NIK/MAP3K14 in hepatocytes orchestrates NASH to hepatocellular carcinoma progression via JAK2/STAT5 inhibition.

OBJECTIVE: Nonalcoholic fatty liver disease (NAFLD) ranges from steatosis to nonalcoholic steatohepatitis (NASH), which often progresses to hepatocellular carcinoma (HCC) through a largely undefined mechanism. NASH and HCC depend on inflammatory signaling, whose master regulator is the NFκB transcription factor family, activated by canonical and non-canonical pathways. METHODS: Here, we investigated non-canonical NFκB-inducing kinase (NIK/MAP3K14) in metabolic NASH, NASH to HCC transition, and DEN-induced HCC. To this end, we performed dietary and chemical interventions in mice that were analyzed via single nucleus sequencing, gene expression and histochemical methods. Ultimately, we verified our mouse results in human patient samples. RESULTS: We revealed that hepatocyte-specific NIK deficiency (NIKLKO) ameliorated metabolic NASH complications and reduced hepatocarcinogenesis, independent of its role in the NFκB pathway. Instead, hepatic NIK attenuated hepatoprotective JAK2/STAT5 signaling that is a prerequisite for NASH and NASH to HCC progression in mice and humans. CONCLUSIONS: Our data suggest NIK-mediated inhibitory JAK2 phosphorylation at serine 633 that might be amenable for future therapeutic interventions in patients.

Mitochondrial metabolism coordinates stage-specific repair processes in macrophages during wound healing.

Wound healing is a coordinated process that initially relies on pro-inflammatory macrophages, followed by a pro-resolution function of these cells. Changes in cellular metabolism likely dictate these distinct activities, but the nature of these changes has been unclear. Here, we profiled early- versus late-stage skin wound macrophages in mice at both the transcriptional and functional levels. We found that glycolytic metabolism in the early phase is not sufficient to ensure productive repair. Instead, by combining conditional disruption of the electron transport chain with deletion of mitochondrial aspartyl-tRNA synthetase, followed by single-cell sequencing analysis, we found that a subpopulation of early-stage wound macrophages are marked by mitochondrial ROS (mtROS) production and HIF1α stabilization, which ultimately drives a pro-angiogenic program essential for timely healing. In contrast, late-phase, pro-resolving wound macrophages are marked by IL-4Rα-mediated mitochondrial respiration and mitohormesis. Collectively, we identify changes in mitochondrial metabolism as a critical control mechanism for macrophage effector functions during wound healing.

Beneficial Metabolic Effects of TREM2 in Obesity Are Uncoupled From Its Expression on Macrophages.

Obesity-induced white adipose tissue (WAT) hypertrophy is associated with elevated adipose tissue macrophage (ATM) content. Overexpression of the triggering receptor expressed on myeloid cells 2 (TREM2) reportedly increases adiposity, worsening health. Paradoxically, using insulin resistance, elevated fat mass, and hypercholesterolemia as hallmarks of unhealthy obesity, a recent report demonstrated that ATM-expressed TREM2 promoted health. Here, we identified that in mice, TREM2 deficiency aggravated diet-induced insulin resistance and hepatic steatosis independently of fat and cholesterol levels. Metabolomics linked TREM2 deficiency with elevated obesity-instigated serum ceramides that correlated with impaired insulin sensitivity. Remarkably, while inhibiting ceramide synthesis exerted no influences on TREM2-dependent ATM remodeling, inflammation, or lipid load, it restored insulin tolerance, reversing adipose hypertrophy and secondary hepatic steatosis of TREM2-deficient animals. Bone marrow transplantation experiments revealed unremarkable influences of immune cell-expressed TREM2 on health, instead demonstrating that WAT-intrinsic mechanisms impinging on sphingolipid metabolism dominate in the systemic protective effects of TREM2 on metabolic health.

Identification of ALK in Thinness.

There is considerable inter-individual variability in susceptibility to weight gain despite an equally obesogenic environment in large parts of the world. Whereas many studies have focused on identifying the genetic susceptibility to obesity, we performed a GWAS on metabolically healthy thin individuals (lowest 6th percentile of the population-wide BMI spectrum) in a uniquely phenotyped Estonian cohort. We discovered anaplastic lymphoma kinase (ALK) as a candidate thinness gene. In Drosophila, RNAi mediated knockdown of Alk led to decreased triglyceride levels. In mice, genetic deletion of Alk resulted in thin animals with marked resistance to diet- and leptin-mutation-induced obesity. Mechanistically, we found that ALK expression in hypothalamic neurons controls energy expenditure via sympathetic control of adipose tissue lipolysis. Our genetic and mechanistic experiments identify ALK as a thinness gene, which is involved in the resistance to weight gain.

PNOCARC Neurons Promote Hyperphagia and Obesity upon High-Fat-Diet Feeding.

Calorie-rich diets induce hyperphagia and promote obesity, although the underlying mechanisms remain poorly defined. We find that short-term high-fat-diet (HFD) feeding of mice activates prepronociceptin (PNOC)-expressing neurons in the arcuate nucleus of the hypothalamus (ARC). PNOCARC neurons represent a previously unrecognized GABAergic population of ARC neurons distinct from well-defined feeding regulatory AgRP or POMC neurons. PNOCARC neurons arborize densely in the ARC and provide inhibitory synaptic input to nearby anorexigenic POMC neurons. Optogenetic activation of PNOCARC neurons in the ARC and their projections to the bed nucleus of the stria terminalis promotes feeding. Selective ablation of these cells promotes the activation of POMC neurons upon HFD exposure, reduces feeding, and protects from obesity, but it does not affect food intake or body weight under normal chow consumption. We characterize PNOCARC neurons as a novel ARC neuron population activated upon palatable food consumption to promote hyperphagia.

Publisher Correction: The metabolite BH4 controls T cell proliferation in autoimmunity and cancer.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

AIF-regulated oxidative phosphorylation supports lung cancer development.

Cancer is a major and still increasing cause of death in humans. Most cancer cells have a fundamentally different metabolic profile from that of normal tissue. This shift away from mitochondrial ATP synthesis via oxidative phosphorylation towards a high rate of glycolysis, termed Warburg effect, has long been recognized as a paradigmatic hallmark of cancer, supporting the increased biosynthetic demands of tumor cells. Here we show that deletion of apoptosis-inducing factor (AIF) in a KrasG12D-driven mouse lung cancer model resulted in a marked survival advantage, with delayed tumor onset and decreased malignant progression. Mechanistically, Aif deletion leads to oxidative phosphorylation (OXPHOS) deficiency and a switch in cellular metabolism towards glycolysis in non-transformed pneumocytes and at early stages of tumor development. Paradoxically, although Aif-deficient cells exhibited a metabolic Warburg profile, this bioenergetic change resulted in a growth disadvantage of KrasG12D-driven as well as Kras wild-type lung cancer cells. Cell-autonomous re-expression of both wild-type and mutant AIF (displaying an intact mitochondrial, but abrogated apoptotic function) in Aif-knockout KrasG12D mice restored OXPHOS and reduced animal survival to the same level as AIF wild-type mice. In patients with non-small cell lung cancer, high AIF expression was associated with poor prognosis. These data show that AIF-regulated mitochondrial respiration and OXPHOS drive the progression of lung cancer.

Non-parenchymal TREM-2 protects the liver from immune-mediated hepatocellular damage.

OBJECTIVE: Liver injury impacts hepatic inflammation in part via Toll-like receptor (TLR) signalling. Triggering receptor expressed on myeloid cells 2 (TREM-2) modulates TLR4-mediated inflammation in bone marrow (BM)-derived macrophages but its function in liver injury is unknown. Here we hypothesised that the anti-inflammatory effects of TREM-2 on TLR signalling may limit hepatic injury. DESIGN: TREM-2 expression was analysed in livers of humans with various forms of liver injury compared with control individuals. Acute and chronic liver injury models were performed in wild type and Trem-2-/- mice. Primary liver cells from both genotypes of mice were isolated for in vitro experiments. RESULTS: TREM-2 was expressed on non-parenchymal hepatic cells and induced during liver injury in mice and man. Mice lacking TREM-2 exhibited heightened liver damage and inflammation during acute and repetitive carbon tetrachloride and acetaminophen (APAP) intoxication, the latter of which TREM-2 deficiency was remarkably associated with worsened survival. Liver damage in Trem-2-/- mice following chronic injury and APAP challenge was associated with elevated hepatic lipid peroxidation and macrophage content. BM transplantation experiments and cellular reactive oxygen species assays revealed effects of TREM-2 in the context of chronic injury depended on both immune and resident TREM-2 expression. Consistent with effects of TREM-2 on inflammation-associated injury, primary hepatic macrophages and hepatic stellate cells lacking TREM-2 exhibited augmented TLR4-driven proinflammatory responses. CONCLUSION: Our data indicate that by acting as a natural brake on inflammation during hepatocellular injury, TREM-2 is a critical regulator of diverse types of hepatotoxic injury.

The metabolite BH4 controls T cell proliferation in autoimmunity and cancer.

Genetic regulators and environmental stimuli modulate T cell activation in autoimmunity and cancer. The enzyme co-factor tetrahydrobiopterin (BH4) is involved in the production of monoamine neurotransmitters, the generation of nitric oxide, and pain1,2. Here we uncover a link between these processes, identifying a fundamental role for BH4 in T cell biology. We find that genetic inactivation of GTP cyclohydrolase 1 (GCH1, the rate-limiting enzyme in the synthesis of BH4) and inhibition of sepiapterin reductase (the terminal enzyme in the synthetic pathway for BH4) severely impair the proliferation of mature mouse and human T cells. BH4 production in activated T cells is linked to alterations in iron metabolism and mitochondrial bioenergetics. In vivo blockade of BH4 synthesis abrogates T-cell-mediated autoimmunity and allergic inflammation, and enhancing BH4 levels through GCH1 overexpression augments responses by CD4- and CD8-expressing T cells, increasing their antitumour activity in vivo. Administration of BH4 to mice markedly reduces tumour growth and expands the population of intratumoral effector T cells. Kynurenine-a tryptophan metabolite that blocks antitumour immunity-inhibits T cell proliferation in a manner that can be rescued by BH4. Finally, we report the development of a potent SPR antagonist for possible clinical use. Our data uncover GCH1, SPR and their downstream metabolite BH4 as critical regulators of T cell biology that can be readily manipulated to either block autoimmunity or enhance anticancer immunity.

VEGF and GLUT1 are highly heritable, inversely correlated and affected by dietary fat intake: Consequences for cognitive function in humans.

OBJECTIVE: Reduction of brain glucose transporter GLUT1 results in severe neurological dysfunction. VEGF is required to restore and maintain brain glucose uptake across the blood brain barrier via GLUT1, which was shown to be acutely diminished in response to a high fat diet (HFD) in mice. The genetic and HFD-related regulation and association of VEGF and GLUT1 (SLC2A1) in humans was investigated in the NUtriGenomic Analysis in Twins (NUGAT) study. METHODS: 92 healthy and non-obese twins were standardized to a high-carbohydrate low-fat diet for 6 weeks before switched to a 6-week HFD under isocaloric conditions. Three clinical investigation days were conducted: after 6 weeks of low-fat diet and after 1 and 6 weeks of HFD. Serum VEGF and other cytokine levels were measured using ELISA. Gene expression in subcutaneous adipose tissue was assessed by quantitative Real-Time PCR. Genotyping was performed using microarray. The Auditory Verbal Learning Task was conducted to measure cognitive performance. RESULTS: In this human study, we showed that the environmental regulation of SLC2A1 expression and serum VEGF by HFD was inversely correlated and both factors showed strong heritability (>90%). In response to the HFD containing 45% fat, serum VEGF levels increased (P = 0.002) while SLC2A1 mRNA expression in adipose tissue decreased (P = 0.001). Higher BMI was additionally associated with lower SLC2A1 expression. AA-genotypes of the rs9472159 polymorphism, which explained ∼39% of the variation in circulating VEGF concentrations, showed significantly reduced serum VEGF levels (P = 6.4 × 10-11) but higher SLC2A1 expression (P = 0.009) in adipose tissue compared to CC/CA-genotypes after 6 weeks of HFD. Memory performance in AA-genotypes declined in response to the HFD compared to CC- and CA-genotypes. CONCLUSIONS: The results provide evidence to suggest the translatability of the dietary regulation of VEGF and GLUT1 from mouse models to humans. Our data demonstrate that HFD induces a genetically determined and correlated decrease of GLUT1 and increase of VEGF which may affect memory performance. CLINICAL TRIAL REGISTRATION NUMBER: NCT01631123.

RANK rewires energy homeostasis in lung cancer cells and drives primary lung cancer.

Lung cancer is the leading cause of cancer deaths. Besides smoking, epidemiological studies have linked female sex hormones to lung cancer in women; however, the underlying mechanisms remain unclear. Here we report that the receptor activator of nuclear factor-kB (RANK), the key regulator of osteoclastogenesis, is frequently expressed in primary lung tumors, an active RANK pathway correlates with decreased survival, and pharmacologic RANK inhibition reduces tumor growth in patient-derived lung cancer xenografts. Clonal genetic inactivation of KRasG12D in mouse lung epithelial cells markedly impairs the progression of KRasG12D -driven lung cancer, resulting in a significant survival advantage. Mechanistically, RANK rewires energy homeostasis in human and murine lung cancer cells and promotes expansion of lung cancer stem-like cells, which is blocked by inhibiting mitochondrial respiration. Our data also indicate survival differences in KRasG12D -driven lung cancer between male and female mice, and we show that female sex hormones can promote lung cancer progression via the RANK pathway. These data uncover a direct role for RANK in lung cancer and may explain why female sex hormones accelerate lung cancer development. Inhibition of RANK using the approved drug denosumab may be a therapeutic drug candidate for primary lung cancer.

Hepatic Cholesterol-25-Hydroxylase Overexpression Improves Systemic Insulin Sensitivity in Mice.

Obesity is a major risk factor for several diseases including diabetes, heart disease, and some forms of cancer and due to its rapidly increasing prevalence it has become one of the biggest problems medicine is facing today. All the more surprising, a substantial percentage of obese patients are metabolically healthy when classified based on insulin resistance and systemic inflammation. Oxysterols are naturally occurring molecules that play important role in various metabolic and inflammatory processes and their levels are elevated in patients suffering from obesity and diabetes. 25-Hydroxycholesterol (25-OHC) is produced in cells from cholesterol by the enzyme cholesterol 25-hydroxylase (Ch25h) and is involved in lipid metabolism, inflammatory processes, and cell proliferation. Here, we investigated the role of hepatic Ch25h in the transition from metabolically healthy obesity to insulin resistance and diabetes. Using several different experimental approaches, we demonstrated the significance of Ch25h on the border of "healthy" and "diseased" states of obesity. Adenovirus-mediated Ch25h overexpression in mice improved glucose tolerance and insulin sensitivity and lowered HOMA-IR. Our data suggest that low hepatic Ch25h levels could be considered a risk marker for unhealthy obesity.

Myeloid-Cell-Derived VEGF Maintains Brain Glucose Uptake and Limits Cognitive Impairment in Obesity.

High-fat diet (HFD) feeding induces rapid reprogramming of systemic metabolism. Here, we demonstrate that HFD feeding of mice downregulates glucose transporter (GLUT)-1 expression in blood-brain barrier (BBB) vascular endothelial cells (BECs) and reduces brain glucose uptake. Upon prolonged HFD feeding, GLUT1 expression is restored, which is paralleled by increased expression of vascular endothelial growth factor (VEGF) in macrophages at the BBB. In turn, inducible reduction of GLUT1 expression specifically in BECs reduces brain glucose uptake and increases VEGF serum concentrations in lean mice. Conversely, myeloid-cell-specific deletion of VEGF in VEGF(Δmyel) mice impairs BBB-GLUT1 expression, brain glucose uptake, and memory formation in obese, but not in lean mice. Moreover, obese VEGF(Δmyel) mice exhibit exaggerated progression of cognitive decline and neuroinflammation on an Alzheimer's disease background. These experiments reveal that transient, HFD-elicited reduction of brain glucose uptake initiates a compensatory increase of VEGF production and assign obesity-associated macrophage activation a homeostatic role to restore cerebral glucose metabolism, preserve cognitive function, and limit neurodegeneration in obesity.

Ecdysone and mediator change energy metabolism to terminate proliferation in Drosophila neural stem cells.

Stem cells are highly abundant during early development but become a rare population in most adult organs. The molecular mechanisms causing stem cells to exit proliferation at a specific time are not well understood. Here, we show that changes in energy metabolism induced by the steroid hormone ecdysone and the Mediator initiate an irreversible cascade of events leading to cell-cycle exit in Drosophila neural stem cells. We show that the timely induction of oxidative phosphorylation and the mitochondrial respiratory chain are required in neuroblasts to uncouple the cell cycle from cell growth. This results in a progressive reduction in neuroblast cell size and ultimately in terminal differentiation. Brain tumor mutant neuroblasts fail to undergo this shrinkage process and continue to proliferate until adulthood. Our findings show that cell size control can be modified by systemic hormonal signaling and reveal a unique connection between metabolism and proliferation in stem cells.

Heme oxygenase-1 drives metaflammation and insulin resistance in mouse and man.

Obesity and diabetes affect more than half a billion individuals worldwide. Interestingly, the two conditions do not always coincide and the molecular determinants of "healthy" versus "unhealthy" obesity remain ill-defined. Chronic metabolic inflammation (metaflammation) is believed to be pivotal. Here, we tested a hypothesized anti-inflammatory role for heme oxygenase-1 (HO-1) in the development of metabolic disease. Surprisingly, in matched biopsies from "healthy" versus insulin-resistant obese subjects we find HO-1 to be among the strongest positive predictors of metabolic disease in humans. We find that hepatocyte and macrophage conditional HO-1 deletion in mice evokes resistance to diet-induced insulin resistance and inflammation, dramatically reducing secondary disease such as steatosis and liver toxicity. Intriguingly, cellular assays show that HO-1 defines prestimulation thresholds for inflammatory skewing and NF-κB amplification in macrophages and for insulin signaling in hepatocytes. These findings identify HO-1 inhibition as a potential therapeutic strategy for metabolic disease.

Gene expression profile of vascular endothelial growth factor (VEGF) and its receptors in various cell types of the canine lymph node using laser capture microdissection (LCM).

The role of VEGF and its receptors has extensively been studied in tumours. In contrast, the presence and function of VEGF in normal tissues like the lymph node has not been given much attention until now. To study the expression of VEGF, VEGFR-1, VEGFR-2 and VEGFR-3 in the heterogenous cell population of the canine lymph node, laser capture microdissection was used to isolate pure cell fractions of macrophages, lymphocytes, endothelial cells, and capsule cells of the canine lymph node. To clarify if macrophages take up VEGF from the environment or express VEGF, VEGFR-1, VEGFR-2 or VEGFR-3 themselves, the mRNA expression was studied by real-time RT-PCR. After RNA isolation and subsequent analysis with the Agilent 2100 Bioanalyzer only RNA samples with appropriate RNA integrity were used for real-time PCR. For the accurate relative quantification of mRNA expression levels several reference genes were evaluated. It was shown that the reference genes HPRT1 and B2M serve as reliable reference genes for gene expression studies in the canine lymph node. Expression data analysis revealed no significant difference in VEGF expression levels between endothelial cells and the other investigated cells. VEGFR-1 expression was significantly lower in lymphocytes. Also macrophages showed a highly significant lower expression of VEGFR-1 compared to endothelial cells. In addition, the VEGFR-2 expression in lymphocytes and macrophages was significantly lower in comparison to endothelial cells. We were not able to detect VEGFR-3 mRNA in the lymphocyte cell population, in macrophages and cells of the lymph node capsule VEGFR-3 was expressed at very low levels. It was shown that laser capture microdissection in combination with quantitative real-time PCR is a valuable tool for studying the expression patterns of specific cells in their microenvironment. Our results support the hypothesis that VEGF and its receptors have other biological roles besides stimulating angiogenesis in the normal lymph node. These biological functions need to be clarified in further studies.

Transcriptome analysis of human cancer reveals a functional role of heme oxygenase-1 in tumor cell adhesion.

BACKGROUND: Heme Oxygenase-1 (HO-1) is expressed in many cancers and promotes growth and survival of neoplastic cells. Recently, HO-1 has been implicated in tumor cell invasion and metastasis. However, the molecular mechanisms underlying these biologic effects of HO-1 remain largely unknown. To identify a common mechanism of action of HO-1 in cancer, we determined the global effect of HO-1 on the transcriptome of multiple tumor entities and identified a universal HO-1-associated gene expression signature. RESULTS: Genome-wide expression profiling of Heme Oxygenase-1 expressing versus HO-1 silenced BeWo choriocarcinoma cells as well as a comparative meta-profiling of the preexisting expression database of 190 human tumors of 14 independent cancer types led to the identification of 14 genes, the expression of which correlated strongly and universally with that of HO-1 (P = 0.00002). These genes included regulators of cell plasticity and extracellular matrix (ECM) remodeling (MMP2, ADAM8, TGFB1, BGN, COL21A1, PXDN), signaling (CRIP2, MICB), amino acid transport and glycosylation (SLC7A1 and ST3GAL2), estrogen and phospholipid biosynthesis (AGPAT2 and HSD17B1), protein stabilization (IFI30), and phosphorylation (ALPPL2). We selected PXDN, an adhesion molecule involved in ECM formation, for further analysis and functional characterization. Immunofluorescence and Western blotting confirmed the positive correlation of expression of PXDN and HO-1 in BeWo cancer cells as well as co-localization of these two proteins in invasive extravillous trophoblast cells. Modulation of HO-1 expression in both loss-of and gain-of function cell models (BeWo and 607B melanoma cells, respectively) demonstrated a direct relationship of HO-1 expression with cell adhesion to Fibronectin and Laminin coated wells. The adhesion-promoting effects of HO-1 were dependent on PXDN expression, as loss of PXDN in HO-1 expressing BeWo and 607B cells led to reduced cell attachment to Laminin and Fibronectin coated wells. CONCLUSIONS: Collectively, our results show that HO-1 expression determines a distinct 'molecular signature' in cancer cells, which is enriched in genes associated with tumorigenesis. The protein network downstream of HO-1 modulates adhesion, signaling, transport, and other critical cellular functions of neoplastic cells and thus promotes tumor cell growth and dissemination.