Final Report Summary - STRESSMETABOL (Stress signaling mechanisms in metabolism and inflammation and related disorders)
The laboratory focuses on cellular and molecular mechanisms underlying adaptation to environmental stress in mammalian cells, focusing on nutritional and inflammatory stress. Different cell types employ unique evolutionary programmed strategies to cope with such stresses. Importantly, there is pivotal evidence that crosstalks and overlaps between canonical immune and metabolic pathways exist. While malnutrition compromises proper immune responses, overnutrition activates them. On the other hand, it is well known that acute and chronic inflammatory conditions can impact on whole body metabolism. Deregulation of these interlinked pathways and processes confer human metabolic and inflammatory diseases, respectively. For these reasons, we employ complementary approaches using different cellular model systems in vitro and in vivo.
a. The pancreatic β cell : The β cell harbors an exemplary nutrient sensing machinery that is coupled to secretion of insulin, a major anabolic hormone. Glucose, amino acids and lipids all can stimulate insulin release after a meal. In the absence of such nutrients during fasting, insulin secretion is attenuated. While mechanisms governing regulation of insulin secretion in response to nutrients have been widely investigated, relatively little is known about how nutrients impact on other basic cellular processes such as insulin granule biosynthesis, autophagy and cellular growth, deregulation of all of which have been demonstrated to be involved in β cell failure in type 2 diabetes (T2D). For these reasons, we have chosen to enter this particular research niche in the β cell field.
We have recently discovered a new nutrient-dependent control mechanism governing insulin granule formation. Protein Kinase D (PKD) at the Golgi is activated upon nutrients to phosphorylate Arfaptin-1, a Bin-Amphiphysin-Rvs (BAR) domain containing protein. Upon phosphorylation, Arfaptin-1 is released from the neck of budding insulin granules allowing scission at the Golgi to occur. Interfering with this mechanism affects replenishment of the releasable insulin granule pool and as a consequence insulin secretion.
A prevailing concept in cell biology is that enhanced degradation of cellular components through autophagy can counteract energy depletion caused by shortage of environmental nutrients. We recently discovered that β cells employ a very distinct and so far unknown mechanism to adapt to nutrient depletion. β cells induce specific degradation of newly formed insulin granules at lysosomes and suppress autophagy upon nutrient withdrawal in a PKD-dependent manner. Insulin granule degradation allows for generation of intracellular nutrients and in the same time avoids release of these granules. In fact, secreting insulin would have detrimental consequences in living organisms in times of fasting as insulin lowers nutrients in the blood circulation. Importantly, we have now evidence that degradation of insulin granules is dramatically enhanced in beta cells of diabetic islets (unpublished data). This does not only lead to loss of insulin but also to chronic suppression of autophagy. Autophagy is indeed important to maintain β cell function, in particular in a situation of high insulin demands such as in T2D. Thus, this mechanism represents an important evolutionary adaption for nutrient-deprived β cells. However, its deregulation may actually contribute to β cell failure in T2D. Our findings may thus change the current paradigm in which decreased β cell survival and/or dedifferentiation mainly accounted for insulin loss in T2D.
b. The hepatocyte : The hepatocyte represents a major source of energy metabolites in times environmental nutrients are lacking. While direct sensing of nutrients is still elusive, signaling in response to hormonal inputs is relatively well established in hepatocytes. Regulation of hepatic fasting and feeding pathways requires fast cellular signaling mechanisms through posttranslational modifications. Kinase-dependent phosphorylation has been widely explored. In contrast, ubiquitylation as a way to engage signaling events independent of protein degradation is very poorly assessed in this context. We therefore conducted a global proteomic analysis to identify metabolic pathways modified by ubiquitylation. To this end, we used livers of mice subjected to a fasting – refeeding protocol. So far, we explored more in detail one interesting hit, complement C3 that is produced in and secreted from hepatocytes to exert innate immune functions. We discovered that C3 under feeding conditions exited from the endoplasmic reticulum (ER) and was ubiquitylated to trigger metabolic signaling at lysosomes. We also found that C3 release from hepatocytes was nutrient-dependent. This work thus uncovered how nutrients may impact on immune and metabolic functions of C3.
c. The macrophage : The inflammasome multiprotein complexes in macrophages sense sterile tissue damage and infectious agents to propagate innate immune responses. The NLRP3 inflammasome is unique in the sense that it is capable of detecting a broad variety of stress agents. However, mechanisms leading to the assembly and activation of the Nlrp3 inflammasome remain elusive. Recruitment of Nlrp3 to mitochondria-associated endoplasmic reticulum membranes (MAMs) and its activation by MAM-derived effector molecules is an emerging concept. We showed that the Golgi is another key element in the Nlrp3 inflammasome activation cascade. In response to inflammasome activators, production of diacylglycerol (DAG) rapidly increased at the Golgi in a phospholipase C-dependent manner. When PKD, a key effector protein of DAG at the Golgi, oligomerized Nlrp3 remained stuck at MAMs close to the Golgi blocking its activation. PKD phosphorylated oligomerized Nlrp3 to release it from MAMs, which was required for subsequent inflammasome maturation. Inhibition of PKD in blood mononuclear cells from human cryopyrin-associated periodic syndrome (CAPS) patients, carrying auto-activatory mutations in the Nlrp3 gene, attenuated inflammasome activation. This work thus uncovered that Golgi-mediated signaling is important for Nlrp3 inflammasome activation.
a. The pancreatic β cell : The β cell harbors an exemplary nutrient sensing machinery that is coupled to secretion of insulin, a major anabolic hormone. Glucose, amino acids and lipids all can stimulate insulin release after a meal. In the absence of such nutrients during fasting, insulin secretion is attenuated. While mechanisms governing regulation of insulin secretion in response to nutrients have been widely investigated, relatively little is known about how nutrients impact on other basic cellular processes such as insulin granule biosynthesis, autophagy and cellular growth, deregulation of all of which have been demonstrated to be involved in β cell failure in type 2 diabetes (T2D). For these reasons, we have chosen to enter this particular research niche in the β cell field.
We have recently discovered a new nutrient-dependent control mechanism governing insulin granule formation. Protein Kinase D (PKD) at the Golgi is activated upon nutrients to phosphorylate Arfaptin-1, a Bin-Amphiphysin-Rvs (BAR) domain containing protein. Upon phosphorylation, Arfaptin-1 is released from the neck of budding insulin granules allowing scission at the Golgi to occur. Interfering with this mechanism affects replenishment of the releasable insulin granule pool and as a consequence insulin secretion.
A prevailing concept in cell biology is that enhanced degradation of cellular components through autophagy can counteract energy depletion caused by shortage of environmental nutrients. We recently discovered that β cells employ a very distinct and so far unknown mechanism to adapt to nutrient depletion. β cells induce specific degradation of newly formed insulin granules at lysosomes and suppress autophagy upon nutrient withdrawal in a PKD-dependent manner. Insulin granule degradation allows for generation of intracellular nutrients and in the same time avoids release of these granules. In fact, secreting insulin would have detrimental consequences in living organisms in times of fasting as insulin lowers nutrients in the blood circulation. Importantly, we have now evidence that degradation of insulin granules is dramatically enhanced in beta cells of diabetic islets (unpublished data). This does not only lead to loss of insulin but also to chronic suppression of autophagy. Autophagy is indeed important to maintain β cell function, in particular in a situation of high insulin demands such as in T2D. Thus, this mechanism represents an important evolutionary adaption for nutrient-deprived β cells. However, its deregulation may actually contribute to β cell failure in T2D. Our findings may thus change the current paradigm in which decreased β cell survival and/or dedifferentiation mainly accounted for insulin loss in T2D.
b. The hepatocyte : The hepatocyte represents a major source of energy metabolites in times environmental nutrients are lacking. While direct sensing of nutrients is still elusive, signaling in response to hormonal inputs is relatively well established in hepatocytes. Regulation of hepatic fasting and feeding pathways requires fast cellular signaling mechanisms through posttranslational modifications. Kinase-dependent phosphorylation has been widely explored. In contrast, ubiquitylation as a way to engage signaling events independent of protein degradation is very poorly assessed in this context. We therefore conducted a global proteomic analysis to identify metabolic pathways modified by ubiquitylation. To this end, we used livers of mice subjected to a fasting – refeeding protocol. So far, we explored more in detail one interesting hit, complement C3 that is produced in and secreted from hepatocytes to exert innate immune functions. We discovered that C3 under feeding conditions exited from the endoplasmic reticulum (ER) and was ubiquitylated to trigger metabolic signaling at lysosomes. We also found that C3 release from hepatocytes was nutrient-dependent. This work thus uncovered how nutrients may impact on immune and metabolic functions of C3.
c. The macrophage : The inflammasome multiprotein complexes in macrophages sense sterile tissue damage and infectious agents to propagate innate immune responses. The NLRP3 inflammasome is unique in the sense that it is capable of detecting a broad variety of stress agents. However, mechanisms leading to the assembly and activation of the Nlrp3 inflammasome remain elusive. Recruitment of Nlrp3 to mitochondria-associated endoplasmic reticulum membranes (MAMs) and its activation by MAM-derived effector molecules is an emerging concept. We showed that the Golgi is another key element in the Nlrp3 inflammasome activation cascade. In response to inflammasome activators, production of diacylglycerol (DAG) rapidly increased at the Golgi in a phospholipase C-dependent manner. When PKD, a key effector protein of DAG at the Golgi, oligomerized Nlrp3 remained stuck at MAMs close to the Golgi blocking its activation. PKD phosphorylated oligomerized Nlrp3 to release it from MAMs, which was required for subsequent inflammasome maturation. Inhibition of PKD in blood mononuclear cells from human cryopyrin-associated periodic syndrome (CAPS) patients, carrying auto-activatory mutations in the Nlrp3 gene, attenuated inflammasome activation. This work thus uncovered that Golgi-mediated signaling is important for Nlrp3 inflammasome activation.