Gene_locus Report for: human-ABHD15

OBJECTIVE: Insulin suppresses adipose tissue lipolysis after a meal, playing a key role in metabolic homeostasis. This is mediated via the kinase Akt and its substrate phosphodiesterase 3B (PDE3B). Once phosphorylated and activated, PDE3B hydrolyses cAMP leading to the inactivation of cAMP-dependent protein kinase (PKA) and suppression of lipolysis. However, several gaps have emerged in this model. Here we investigated the role of the PDE3B-interacting protein, alpha/beta-hydrolase ABHD15 in this process. METHODS: Lipolysis, glucose uptake, and signaling were assessed in ABHD15 knock down and knock out adipocytes and fat explants in response to insulin and/or beta-adrenergic receptor agonist. Glucose and fatty acid metabolism were determined in wild type and ABHD15(-/-) littermate mice. RESULTS: Deletion of ABHD15 in adipocytes resulted in a significant defect in insulin-mediated suppression of lipolysis with no effect on insulin-mediated glucose uptake. ABHD15 played a role in suppressing PKA signaling as phosphorylation of the PKA substrate Perilipin-1 remained elevated in response to insulin upon ABHD15 deletion. ABHD15(-/-) mice had normal glucose metabolism but defective fatty acid metabolism: plasma fatty acids were elevated upon fasting and in response to insulin, and this was accompanied by elevated liver triglycerides upon beta-adrenergic receptor activation. This is likely due to hyperactive lipolysis as evident by the larger triglyceride depletion in brown adipose tissue in these mice. Finally, ABHD15 protein levels were reduced in adipocytes from mice fed a Western diet, further implicating this protein in metabolic homeostasis. CONCLUSIONS: Collectively, ABHD15 regulates adipocyte lipolysis and liver lipid accumulation, providing novel therapeutic opportunities for modulating lipid homeostasis in disease.

Our knowledge about adipocyte metabolism and development is steadily growing, yet many players are still undefined. Here, we show that alpha/beta-hydrolase domain containing protein 15 (Abhd15) is a direct and functional target gene of peroxisome proliferator-activated receptor gamma (PPARgamma), the master regulator of adipogenesis. In line, Abhd15 is mainly expressed in brown and white adipose tissue and strongly upregulated during adipogenesis in various murine and human cell lines. Stable knockdown of Abhd15 in 3T3-L1 cells evokes a striking differentiation defect, as evidenced by low lipid accumulation and decreased expression of adipocyte marker genes. In preconfluent cells, knockdown of Abhd15 leads to impaired proliferation, which is caused by apoptosis, as we see an increased SubG1 peak, caspase 3/7 activity, and BAX protein expression as well as a reduction in anti-apoptotic BCL-2 protein. Furthermore, apoptosis-inducing amounts of palmitic acid evoke a massive increase of Abhd15 expression, proposing an apoptosis-protecting role for ABHD15. On the other hand, in mature adipocytes physiological (i.e. non-apoptotic) concentrations of palmitic acid down-regulate Abhd15 expression. Accordingly, we found that the expression of Abhd15 in adipose tissue is reduced in physiological situations with high free fatty acid levels, like high-fat diet, fasting, and aging as well as in genetically obese mice. Collectively, our results position ABHD15 as an essential component in the development of adipocytes as well as in apoptosis, thereby connecting two substantial factors in the regulation of adipocyte number and size. Together with its intricate regulation by free fatty acids, ABHD15 might be an intriguing new target in obesity and diabetes research.

We have previously described a novel putative 47kDa substrate for the protein kinase Akt (designated AS47) in 3T3-L1 adipocytes. In the present study, we have found by co-immunoprecipitation that AS47 was associated with cyclic nucleotide phosphodiesterase 3B (PDE3B) in lysates of 3T3-L1 adipocytes. The patterns of expression of AS47 and PDE3B upon 3T3-L1 adipocyte differentiation, among mouse tissues, and in adipocytes with and without the transcription factor C/EBPalpha were virtually coincident. Partial knockdown of AS47 in 3T3-L1 adipocytes with shRNA resulted in a similar reduction in PDE3B protein. These results indicate that AS47 exists in a complex with PDE3B in adipocytes and that the amount of AS47 protein regulates the amount of PDE3B.

OBJECTIVE: Insulin suppresses adipose tissue lipolysis after a meal, playing a key role in metabolic homeostasis. This is mediated via the kinase Akt and its substrate phosphodiesterase 3B (PDE3B). Once phosphorylated and activated, PDE3B hydrolyses cAMP leading to the inactivation of cAMP-dependent protein kinase (PKA) and suppression of lipolysis. However, several gaps have emerged in this model. Here we investigated the role of the PDE3B-interacting protein, alpha/beta-hydrolase ABHD15 in this process. METHODS: Lipolysis, glucose uptake, and signaling were assessed in ABHD15 knock down and knock out adipocytes and fat explants in response to insulin and/or beta-adrenergic receptor agonist. Glucose and fatty acid metabolism were determined in wild type and ABHD15(-/-) littermate mice. RESULTS: Deletion of ABHD15 in adipocytes resulted in a significant defect in insulin-mediated suppression of lipolysis with no effect on insulin-mediated glucose uptake. ABHD15 played a role in suppressing PKA signaling as phosphorylation of the PKA substrate Perilipin-1 remained elevated in response to insulin upon ABHD15 deletion. ABHD15(-/-) mice had normal glucose metabolism but defective fatty acid metabolism: plasma fatty acids were elevated upon fasting and in response to insulin, and this was accompanied by elevated liver triglycerides upon beta-adrenergic receptor activation. This is likely due to hyperactive lipolysis as evident by the larger triglyceride depletion in brown adipose tissue in these mice. Finally, ABHD15 protein levels were reduced in adipocytes from mice fed a Western diet, further implicating this protein in metabolic homeostasis. CONCLUSIONS: Collectively, ABHD15 regulates adipocyte lipolysis and liver lipid accumulation, providing novel therapeutic opportunities for modulating lipid homeostasis in disease.

Elevated circulating fatty acids (FAs) contribute to obesity-associated metabolic complications, but the mechanisms by which insulin suppresses lipolysis are poorly understood. We show that alpha/beta-hydrolase domain-containing 15 (ABHD15) is required for the anti-lipolytic action of insulin in white adipose tissue (WAT). Neither insulin nor glucose treatments can suppress FA mobilization in global and conditional Abhd15-knockout (KO) mice. Accordingly, insulin signaling is impaired in Abhd15-KO adipocytes, as indicated by reduced AKT phosphorylation, glucose uptake, and de novo lipogenesis. In vitro data reveal that ABHD15 associates with and stabilizes phosphodiesterase 3B (PDE3B). Accordingly, PDE3B expression is decreased in the WAT of Abhd15-KO mice, mechanistically explaining increased protein kinase A (PKA) activity, hormone-sensitive lipase (HSL) phosphorylation, and undiminished FA release upon insulin signaling. Ultimately, Abhd15-KO mice develop insulin resistance. Notably, ABHD15 expression is decreased in humans with obesity and diabetes compared to humans with obesity and normal glucose tolerance, identifying ABHD15 as a potential therapeutic target to mitigate insulin resistance.

Our knowledge about adipocyte metabolism and development is steadily growing, yet many players are still undefined. Here, we show that alpha/beta-hydrolase domain containing protein 15 (Abhd15) is a direct and functional target gene of peroxisome proliferator-activated receptor gamma (PPARgamma), the master regulator of adipogenesis. In line, Abhd15 is mainly expressed in brown and white adipose tissue and strongly upregulated during adipogenesis in various murine and human cell lines. Stable knockdown of Abhd15 in 3T3-L1 cells evokes a striking differentiation defect, as evidenced by low lipid accumulation and decreased expression of adipocyte marker genes. In preconfluent cells, knockdown of Abhd15 leads to impaired proliferation, which is caused by apoptosis, as we see an increased SubG1 peak, caspase 3/7 activity, and BAX protein expression as well as a reduction in anti-apoptotic BCL-2 protein. Furthermore, apoptosis-inducing amounts of palmitic acid evoke a massive increase of Abhd15 expression, proposing an apoptosis-protecting role for ABHD15. On the other hand, in mature adipocytes physiological (i.e. non-apoptotic) concentrations of palmitic acid down-regulate Abhd15 expression. Accordingly, we found that the expression of Abhd15 in adipose tissue is reduced in physiological situations with high free fatty acid levels, like high-fat diet, fasting, and aging as well as in genetically obese mice. Collectively, our results position ABHD15 as an essential component in the development of adipocytes as well as in apoptosis, thereby connecting two substantial factors in the regulation of adipocyte number and size. Together with its intricate regulation by free fatty acids, ABHD15 might be an intriguing new target in obesity and diabetes research.

We have previously described a novel putative 47kDa substrate for the protein kinase Akt (designated AS47) in 3T3-L1 adipocytes. In the present study, we have found by co-immunoprecipitation that AS47 was associated with cyclic nucleotide phosphodiesterase 3B (PDE3B) in lysates of 3T3-L1 adipocytes. The patterns of expression of AS47 and PDE3B upon 3T3-L1 adipocyte differentiation, among mouse tissues, and in adipocytes with and without the transcription factor C/EBPalpha were virtually coincident. Partial knockdown of AS47 in 3T3-L1 adipocytes with shRNA resulted in a similar reduction in PDE3B protein. These results indicate that AS47 exists in a complex with PDE3B in adipocytes and that the amount of AS47 protein regulates the amount of PDE3B.

Akt is a key insulin-activated protein kinase. We searched for Akt substrates in 3T3-L1 adipocytes by means of immunoprecipitation with an Akt phosphomotif-specific antibody (PAS antibody). Four insulin-elicited phosphoproteins were isolated and identified by mass spectrometry. The identity of each protein was established by isolating the protein from lysates of untreated and insulin-treated adipocytes with an antibody specific for the protein and showing that the PAS antibody reacted only with the protein in the immunoprecipitate from insulin-treated cells. These proteins have sizes of 47, 75, 105, and 250 kDa on SDS PAGE, and have been designated pp47, 75, 105, and 250. The effect of inhibitors on the phosphorylation of the proteins, the identified sites of phosphorylation, and in vitro phosphorylation by recombinant Akt further indicated that pp47, 105, and 250 are likely to be Akt substrates, whereas pp75 may not be. pp47 and 105 are novel proteins with no known or predicted function. pp75 was previously found as a protein that associated with the colony-stimulating factor receptor, designated as Fms-interacting protein. pp250 is a novel protein with a predicted GTPase activating protein (GAP) domain for Rheb and/or Rap at its carboxy terminus. The subcellular and tissue distributions of the four proteins were determined.