dFOXO Regulates Transcription of a Drosophila Acid Lipase

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Abstract

Insulin resistance is a major feature of pathological states such as obesity and diabetes. A consequence of insulin resistance is enhanced lipolysis, which causes excessive release of free fatty acids and deregulates fatty acid homeostasis. The transcription factor FOXO1 has a central role in the regulation of glucose levels by insulin: reduced insulin signaling causes FOXO1 activation, which increases hepatic glucose production by activating transcription of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase mRNAs. Our results suggest an additional role for FOXO transcription factors: the regulation of lipid homeostasis by insulin. Here, we show that in flies, dFOXO regulates lipase 4 (dLip4), a Drosophila homologue of human acid lipases. dFOXO binds and activates the dLip4 promoter, in vitro and in vivo, and regulates dLip4 expression. In addition, dLip4 mRNA expression in flies is dependent on dFOXO. Our results support a model where dFOXO acts as a key modulator of lipid metabolism by insulin signaling and integrates insulin responses to glucose and lipid homeostasis.

Introduction

Obesity and diabetes are among the major causes of morbidity and mortality worldwide. Obesity is associated with an increased risk of developing insulin resistance and type 2 diabetes,1 and it derives from a chronic imbalance between energy intake (food consumption) and expenditure (basal metabolism, physical activity, etc.) (for a review, see Ref. 2). Excess energy is stored in the form of triacylglycerides (TAGs) as lipid droplets in the adipose tissue and other tissues, and under food deprivation, energy can be mobilized as free fatty acids (FFAs) to keep the energy supply constant. Lipases are the key enzymes that regulate TAG metabolism. Lipases are involved in a broad variety of reactions and differ in their amino acid sequence, tissue specificity, and cellular compartmentalization. The acid lipase family includes digestive lipases such as gastric lipase, lingual lipase, and gastric esterase as well as lysosomal acid lipases (LALs), which are lysosomal enzymes crucial for the intracellular hydrolysis of cholesteryl esters (CEs) and triglycerides. The neutral lipase family includes the lipoprotein lipase, which functions in the catabolism of triglyceride-rich lipoproteins in the circulation. Finally, the hormone-sensitive lipases are involved in mobilization of triglycerides in adipose tissues and have a critical role in regulating FFA metabolism and energy supply.

Lack of certain lipase activities may have catastrophic consequences. For example, LAL-deficient mice have altered lipid metabolism; they accumulate TAGs and CEs in the liver, spleen, and small intestine. These mice show that plasma FFA and CE significantly increased; they have higher food intake and develop mild insulin resistance. In addition, their white adipose tissue disappears at 6–8 months of age.3 In humans, Wolman disease and CE storage disease are caused by deficiencies in the lipase A (LAL) gene. They are both rare genetic disorders that result from excessive storage of CEs and triglycerides in tissues across the body, underscoring the importance of proper LAL function in metabolism.

A major molecular pathway regulating energy balance is the insulin signaling pathway. Insulin stimulates adipose tissue fat storage and suppresses fat mobilization (lipolysis), leading to storage of energy in the fed state. In mammals, FFAs are elevated in insulin-resistant states,4 and it has been suggested that central adiposity, by increasing circulating FFA, promotes the development of insulin resistance in the liver.5 This is due to the ability of FFA to interfere with glucose utilization,6 causing overproduction of glucose. Therefore, proper insulin signaling is required for an adequate regulation of TAG metabolism; both processes are tightly regulated, and deregulation of one of them impinges on the other.

FOXO transcription factors are key regulators in the insulin signaling cascade, which is largely conserved in metazoans.7,8 Among them, FOXO1 has been shown to be a central regulator of the metabolic responses to insulin. Thus, FOXO1 regulates expression of insulin-like growth factor binding protein-1,9 phosphoenolpyruvate carboxykinase,10 and glucose-6-phosphatase.11 In addition, FOXO1 has also been implicated in myoblast12 and adipocyte differentiation13 and pancreatric β-cell growth,14,15 cells that are all pivotal in the regulation of glucose metabolism by insulin. In flies, dFOXO, the fly orthologue of FOXO1, has also been shown to regulate insulin signaling,16,17 having an effect in metabolism, life span, and fertility.18, 19 Interestingly, links between dFOXO/FOXO1 and lipid metabolism have been established in worms,20 flies,21 and mammals.22,23 However, although these results suggest that dFOXO/FOXO1 could affect lipid metabolism, the molecular mechanisms are unknown.

Here, we show that in flies, dFOXO regulates lipase 4 (dLip4), a Drosophila homologue of human acid lipases. We show that dFOXO binds and activates the dLip4 promoter, in vitro and in vivo, regulating dLip4 expression. In addition, dLip4 mRNA expression in vivo is dependent on dFOXO. Our results indicate that dFOXO is a key modulator of lipid metabolism by insulin signaling and integrates insulin responses to glucose and lipid homeostasis.

Section snippets

dFOXO upregulates dLip4 in S2 cells

dLip4 (CG6113) was found in a screening for dFOXO targets in Drosophila S2 cells.16 dLip4 mRNA was upregulated 20-fold in cells overexpressing dFOXO A3 (a constitutively active form of dFOXO that is insensitive to insulin repression16), suggesting that dLip4 is a target of dFOXO. This result from microarrays was independently confirmed by quantitating dLip4 mRNA by quantitative polymerase chain reaction (qPCR). dLip4 mRNA was upregulated several fold in S2 cells stably transfected with dFOXO A3

Discussion

Our results show that Drosophila dLip4 belongs to the protein family of acid lipases, which includes LAL, gastric lipase, and endothelial lipase. dLip4 is a direct target of dFOXO regulation: results from luciferase reporter assays, band-shift analyses, and ChIP experiments show that transcription of dLip4 is directly activated by dFOXO binding to the dLip4 promoter. In addition, dFOXO is necessary for dLip4 expression in flies. When flies are starved for 4 days, dLip4 mRNA is upregulated, and

Constructs and Drosophila strains

Clone RE12242 contains the complete dLip4 open reading frame.

A 2.3-kb-long dLip4 promoter fragment was cloned in pGL3-basic vector (Promega), which has a firefly luciferase reporter gene (luc). The dLip4 promoter was amplified by PCR (F primer, TATTCCATGGGATCTATGGCTCGGCGGATG; R primer, TTATGCTAGCGTTGGTGAGTTCAAAGGGCGGG) and cloned in the pGL3-basic vector between the NcoI and NheI sites. Shorter versions of this promoter were produced by endonucleases HindIII, NsiI, and NdeI. All four constructs

Acknowledgements

We are grateful to Jaakko Mattila and Johanna Seppäläinen for suggestions and comments on the manuscript. This work was supported by grants to O.P. from the Finnish Diabetes Association, the Sigrid Juselius Foundation, the Novo Nordisk Foundation, and the Biocentrum Helsinki. The authors declare that they have no competing financial interests.

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    1

    Present addresses: T. Vihervaara, National Public Health Institute, Biomedicum, Helsinki, Finland; O. Puig, Molecular Profiling, Merck and Co., Inc., Rahway, NJ, USA.

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