InteractiveFly: GeneBrief

Neural Lazarillo: Biological Overview | References

Metabolic homeostasis in metazoans is regulated by endocrine control of insulin/IGF signaling (IIS) activity. Stress and inflammatory signaling pathways -- such as Jun-N-terminal Kinase (JNK) signaling -- repress IIS, curtailing anabolic processes to promote stress tolerance and extend lifespan. While this interaction constitutes an adaptive response that allows managing energy resources under stress conditions, excessive JNK activity in adipose tissue of vertebrates has been found to cause insulin resistance, promoting type II diabetes. Thus, the interaction between JNK and IIS has to be tightly regulated to ensure proper metabolic adaptation to environmental challenges. This study identified a new regulatory mechanism by which JNK influences metabolism systemically. JNK signaling is required for metabolic homeostasis in flies and that this function is mediated by the Drosophila Lipocalin family member Neural Lazarillo (NLaz), a homologue of vertebrate Apolipoprotein D (ApoD) and Retinol Binding Protein 4 (RBP4). Lipocalins are emerging as central regulators of peripheral insulin sensitivity and have been implicated in metabolic diseases. NLaz is transcriptionally regulated by JNK signaling and is required for JNK-mediated stress and starvation tolerance. Loss of NLaz function reduces stress resistance and lifespan, while its over-expression represses growth, promotes stress tolerance and extends lifespan -- phenotypes that are consistent with reduced IIS activity. Accordingly, this study found that NLaz represses IIS activity in larvae and adult flies. The results show that JNK-NLaz signaling antagonizes IIS and is critical for metabolic adaptation of the organism to environmental challenges. The JNK pathway and Lipocalins are structurally and functionally conserved, suggesting that similar interactions represent an evolutionarily conserved system for the control of metabolic homeostasis (Hull-Thompson, 2009).

System-wide coordination of cellular energy consumption and storage is crucial to maintain metabolic homeostasis in multicellular organisms. It is becoming increasingly apparent that endocrine mechanisms that are required for this coordination impact the long-term health of adult animals and significantly influence lifespan and environmental stress tolerance. Insulin/IGF signaling (IIS) is central to this regulation, as loss of Insulin signaling activity impairs metabolic homeostasis, but induces stress tolerance and increases lifespan in a variety of model organisms. Interestingly, environmental stress and cellular damage can systemically repress IIS activity, suggesting the existence of adaptive response mechanisms by which metazoans manage energy resources in times of need. The mechanism(s) and mediators of this endocrine regulatory system are only beginning to be understood (Hull-Thompson, 2009).

Studies in flies and worms have recently identified the stress-responsive Jun-N-terminal Kinase (JNK) signaling pathway as an important component of such an adaptive metabolic response to stress. JNK activation, which can be induced by a variety of environmental stressors, including oxidative stress, represses IIS activity, extending lifespan but limiting growth. Interestingly, similar effects of JNK signaling are also observed in mammals, in which it represses Insulin signal transduction by various mechanisms, including an inhibitory phosphorylation of Ser-307 of the insulin receptor substrate, as well as activation of the transcription factor FoxO. This inhibition contributes to Insulin resistance and the metabolic syndrome in obese mice, suggesting that chronic inflammatory processes (which result in activation of JNK signaling) are central to the etiology of metabolic diseases in obese individuals (Hull-Thompson, 2009).

Endocrine interactions between Insulin-producing and various Insulin-responsive tissues are likely to coordinate the adaptive metabolic response described above. JNK-mediated activation of Foxo in Insulin Producing Cells (IPCs) of flies, for example, represses the expression of insulin-like peptide 2 (dilp2), regulating growth and longevity. At the same time, Foxo activation in the fatbody results in lifespan extension, presumably by an endocrine mechanism that feeds back to IPCs (Hull-Thompson, 2009).

Adipose tissue is increasingly being recognized as an important regulator of metabolic homeostasis. It secretes a variety of so-called adipokines, including the inflammatory cytokine TNF-alpha (Hotamisligil, 2006). TNF-alpha activates JNK signaling, contributing to JNK-mediated insulin resistance in mouse models for obesity. JNK activation in adipose tissue further induces expression of IL-6, which specifically induces Insulin resistance in the liver. While the chronic inhibition of insulin signaling by adipose-derived inflammatory cytokines thus has deleterious effects in obese individuals, it is likely that such endocrine interactions have evolved to govern metabolic homeostasis systemically in an adaptive manner (Hotamisligil, 2006). Supporting this view, adipose tissue is an important regulator of lifespan in worms, flies, and mice, and it is emerging that systemic inhibition of Insulin signaling by adipose-derived factors is involved in this effect (Hull-Thompson, 2009).

An endocrine role for adipose tissue in metabolic regulation has further been demonstrated in mice with adipose-specific deletion of the glucose transporter GLUT4, in which secretion of the Lipocalin family member RBP4 from fat cells induces insulin resistance throughout the organism (Wang, 2005; Abel, 2001; Yang, 2005). Such an endocrine system is expected to be adaptive, since it preserves glucose for only the most essential functions during starvation or environmental stress. At the same time, mis-regulation of this system is likely to contribute to metabolic diseases like type II diabetes. Accordingly, increased serum levels of RBP4 are found in obese and diabetic individuals (Graham, 2006), and polymorphisms in the rbp4 locus are associated with type II diabetes (Munkhtulga, 2006; Hull-Thompson, 2009 and references therein).

The Lipocalins are a large family of mostly secreted proteins that bind small hydrophobic ligands (Flower, 1996; Åkerström, 2006). Lipocalin family members are characterized by a low sequence similarity (reflecting diversification of biological functions), but a highly conserved tertiary protein structure and similar exon/intron structures of their genes (Ganfornina, 2000; Sanchez, 2003). Recent studies implicate various Lipocalins in the regulation of systemic insulin action and of stress responses (Yang, 2005, vanDam, 2007; Yan, 2007; Ganfornina, 2008; Muffat, 2008). Interestingly, the neuroprotective Lipocalin ApoD is strongly induced in aging mice, rhesus macaques and humans, suggesting evolutionarily conserved regulation of this gene, an it induces insulin resistance when overexpressed in the mouse brain (Hull-Thompson, 2009 and references therein).

The Drosophila genome contains three Lipocalin genes: NLaz, GLaz, and karl. Unlike the protein Lazarillo in more ancient insect lineages, which is GPI-anchored to the cell membrane of neurons (Ganfornina, 1995), all Drosophila Lipocalins are secreted extracellular proteins, like ApoD and all other vertebrate Lipocalins. Recent studies have identified an important role for GLaz in stress resistance and lifespan control as well as in the regulation of lipid storage (Walker, 2006; Sanchez, 2006). While the function of NLaz remains unclear, in situ hybridization in Drosophila embryos shows that it is expressed in a subset of neuronal cells, and, interestingly, in the developing fat body (Sanchez, 2006), indicating a potential role in the systemic regulation of metabolism (Hull-Thompson, 2009).

This study shows that NLaz transcription is induced by oxidative stress and by JNK signaling in the fat body, influencing metabolic homeostasis in the fly. Importantly, NLaz induces stress and starvation tolerance downstream of JNK signaling, and negatively regulates Insulin signaling, disrupting glucose homeostasis, repressing growth, and extending lifespan. The results thus indicate that induction of NLaz mediates the antagonistic interaction between JNK and Insulin signaling in flies, acting as part of a stress response mechanism that adjusts metabolism and growth in response to environmental insults (Hull-Thompson, 2009).

The findings support a role for JNK-mediated NLaz induction in the fatbody as a central part of an adaptive endocrine system that coordinates metabolism in response to environmental stress by regulating insulin sensitivity of peripheral tissues. Recent studies have highlighted the role of adipose-derived endocrine factors in such adaptive responses. For example, reducing IIS activity or over-expressing Foxo specifically in adipose tissue leads to lifespan extension and stress tolerance in flies, mice and worms, presumably mediated by systemic repression of IIS. Furthermore, amino acid deprivation of Drosophila fat body cells leads to marked decreases in PI3K activity in wing imaginal discs and in the epidermis. In vertebrates, in contrast, excessive JNK activation in adipose tissue induces insulin resistance in the periphery, promoting Type II diabetes. The current results implicate NLaz as a mediator of such systemic repression of IIS activity by adipose tissue (Hull-Thompson, 2009).

JNK-mediated repression of IIS in flies is thus not only mediated by its function in IPCs, where it represses dilp2 transcription , but also by adipose-specific induction of NLaz, which then inhibits IIS activity in insulin target tissues. This dual antagonism of IIS by JNK is intriguing, as it indicates that adaptive regulation of metabolism requires coordinated control of both insulin-like peptide production and peripheral insulin sensitivity. How the relative contribution of these effects regulates the organism's metabolic homeostasis, stress resistance and lifespan, is an interesting question that will require further investigation (Hull-Thompson, 2009).

Vertebrate Lipocalins have also been implicated in the modulation of insulin action, and recent studies suggest a protective role of these molecules under diverse stress conditions. This function of Lipocalins thus emerges as an evolutionarily conserved adaptive mechanism, and this work integrates this mechanism into the known antagonism between JNK and IIS. Based on the evolutionary conservation of this antagonism it is tempting to speculate that vertebrate Lipocalins also act as effectors of JNK in the regulation of systemic insulin sensitivity, with important implications for potential therapeutic targeting of these molecules (Hull-Thompson, 2009).

While generally promoting metabolic homeostasis and stress tolerance, functional specialization of different Lipocalin family members is expected due to their high sequence divergence. Accordingly, the data show that the Lipocalins present in Drosophila differ in regulation and function. While NLaz and GLaz both regulate stress sensitivity, only NLaz was found to be regulated by JNK signaling. Regulation of Karl, on the other hand, does not influence starvation tolerance (as NLaz does), but promotes resistance against infection by E. faecalis. Further investigation of this diversification of Lipocalin function promises to provide important insight into the systemic regulation of adaptation to diverse environmental challenges. Of particular interest will be to assess the role of Karl as a potential regulator of IIS during infection. Infection with Mycobacterium Marinum can result in significant repression of IIS activity, leading to phenotypes similar to wasting disease. It is intriguing to speculate that excessive JNK-induced Karl expression may cause this pathology (Hull-Thompson, 2009).

In humans, dysregulation of Lipocalins has been correlated with obesity, insulin resistance, and type II diabetes. The cause for this mis-regulation of Lipocalin expression remains unclear, however. The current results implicate JNK signaling, which is activated chronically in obese conditions, as a possible cause. The finding that mammalian lipocalin-2, which impairs insulin action, is induced by the JNK activator TNFalpha, is especially intriguing. Additional studies in vertebrates, as well as in the Drosophila model, will provide further insight into the physiological role of Lipocalins, their regulation by stress signaling, as well as their interaction with Insulin signaling. As Lipocalins are secreted molecules that bind hydrophobic ligands, it is further crucial to identify their physiological ligands in an effort to understand the mechanism(s) by which IIS activity is antagonized by Lipocalins. Such insight promises to provide a deeper understanding of the coordination of metabolic adaptation in metazoans as well as of the etiology of diabetes and other metabolic diseases (Hull-Thompson, 2009).

Sex-dependent modulation of longevity by two Drosophila homologues of human Apolipoprotein D, GLaz and NLaz

Apolipoprotein D (ApoD), a member of the Lipocalin family, is the gene most up-regulated with age in the mammalian brain. Its expression strongly correlates with aging-associated neurodegenerative and metabolic diseases. Two homologues of ApoD expressed in the Drosophila brain, Glial Lazarillo (GLaz) and Neural Lazarillo (NLaz), are known to alter longevity in male flies. However, sex differences in the aging process have not been explored so far for these genes. This study demonstrates that NLaz alters lifespan in both sexes, but unexpectedly the lack of GLaz influences longevity in a sex-specific way, reducing longevity in males but not in females. While NLaz has metabolic functions similar to ApoD, the regulation of GLaz expression upon aging is the closest to ApoD in the aging brain. A multivariate analysis of physiological parameters relevant to lifespan modulation uncovers both common and specialized functions for the two Lipocalins, and reveals that changes in protein homeostasis account for the observed sex-specific patterns of longevity. The response to oxidative stress and accumulation of lipid peroxides are among their common functions, while the transcriptional and behavioral response to starvation, the pattern of daily locomotor activity, storage of fat along aging, fertility, and courtship behavior differentiate NLaz from GLaz mutants. Food composition is an important environmental parameter influencing stress resistance and reproductive phenotypes of both Lipocalin mutants. Since ApoD shares many properties with the common ancestor of invertebrate Lipocalins, this global comparison with both GLaz and NLaz should be of use in understanding the complex functions of ApoD in mammalian aging and neurodegeneration (Ruiz, 2011).

This work highlights that the two Drosophila Lipocalins expressed in the nervous system have both functional redundancies and specializations. The response to oxidative stress and accumulation of lipid peroxides are among their common functions, while the transcriptional and behavioral response to starvation, the pattern of daily locomotor activity, storage of fat along aging, fertility, and courtship behavior differentiate NLaz from GLaz mutants. This framework is guiding current research, as more details need to be elucidated. However, it can already be asked how many of these shared or unique functions are conserved in the mammalian homologues also expressed in the brain (Ruiz, 2011).

The basal position of ApoD in the phylogenetic tree of vertebrate Lipocalins suggests that ApoD shares many properties with the common ancestor of invertebrate Lipocalins. However, it has to be taken into account that neither GLaz nor NLaz is a true orthologue of ApoD. Molecular phylogenetic analyses strongly suggest that the Drosophila Lipocalins originated from an independent duplication event, taking place within the invertebrate lineage. Subsequently, the resulting genes have diverged both in their protein coding sequence and their regulatory sequences (Ruiz, 2011).

Since ApoD in the adult mammalian brain is expressed mainly in glial cells, one might be tempted to directly conclude that GLaz is the closest Drosophila homologue. Furthermore, the expression data reported in this study strongly suggest that GLaz regulation through aging is most similar to the robust increase of mammalian ApoD in the aged brain (de Magalhaes; Loerch, 2008). Interestingly, ApoD is most similar to GLaz in protein sequence, but to NLaz in the intron-exon structure of the gene. ApoD has been shown to be up-regulated by oxidative stress in astrocytes, and this induction is mediated through the JNK pathway (Bajo-Grañeras, Ganfornina and Sanchez, unpublished observations cited in Ruiz, 2011), comparable to the NLaz JNK-mediated induction by stress in Drosophila. Thus, if the Drosophila data is extrapolated to learn about the functions of ApoD in mammalian aging and neurodegeneration the global comparison with both GLaz and NLaz, as reported in this study, must be taken into account (Ruiz, 2011).

To understand the multigenic control of aging, it must be taken into account that a layer of complexity is added due to the fact that each gene has pleiotropic effects, and each one has differing degrees of specialization or redundancy with members of the same gene family. This fact represents a daunting complication for the task of predicting the actions of putative anti-aging or anti-neurodegeneration drugs. However, complexity should not prevent investigating until a comprehensive understanding of the aging process is available (Ruiz, 2011).

Search PubMed for articles about Drosophila Neural Lazarillo

Abel, E. D., et al. (2001). Adipose-selective targeting of the GLUT4 gene impairs insulin action in muscle and liver. Nature 409: 729-733. PubMed Citation: 11217863

Åkerström, B., Borregaard, N., Flower, D. R. and Salier, J. P. (2006). Austin, TX: Lipocalins, Landes Bioscience.

de Magalhaes, J. P. Curado, J. and Church, G. M. (2009). Meta-analysis of age-related gene expression profiles identifies common signatures of aging. Bioinformatics 25: 875-881. PubMed Citation: 19189975

Flower, D. R. (1996). The lipocalin protein family: structure and function. Biochem J. 318 (Pt 1): 1-14. PubMed Citation: 8761444

Ganfornina, M. D., Sanchez, D. and Bastiani, M. J. (1995). Lazarillo, a new GPI-linked surface lipocalin, is restricted to a subset of neurons in the grasshopper embryo. Development 121: 123-134. PubMed Citation: 7867494

Ganfornina, M. D., Gutierrez, G., Bastiani, M. and Sanchez, D. (2000). A phylogenetic analysis of the lipocalin protein family. Mol. Biol. Evol. 17: 114-126. PubMed Citation: 10666711

Ganfornina, M. D., et al. (2008). Apolipoprotein D is involved in the mechanisms regulating protection from oxidative stress. Aging Cell 7(4): 506-15. PubMed Citation: 18419796

Graham, T. E., et al. (2006). Retinol-binding protein 4 and insulin resistance in lean, obese, and diabetic subjects. N. Engl. J. Med. 354: 2552-2563. PubMed Citation: 16775236

Hotamisligil, G. S. (2006). Inflammation and metabolic disorders. Nature 444: 860-867. PubMed Citation: 17167474

Hull-Thompson, J., Muffat, J., Sanchez, D., Walker, D. W., Benzer, S., Ganfornina, M. D. and Jasper, H. (2009). Control of metabolic homeostasis by stress signaling is mediated by the lipocalin NLaz. PLoS Genet. 5(4): e1000460. PubMed Citation: 19390610

Loerch, P. M., et al. Evolution of the aging brain transcriptome and synaptic regulation, PLoS ONE 3: e3329. PubMed Citation: 18830410

Muffat, J., Walker, D. W. and Benzer, S. (2008). Human ApoD, an apolipoprotein up-regulated in neurodegenerative diseases, extends lifespan and increases stress resistance in Drosophila. Proc. Natl. Acad. Sci. 105: 7088-7093. PubMed Citation: 18458334

Munkhtulga, L., et al. (2006). Identification of a regulatory SNP in the retinol binding protein 4 gene associated with type 2 diabetes in Mongolia. Hum Genet. 120(6): 879-88. PubMed Citation: 17006670

Ruiz, M., Sanchez, D., Canal, I., Acebes, A. and Ganfornina, M. D. (2011). Sex-dependent modulation of longevity by two Drosophila homologues of human Apolipoprotein D, GLaz and NLaz. Exp. Gerontol. 46(7): 579-89. PubMed Citation: 21376794

Sanchez, D., Ganfornina, M. D., Gutierrez, G. and Marin, A. (2003). Exon-intron structure and evolution of the Lipocalin gene family. Mol. Biol. Evol. 20: 775-783. PubMed Citation: 12679526

Sanchez, D., et al. (2006). Loss of glial lazarillo, a homolog of apolipoprotein D, reduces lifespan and stress resistance in Drosophila. Curr. Biol. 16: 680-686. PubMed Citation: 16581513

van Dam, R. M. and Hu, F. B. (2007). Lipocalins and insulin resistance: etiological role of retinol-binding protein 4 and lipocalin-2? Clin Chem 53: 5-7. PubMed Citation: 17202496

Walker, D. W., Muffat, J., Rundel, C. and Benzer, S. (2006). Overexpression of a Drosophila homolog of apolipoprotein D leads to increased stress resistance and extended lifespan. Curr. Biol. 16: 674-679. PubMed Citation: 16581512

Wang, M. C., Bohmann, D. and Jasper, H. (2005). JNK extends life span and limits growth by antagonizing cellular and organism-wide responses to insulin signaling. Cell 121: 115-125. PubMed Citation: 15820683

Yan, Q. W., et al. (2007). The adipokine lipocalin 2 is regulated by obesity and promotes insulin resistance. Diabetes 56: 2533-2540. PubMed Citation: 17639021

Yang, Q., et al. (2005). Serum retinol binding protein 4 contributes to insulin resistance in obesity and type 2 diabetes. Nature 436: 356-362. PubMed Citation: 16034410

date revised: 28 December 2011

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