Our research plan covered three specific aims:
1. Identification of cellular and molecular mechanisms of Protein kinase D (Pkd) 1 and 2 in adipocytes.
2. Uncovering of Pkds’ functions in the regulation of liver metabolism.
3. Identification of a novel, non-canonical signaling modules (phosphatases, E3 ligases, and DUBs) in the regulation of Pkd-dependent and independent lipid metabolism.
Obesity is associated with the accumulation of lipids and lipid precursors such as diacylglycerol (DAG) in multiple peripheral organs. These can aberrantly induce signaling cascades that suppress peripheral glucose uptake, promote lipogenesis and increase the output of free fatty acids (lipolysis) from adipocytes. Protein kinase D family members (Pkd1, 2, and 3) are DAG and Protein kinase C effectors that integrate multiple nutritional and hormonal inputs. We utilized targeted approach to elucidate the role of the individual PKDs in the regulation glucose and lipid metabolism (Aim 1 and 2).
In the framework of the SiCMetabol grant we identified PKD1 as a crucial factor promoting accumulation of lipids in adipocytes and the development of obesity as well as T2D. We showed that PKD1 promotes lipogenesis by inhibiting AMPK activity. Moreover, PKD1 suppresses adaptive thermogenesis by decreasing the expression of thermogenic genes as well as mitochondrial content and dynamics (Loeffler M et al, EMBO J., 2018). We also showed that expression of PKD3 is largely restricted to liver, where it PKD3 suppresses AKT- and mTOR-dependent signaling in hepatocytes and therefore deletion of PKD3 protects against diet-induced insulin resistance (Mayer A et al, Science Signaling, 2019). Further analysis suggested that PKD3 promotes PKA activity in the liver (Loza-Valdes A et al, Life Science Alliance, 2021). We also demonstrated that PKD2 does not affect neither thermogenesis in adipocytes nor liver metabolism. However, similarly to PKD1, ablation of PKD2 in mice results in resistance to a high-fat diet (HFD)-induced obesity and T2D since ablation of PKD2 activity leads to reduced lipid absorption in the intestine, changes in physical properties of the faces, and an elevated amount of energy excreted by mice. Furthermore, PKD2 phosphorylates APOA4 to promote chylomicron size and therefore TG output from enterocytes. PKD2 deficiency in the intestine is also associated with an improved microbiota diversity and the presence of bacterial species which are protecting from diabetes. Importantly, targeting PKD2 with small molecule inhibitors results in the amelioration of obesity and prevents the development of T2D. Finally, we showed that the degree of PKD2 phosphorylation in the intestine of the patients who were subjected to bariatric surgery correlates with the TG levels in the blood, and the levels of glycated hemoglobin (HbA1c) which is a predictor of diabetes, confirming that our findings are also relevant to human subjects (Trujillo-Viera J et al, EMBO Mol Med, 2021). This work served as a base for the patent application on the usage of PKD-inhibitors for treatment of obesity and diabetes (European Patent Application No. 21461519.7). We have also summarized current state of knowledge about the impact of DAG-activated Protein kinase C (PKC) and Protein kinase D (PKD) isoforms on the regulation of glucose and lipid handling in the liver, adipose tissue, skeletal muscles, heart, immune cells, pancreatic β cells, and central nervous system and their mode of action in these organs. We also described their implication in the development of obesity and diabetes. Finally, we discussed the potential of targeting PKCs and PKDs for the treatment of metabolic diseases (Kolczynska K et al, Lipids Health Dis., 2020).
We also performed siRNA-base high throughput screens to identify components of ubiquitination machinery which regulate adipocyte function (Aim 3). Most probably due to the high redundancy between different E3 ligases as well as DUBs, the specific components have not been identified. However, a complementary biochemical approach revealed a number of differentially ubiquitinated proteins during the induction of lipolysis. These data will be published soon.
As in-lab collaboration, ERC-funded scientists helped in the project where we performed high-throughput screening to identify kinases regulating adipocyte function (lipolysis). We showed that a surprisingly high number of kinases (nearly 60) co-regulates this process. Silencing of Extracellular regulated kinase 3 (ERK3), also known as MAPK6, resulted in the strongest suppression of the lipolysis. We showed that at resting condition ERK3 is a highly unstable, but upon β-adrenergic stimulation it forms a complex with another kinase, MAP Kinase-Activated Protein Kinase 5 (MK5). This stabilizes both kinases and promotes their activity to drive lipolysis by promoting ATGL transcription. Deletion of ERK3 in adipose tissue of mice results not only in suppression of β-adrenergic-induced lipolysis but also in conversion of white into beige adipocytes and consequently elevated energy expenditure and resistance to obesity (El-Merahbi R et al, Genes&Dev, 2020).
