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Protein kinase D1 deletion in adipocytes enhances energy dissipation and protects against adiposity
(2018)
Nutrient overload in combination with decreased energy dissipation promotes obesity and diabetes. Obesity results in a hormonal imbalance, which among others, activates G-protein coupled receptors utilizing diacylglycerol (DAG) as secondary messenger. Protein kinase D1 (PKD1) is a DAG effector which integrates multiple nutritional and hormonal inputs, but its physiological role in adipocytes is unknown. Here, we show that PKD1 promotes lipogenesis and suppresses mitochondrial fragmentation, biogenesis, respiration, and energy dissipation in an AMP-activated protein kinase (AMPK)-dependent manner. Moreover, mice lacking PKD1 in adipocytes are resistant to diet-induced obesity due to elevated energy expenditure. Beiging of adipocytes promotes energy expenditure and counteracts obesity. Consistently, deletion of PKD1 promotes expression of the β3-adrenergic receptor (ADRB3) in a CCAAT/enhancerbinding protein (C/EBP)-α and δ-dependent manner, which leads to the elevated expression of beige markers in adipocytes and subcutaneous adipose tissue. Finally, deletion of PKD1 in adipocytes improves insulin sensitivity and ameliorates liver steatosis. Thus, loss of PKD1 in adipocytes increases energy dissipation by several complementary mechanisms and might represent an attractive strategy to treat obesity and its related complications.
Background
Bone morphogenetic protein (BMP)-2 and growth and differentiation factor (GDF)-5 are two related transforming growth factor (TGF)-β family members with important functions in embryonic development and tissue homeostasis. BMP-2 is best known for its osteoinductive properties whereas GDF-5—as evident from its alternative name, cartilage derived morphogenetic protein 1—plays an important role in the formation of cartilage. In spite of these differences both factors signal by binding to the same subset of BMP receptors, raising the question how these different functionalities are generated. The largest difference in receptor binding is observed in the interaction with the type I receptor BMPR-IA. GDF-5, in contrast to BMP-2, shows preferential binding to the isoform BMPR-IB, which is abrogated by a single amino acid (A57R) substitution. The resulting variant, GDF-5 R57A, represents a “BMP-2 mimic” with respect to BMP receptor binding. In this study we thus wanted to analyze whether the two growth factors can induce distinct signals via an identically composed receptor.
Results
Unexpectedly and dependent on the cellular context, GDF-5 R57A showed clear differences in its activity compared to BMP-2. In ATDC-5 cells, both ligands induced alkaline phosphatase (ALP) expression with similar potency. But in C2C12 cells, the BMP-2 mimic GDF-5 R57A (and also wild-type GDF-5) clearly antagonized BMP-2-mediated ALP expression, despite signaling in both cell lines occurring solely via BMPR-IA. The BMP-2- antagonizing properties of GDF-5 and GDF-5 R57A could also be observed in vivo when implanting BMP-2 and either one of the two GDF-5 ligands simultaneously at heterotopic sites.
Conclusions
Although comparison of the crystal structures of the GDF-5 R57A:BMPR-IAEC- and BMP-2:BMPR-IAEC complex revealed small ligand-specific differences, these cannot account for the different signaling characteristics because the complexes seem identical in both differently reacting cell lines. We thus predict an additional component, most likely a not yet identified GDF-5-specific co-receptor, which alters the output of the signaling complexes. Hence the presence or absence of this component then switches GDF-5′s signaling capabilities to act either similar to BMP-2 or as a BMP-2 antagonist. These findings might shed new light on the role of GDF-5, e.g., in cartilage maintenance and/or limb development in that it might act as an inhibitor of signaling events initiated by other BMPs.
Deregulated expression of MYC is a driver of colorectal carcinogenesis, necessitating novel strategies to inhibit MYC function. The ubiquitin ligase HUWE1 (HECTH9, ARF-BP1, MULE) associates with both MYC and the MYC-associated protein MIZ1. We show here that HUWE1 is required for growth of colorectal cancer cells in culture and in orthotopic xenograft models. Using high-throughput screening, we identify small molecule inhibitors of HUWE1, which inhibit MYC-dependent transactivation in colorectal cancer cells, but not in stem and normal colon epithelial cells. Inhibition of HUWE1 stabilizes MIZ1. MIZ1 globally accumulates on MYC target genes and contributes to repression of MYC-activated target genes upon HUWE1 inhibition. Our data show that transcriptional activation by MYC in colon cancer cells requires the continuous degradation of MIZ1 and identify a novel principle that allows for inhibition of MYC function in tumor cells.
Hepatic activation of protein kinase C (PKC) isoforms by diacylglycerol (DAG) promotes insulin resistance and contributes to the development of type 2 diabetes (T2D). The closely related protein kinase D (PKD) isoforms act as effectors for DAG and PKC. Here, we showed that PKD3 was the predominant PKD isoform expressed in hepatocytes and was activated by lipid overload. PKD3 suppressed the activity of downstream insulin effectors including the kinase AKT and mechanistic target of rapamycin complex 1 and 2 (mTORC1 and mTORC2). Hepatic deletion of PKD3 in mice improved insulin-induced glucose tolerance. However, increased insulin signaling in the absence of PKD3 promoted lipogenesis mediated by SREBP (sterol regulatory element-binding protein) and consequently increased triglyceride and cholesterol content in the livers of PKD3-deficient mice fed a high-fat diet. Conversely, hepatic-specific overexpression of a constitutively active PKD3 mutant suppressed insulin-induced signaling and caused insulin resistance. Our results indicate that PKD3 provides feedback on hepatic lipid production and suppresses insulin signaling. Therefore, manipulation of PKD3 activity could be used to decrease hepatic lipid content or improve hepatic insulin sensitivity.
Targeting the intrinsic metabolism of immune or tumor cells is a therapeutic strategy in autoimmunity, chronic inflammation or cancer. Metabolite repair enzymes may represent an alternative target class for selective metabolic inhibition, but pharmacological tools to test this concept are needed. Here, we demonstrate that phosphoglycolate phosphatase (PGP), a prototypical metabolite repair enzyme in glycolysis, is a pharmacologically actionable target. Using a combination of small molecule screening, protein crystallography, molecular dynamics simulations and NMR metabolomics, we discover and analyze a compound (CP1) that inhibits PGP with high selectivity and submicromolar potency. CP1 locks the phosphatase in a catalytically inactive conformation, dampens glycolytic flux, and phenocopies effects of cellular PGP-deficiency. This study provides key insights into effective and precise PGP targeting, at the same time validating an allosteric approach to control glycolysis that could advance discoveries of innovative therapeutic candidates.
The transcription factor NRF2 is the major mediator of oxidative stress responses and is closely connected to therapy resistance in tumors harboring activating mutations in the NRF2 pathway. In melanoma, such mutations are rare, and it is unclear to what extent melanomas rely on NRF2. Here we show that NRF2 suppresses the activity of the melanocyte lineage marker MITF in melanoma, thereby reducing the expression of pigmentation markers. Intriguingly, we furthermore identified NRF2 as key regulator of immune-modulating genes, linking oxidative stress with the induction of cyclooxygenase 2 (COX2) in an ATF4-dependent manner. COX2 is critical for the secretion of prostaglandin E2 and was strongly induced by H\(_2\)O\(_2\) or TNFα only in presence of NRF2. Induction of MITF and depletion of COX2 and PGE2 were also observed in NRF2-deleted melanoma cells in vivo. Furthermore, genes corresponding to the innate immune response such as RSAD2 and IFIH1 were strongly elevated in absence of NRF2 and coincided with immune evasion parameters in human melanoma datasets. Even in vitro, NRF2 activation or prostaglandin E2 supplementation blunted the induction of the innate immune response in melanoma cells. Transcriptome analyses from lung adenocarcinomas indicate that the observed link between NRF2 and the innate immune response is not restricted to melanoma.
Strains of the food-borne pathogen Listeria (L.) monocytogenes have diverse virulence potential. This study focused on the virulence of three outbreak strains: the CC1 strain PF49 (serovar 4b) from a cheese-associated outbreak in Switzerland, the clinical CC2 strain F80594 (serovar 4b), and strain G6006 (CC3, serovar 1/2a), responsible for a large gastroenteritis outbreak in the USA due to chocolate milk. We analysed the genomes and characterized the virulence in vitro and in vivo. Whole-genome sequencing revealed a high conservation of the major virulence genes. Minor deviations of the gene contents were found in the autolysins Ami, Auto, and IspC. Moreover, different ActA variants were present. Strain PF49 and F80594 showed prolonged survival in the liver of infected mice. Invasion and intracellular proliferation were similar for all strains, but the CC1 and CC2 strains showed increased spreading in intestinal epithelial Caco2 cells compared to strain G6006. Overall, this study revealed long-term survival of serovar 4b strains F80594 and PF49 in the liver of mice. Future work will be needed to determine the genes and molecular mechanism behind the long-term survival of L. monocytogenes strains in organs.
Altered metabolic processes contribute to carcinogenesis by modulating proliferation, survival and differentiation. Tumours are composed of different cell populations, with cancer stem-like cells being one of the most prominent examples. This specific pool of cells is thought to be responsible for cancer growth and recurrence and plays a particularly relevant role in glioblastoma (GBM), the most lethal form of primary brain tumours. Here, we have analysed the transcriptome and metabolome of an established GBM cell line (U87) and a patient-derived GBM stem-like cell line (NCH644) exposed to neurosphere or monolayer culture conditions. By integrating transcriptome and metabolome data, we identified key metabolic pathways and gene signatures that are associated with stem-like and differentiated states in GBM cells, and demonstrated that neurospheres and monolayer cells differ substantially in their metabolism and gene regulation. Furthermore, arginine biosynthesis was identified as the most significantly regulated pathway in neurospheres, although individual nodes of this pathway were distinctly regulated in the two cellular systems. Neurosphere conditions, as opposed to monolayer conditions, cause a transcriptomic and metabolic rewiring that may be crucial for the regulation of stem-like features, where arginine biosynthesis may be a key metabolic pathway. Additionally, TCGA data from GBM patients showed significant regulation of specific components of the arginine biosynthesis pathway, providing further evidence for the importance of this metabolic pathway in GBM.
Multifunctional enzyme, type-1 (MFE1) is a monomeric enzyme with a 2E-enoyl-CoA hydratase and a 3S-hydroxyacyl-CoA dehydrogenase (HAD) active site. Enzyme kinetic data of rat peroxisomal MFE1 show that the catalytic efficiencies for converting the short-chain substrate 2E-butenoyl-CoA into acetoacetyl-CoA are much lower when compared with those of the homologous monofunctional enzymes. The mode of binding of acetoacetyl-CoA (to the hydratase active site) and the very similar mode of binding of NAD\(^+\) and NADH (to the HAD part) are described and compared with those of their monofunctional counterparts. Structural comparisons suggest that the conformational flexibility of the HAD and hydratase parts of MFE1 are correlated. The possible importance of the conformational flexibility of MFE1 for its biocatalytic properties is discussed.
Stroma-infiltrating immune cells, such as tumor-associated macrophages (TAM), play an important role in regulating tumor progression and chemoresistance. These effects are mostly conveyed by secreted mediators, among them several cathepsin proteases. In addition, increasing evidence suggests that stroma-infiltrating immune cells are able to induce profound metabolic changes within the tumor microenvironment. In this study, we aimed to characterize the impact of cathepsins in maintaining the TAM phenotype in more detail. For this purpose, we investigated the molecular effects of pharmacological cathepsin inhibition on the viability and polarization of human primary macrophages as well as its metabolic consequences. Pharmacological inhibition of cathepsins B, L, and S using a novel inhibitor, GB111-NH\(_2\), led to changes in cellular recycling processes characterized by an increased expression of autophagy- and lysosome-associated marker genes and reduced adenosine triphosphate (ATP) content. Decreased cathepsin activity in primary macrophages further led to distinct changes in fatty acid metabolites associated with increased expression of key modulators of fatty acid metabolism, such as fatty acid synthase (FASN) and acid ceramidase (ASAH1). The altered fatty acid profile was associated with an increased synthesis of the pro-inflammatory prostaglandin PGE\(_2\), which correlated with the upregulation of numerous NF\(_k\)B-dependent pro-inflammatory mediators, including interleukin-1 (IL-1), interleukin-6 (IL-6), C-C motif chemokine ligand 2 (CCL2), and tumor necrosis factor-alpha (TNFα). Our data indicate a novel link between cathepsin activity and metabolic reprogramming in macrophages, demonstrated by a profound impact on autophagy and fatty acid metabolism, which facilitates a pro-inflammatory micromilieu generally associated with enhanced tumor elimination. These results provide a strong rationale for therapeutic cathepsin inhibition to overcome the tumor-promoting effects of the immune-evasive tumor micromilieu.