basal and insulin-stimulated glucose uptake was measured in the presence and absence of two distinct pharmacological Panx1 inhibitors. Glucose uptake into cultured 3T3-L1 adipocytes was significantly increased after treatment with insulin; notably, pretreatment with carbenoxolone (CBX) or probenecid (Prob) resulted in significantly blunted insulin-stimulated glucose uptake (Figure 2A). In addition, insulin-stimulated glucose uptake was significantly impaired in adipocytes isolated from AdipPanx1KO mice compared to adipocytes isolated from WT mice (Figure 2B). Since pharmacological as well as genetic inhibition of Panx1 in adipocytes resulted in blunted insulin-stimulated glucose uptake, we hypothesized that Panx1-mediated ATP release was responsible for the effect. Indeed, the addition of exogenous ATP rescued the compromised insulin-stimulated glucose uptake in Panx1-deficient adipocytes (Figure 2B).

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