BACKGROUND: Different strategies to boost cellular NAD+ levels, such as supplementation of NAD+ precursors, or inhibition of NAD+ consumption, are currently under investigation as promising means to promote healthy aging and ameliorate dysfunctional metabolism. CD38 is a NAD+-dependent enzyme that converts NAD+ to different Ca2+-active second messengers, involved in the regulation of different signaling pathways, cell functions and metabolism. TRPM2, is an ion channel that allows Ca2+ influx from the extracellular space toward the cytosol, and is gated by ADPR, one of the molecules produced from the NAD+ degradation. In the context of systemic energy metabolism, brown adipocytes, the parenchymal cells of brown adipose tissue (BAT) as well as beige adipocytes that emerge in white adipose tissue (WAT) depots in response to catabolic conditions, are important to maintain metabolic homeostasis, together with liver. HYPOTHESIS: We aim to understand the functional relevance of CD38 and TRPM2 in the regulation of energy metabolism and NAD(P)(H) levels in BAT, WAT and liver during thermogenesis. METHODS: We used wild type, Cd38-/- and Trpm2-/- mice, exposed to cold temperatures and BAT, WAT and liver were collected. We evaluated mRNA levels by RT-PCR, proteins/enzymes levels by Western blot, FACS analysis and enzymatic activities. NAD(P)(H) levels were determined with cycling assays. Furthermore, we performed a set of in vivo experiments in which O2 consumption, CO2 production and energy expenditure were measured in mice upon thermogenic stimulation. RESULTS: We confirmed that CD38 is a major NAD+-consumer in BAT, WAT and liver: increased NAD+ levels were observed in these tissues from Cd38-/- compared with wild type mice. Interestingly, during cold exposure, a marked downregulation of CD38 expression (as detected at the mRNA, protein and enzymatic level) occurred in BAT, WAT and in liver of wild type mice. As a consequence of CD38 downregulation, an increase in NAD+ levels occurred in BAT. Instead, in WAT, CD38 downregulation was accompanied by a strong increase in NADP(H) levels, likely as a consequence of increased NADK, G6PD and malic enzyme activities. In liver, CD38 downregulation was paralleled by increased NAD(H) levels. NADK activity and NADP+ levels were not significantly modified in liver during cold-exposure. Notably, a marked decrease of NADPH level occurred in liver from both wild type and Cd38-/- mice exposed to a cold temperature, possibly as a consequence of the observed downregulation of the hepatic G6PD activity. Saving G6P from the PPP is in line with the increased activity of the enzyme glucose-6-phosphatase in liver of wild type, but not Cd38-/-, cold-exposed mice, with up-regulated gluconeogenesis. When Cd38-/- mice were kept at 6°C, higher levels of Ucp1 and Pgc-1 in BAT and WAT were revealed, compared with wild type mice. Conversely, when Trpm2-/- mice were exposed to cold temperature, lower levels of these two browning marker genes were detected, compared with wild type mice. In line with this, mice lacking Trpm2 displayed lower respiration rate and energy expenditure, when thermogenesis was induced by cold exposure and CL316,243 (a specific compound triggering adipose tissue activation). Interestingly, during cold exposure, a marked Trpm2 overexpression was observed in WAT and BAT of wild type mice. In addition, ADPR levels and mono/poly-ADPR hydrolases expression were higher in mice exposed to cold, in comparison with wild type mice. CONCLUSION: Taken together, these results demonstrate that CD38, by modulating cellular NAD(P)+ levels, is involved in the regulation of thermogenic responses in cold-activated BAT and WAT. Indeed, CD38 inhibition is being investigated as a possible strategy to ameliorate dysfunctional metabolism, by boosting NAD+ levels and sirtuins’ activity. In addition, TRPM2 plays a pivotal role in BAT and WAT activation. TRPM2 gating, is likely due to alternative pathways that do not include CD38 activity.

The role of CD38 and TRPM2 in adipose tissue and liver during thermogenesis

BENZI, ANDREA
2022-05-11

Abstract

BACKGROUND: Different strategies to boost cellular NAD+ levels, such as supplementation of NAD+ precursors, or inhibition of NAD+ consumption, are currently under investigation as promising means to promote healthy aging and ameliorate dysfunctional metabolism. CD38 is a NAD+-dependent enzyme that converts NAD+ to different Ca2+-active second messengers, involved in the regulation of different signaling pathways, cell functions and metabolism. TRPM2, is an ion channel that allows Ca2+ influx from the extracellular space toward the cytosol, and is gated by ADPR, one of the molecules produced from the NAD+ degradation. In the context of systemic energy metabolism, brown adipocytes, the parenchymal cells of brown adipose tissue (BAT) as well as beige adipocytes that emerge in white adipose tissue (WAT) depots in response to catabolic conditions, are important to maintain metabolic homeostasis, together with liver. HYPOTHESIS: We aim to understand the functional relevance of CD38 and TRPM2 in the regulation of energy metabolism and NAD(P)(H) levels in BAT, WAT and liver during thermogenesis. METHODS: We used wild type, Cd38-/- and Trpm2-/- mice, exposed to cold temperatures and BAT, WAT and liver were collected. We evaluated mRNA levels by RT-PCR, proteins/enzymes levels by Western blot, FACS analysis and enzymatic activities. NAD(P)(H) levels were determined with cycling assays. Furthermore, we performed a set of in vivo experiments in which O2 consumption, CO2 production and energy expenditure were measured in mice upon thermogenic stimulation. RESULTS: We confirmed that CD38 is a major NAD+-consumer in BAT, WAT and liver: increased NAD+ levels were observed in these tissues from Cd38-/- compared with wild type mice. Interestingly, during cold exposure, a marked downregulation of CD38 expression (as detected at the mRNA, protein and enzymatic level) occurred in BAT, WAT and in liver of wild type mice. As a consequence of CD38 downregulation, an increase in NAD+ levels occurred in BAT. Instead, in WAT, CD38 downregulation was accompanied by a strong increase in NADP(H) levels, likely as a consequence of increased NADK, G6PD and malic enzyme activities. In liver, CD38 downregulation was paralleled by increased NAD(H) levels. NADK activity and NADP+ levels were not significantly modified in liver during cold-exposure. Notably, a marked decrease of NADPH level occurred in liver from both wild type and Cd38-/- mice exposed to a cold temperature, possibly as a consequence of the observed downregulation of the hepatic G6PD activity. Saving G6P from the PPP is in line with the increased activity of the enzyme glucose-6-phosphatase in liver of wild type, but not Cd38-/-, cold-exposed mice, with up-regulated gluconeogenesis. When Cd38-/- mice were kept at 6°C, higher levels of Ucp1 and Pgc-1 in BAT and WAT were revealed, compared with wild type mice. Conversely, when Trpm2-/- mice were exposed to cold temperature, lower levels of these two browning marker genes were detected, compared with wild type mice. In line with this, mice lacking Trpm2 displayed lower respiration rate and energy expenditure, when thermogenesis was induced by cold exposure and CL316,243 (a specific compound triggering adipose tissue activation). Interestingly, during cold exposure, a marked Trpm2 overexpression was observed in WAT and BAT of wild type mice. In addition, ADPR levels and mono/poly-ADPR hydrolases expression were higher in mice exposed to cold, in comparison with wild type mice. CONCLUSION: Taken together, these results demonstrate that CD38, by modulating cellular NAD(P)+ levels, is involved in the regulation of thermogenic responses in cold-activated BAT and WAT. Indeed, CD38 inhibition is being investigated as a possible strategy to ameliorate dysfunctional metabolism, by boosting NAD+ levels and sirtuins’ activity. In addition, TRPM2 plays a pivotal role in BAT and WAT activation. TRPM2 gating, is likely due to alternative pathways that do not include CD38 activity.
File in questo prodotto:
File Dimensione Formato  
phdunige_3953369.pdf

accesso aperto

Tipologia: Tesi di dottorato
Dimensione 2.27 MB
Formato Adobe PDF
2.27 MB Adobe PDF Visualizza/Apri

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11567/1081136
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact