Lack of p38 activation in T cells increases IL-35 and protects against obesity by promoting thermogenesis.

Obesity is characterized by low-grade inflammation, energy imbalance and impaired thermogenesis. The role of regulatory T cells (Treg) in inflammation-mediated maladaptive thermogenesis is not well established. Here, we find that the p38 pathway is a key regulator of T cell-mediated adipose tissue (AT) inflammation and browning. Mice with T cells specifically lacking the p38 activators MKK3/6 are protected against diet-induced obesity, leading to an improved metabolic profile, increased browning, and enhanced thermogenesis. We identify IL-35 as a driver of adipocyte thermogenic program through the ATF2/UCP1/FGF21 pathway. IL-35 limits CD8+ T cell infiltration and inflammation in AT. Interestingly, we find that IL-35 levels are reduced in visceral fat from obese patients. Mechanistically, we demonstrate that p38 controls the expression of IL-35 in human and mouse Treg cells through mTOR pathway activation. Our findings highlight p38 signaling as a molecular orchestrator of AT T cell accumulation and function.

Hepatic nutrient and hormone signaling to mTORC1 instructs the postnatal metabolic zonation of the liver.

The metabolic functions of the liver are spatially organized in a phenomenon called zonation, linked to the differential exposure of portal and central hepatocytes to nutrient-rich blood. The mTORC1 signaling pathway controls cellular metabolism in response to nutrients and insulin fluctuations. Here we show that simultaneous genetic activation of nutrient and hormone signaling to mTORC1 in hepatocytes results in impaired establishment of postnatal metabolic and zonal identity of hepatocytes. Mutant hepatocytes fail to upregulate postnatally the expression of Frizzled receptors 1 and 8, and show reduced Wnt/ß-catenin activation. This defect, alongside diminished paracrine Wnt2 ligand expression by endothelial cells, underlies impaired postnatal maturation. Impaired zonation is recapitulated in a model of constant supply of nutrients by parenteral nutrition to piglets. Our work shows the role of hepatocyte sensing of fluctuations in nutrients and hormones for triggering a latent metabolic zonation program.

Myeloid p38 activation maintains macrophage-liver crosstalk and BAT thermogenesis through IL-12-FGF21 axis.

Obesity features excessive fat accumulation in several body tissues and induces a state of chronic low-grade inflammation that contributes to the development of diabetes, steatosis, and insulin resistance. Recent research has shown that this chronic inflammation is crucially dependent on p38 pathway activity in macrophages, suggesting p38 inhibition as a possible treatment for obesity comorbidities. Nevertheless, we report here that lack of p38 activation in myeloid cells worsens high-fat diet-induced obesity, diabetes, and steatosis. Deficient p38 activation increases macrophage IL-12 production, leading to inhibition of hepatic FGF21 and reduction of thermogenesis in the brown fat. The implication of FGF21 in the phenotype was confirmed by its specific deletion in hepatocytes. We also found that IL-12 correlates with liver damage in human biopsies, indicating the translational potential of our results. Our findings suggest that myeloid p38 has a dual role in inflammation and that drugs targeting IL-12 might improve the homeostatic regulation of energy balance in response to metabolic stress.

MKK6 deficiency promotes cardiac dysfunction through MKK3-p38γ/δ-mTOR hyperactivation.

Stress-activated p38 kinases control a plethora of functions, and their dysregulation has been linked to the development of steatosis, obesity, immune disorders, and cancer. Therefore, they have been identified as potential targets for novel therapeutic strategies. There are four p38 family members (p38α, p38ß, p38γ, and p38δ) that are activated by MKK3 and MKK6. Here, we demonstrate that lack of MKK6 reduces the lifespan in mice. Longitudinal study of cardiac function in MKK6 KO mice showed that young mice develop cardiac hypertrophy which progresses to cardiac dilatation and fibrosis with age. Mechanistically, lack of MKK6 blunts p38α activation while causing MKK3-p38γ/δ hyperphosphorylation and increased mammalian target of rapamycin (mTOR) signaling, resulting in cardiac hypertrophy. Cardiac hypertrophy in MKK6 KO mice is reverted by knocking out either p38γ or p38δ or by inhibiting the mTOR pathway with rapamycin. In conclusion, we have identified a key role for the MKK3/6-p38γ/δ pathway in the development of cardiac hypertrophy, which has important implications for the clinical use of p38α inhibitors in the long-term treatment since they might result in cardiotoxicity.

The human heart can increase its size to supply more blood to the body's organs. This process, called hypertrophy, can happen during exercise or be caused by medical conditions, such as high blood pressure or inherited genetic diseases. If hypertrophy is continually driven by illness, this can cause the heart to fail and no longer be able to properly pump blood around the body. For hypertrophy to happen, several molecular changes occur in the cells responsible for contracting the heart, including activation of the p38 pathway. Within this pathway is a p38 enzyme as well as a series of other proteins which are sequentially turned on in response to stress, such as inflammatory molecules or mechanical forces that alter the cell's shape. There are different types of p38 enzyme which have been linked to other diseases, making them a promising target for drug development. However, clinical trials blocking individual members of the p38 family have had disappointing results. An alternative approach is to target other proteins involved in the p38 pathway, such as MKK6, but it is not known what effect this might have. To investigate, Romero-Becerra et al. genetically modified mice to not have any MKK6 protein. As a result, these mice had a shorter lifespan, with hypertrophy developing at a young age that led to heart problems. Romero-Becerra et al. used different mice models to understand why this happened, showing that a lack of MKK6 reduces the activity of a specific member of the p38 family called p38α. However, this blockage boosted a different branch of the pathway which involved two other p38 proteins, p38γ and p38δ. This, in turn, triggered another key pathway called mTOR which also promotes hypertrophy of the heart. These results suggest that drugs blocking MKK6 and p38α could lead to side effects that cause further harm to the heart. A more promising approach for treating hypertrophic heart conditions could be to inhibit p38γ and/or p38δ. However, before this can be fully explored, further work is needed to generate compounds that specifically target these proteins.

p38γ and p38δ regulate postnatal cardiac metabolism through glycogen synthase 1.

During the first weeks of postnatal heart development, cardiomyocytes undergo a major adaptive metabolic shift from glycolytic energy production to fatty acid oxidation. This metabolic change is contemporaneous to the up-regulation and activation of the p38γ and p38δ stress-activated protein kinases in the heart. We demonstrate that p38γ/δ contribute to the early postnatal cardiac metabolic switch through inhibitory phosphorylation of glycogen synthase 1 (GYS1) and glycogen metabolism inactivation. Premature induction of p38γ/δ activation in cardiomyocytes of newborn mice results in an early GYS1 phosphorylation and inhibition of cardiac glycogen production, triggering an early metabolic shift that induces a deficit in cardiomyocyte fuel supply, leading to whole-body metabolic deregulation and maladaptive cardiac pathogenesis. Notably, the adverse effects of forced premature cardiac p38γ/δ activation in neonate mice are prevented by maternal diet supplementation of fatty acids during pregnancy and lactation. These results suggest that diet interventions have a potential for treating human cardiac genetic diseases that affect heart metabolism.

Neutrophil infiltration regulates clock-gene expression to organize daily hepatic metabolism.

Liver metabolism follows diurnal fluctuations through the modulation of molecular clock genes. Disruption of this molecular clock can result in metabolic disease but its potential regulation by immune cells remains unexplored. Here, we demonstrated that in steady state, neutrophils infiltrated the mouse liver following a circadian pattern and regulated hepatocyte clock-genes by neutrophil elastase (NE) secretion. NE signals through c-Jun NH2-terminal kinase (JNK) inhibiting fibroblast growth factor 21 (FGF21) and activating Bmal1 expression in the hepatocyte. Interestingly, mice with neutropenia, defective neutrophil infiltration or lacking elastase were protected against steatosis correlating with lower JNK activation, reduced Bmal1 and increased FGF21 expression, together with decreased lipogenesis in the liver. Lastly, using a cohort of human samples we found a direct correlation between JNK activation, NE levels and Bmal1 expression in the liver. This study demonstrates that neutrophils contribute to the maintenance of daily hepatic homeostasis through the regulation of the NE/JNK/Bmal1 axis.

Every day, the body's biological processes work to an internal clock known as the circadian rhythm. This rhythm is controlled by 'clock genes' that are switched on or off by daily physical and environmental cues, such as changes in light levels. These daily rhythms are very finely tuned, and disturbances can lead to serious health problems, such as diabetes or high blood pressure. The ability of the body to cycle through the circadian rhythm each day is heavily influenced by the clock of one key organ: the liver. This organ plays a critical role in converting food and drink into energy. There is evidence that neutrophils ­ white blood cells that protect the body by being the first response to inflammation ­ can influence how the liver performs its role in obese people, by for example, releasing a protein called elastase. Additionally, the levels of neutrophils circulating in the blood change following a daily pattern. Crespo, González-Terán et al. wondered whether neutrophils enter the liver at specific times of the day to control liver's daily rhythm. Crespo, González-Terán et al. revealed that neutrophils visit the liver in a pattern that peaks when it gets light and dips when it gets dark by counting the number of neutrophils in the livers of mice at different times of the day. During these visits, neutrophils secreted elastase, which activated a protein called JNK in the cells of the mice's liver. This subsequently blocked the activity of another protein, FGF21, which led to the activation of the genes that allow cells to make fat molecules for storage. JNK activation also switched on the clock gene, Bmal1, ultimately causing fat to build up in the mice's liver. Crespo, González-Terán et al. also found that, in samples from human livers, the levels of elastase, the activity of JNK, and whether the Bmal1 gene was switched on were tightly linked. This suggests that neutrophils may be controlling the liver's rhythm in humans the same way they do in mice. Overall, this research shows that neutrophils can control and reset the liver's daily rhythm using a precisely co-ordinated series of molecular changes. These insights into the liver's molecular clock suggest that elastase, JNK and BmaI1 may represent new therapeutic targets for drugs or smart medicines to treat metabolic diseases such as diabetes or high blood pressure.

JNK-mediated disruption of bile acid homeostasis promotes intrahepatic cholangiocarcinoma.

Metabolic stress causes activation of the cJun NH2-terminal kinase (JNK) signal transduction pathway. It is established that one consequence of JNK activation is the development of insulin resistance and hepatic steatosis through inhibition of the transcription factor PPARα. Indeed, JNK1/2 deficiency in hepatocytes protects against the development of steatosis, suggesting that JNK inhibition represents a possible treatment for this disease. However, the long-term consequences of JNK inhibition have not been evaluated. Here we demonstrate that hepatic JNK controls bile acid production. We found that hepatic JNK deficiency alters cholesterol metabolism and bile acid synthesis, conjugation, and transport, resulting in cholestasis, increased cholangiocyte proliferation, and intrahepatic cholangiocarcinoma. Gene ablation studies confirmed that PPARα mediated these effects of JNK in hepatocytes. This analysis highlights potential consequences of long-term use of JNK inhibitors for the treatment of metabolic syndrome.

p38γ is essential for cell cycle progression and liver tumorigenesis.

The cell cycle is a tightly regulated process that is controlled by the conserved cyclin-dependent kinase (CDK)-cyclin protein complex1. However, control of the G0-to-G1 transition is not completely understood. Here we demonstrate that p38 MAPK gamma (p38γ) acts as a CDK-like kinase and thus cooperates with CDKs, regulating entry into the cell cycle. p38γ shares high sequence homology, inhibition sensitivity and substrate specificity with CDK family members. In mouse hepatocytes, p38γ induces proliferation after partial hepatectomy by promoting the phosphorylation of retinoblastoma tumour suppressor protein at known CDK target residues. Lack of p38γ or treatment with the p38γ inhibitor pirfenidone protects against the chemically induced formation of liver tumours. Furthermore, biopsies of human hepatocellular carcinoma show high expression of p38γ, suggesting that p38γ could be a therapeutic target in the treatment of this disease.

Adiponectin accounts for gender differences in hepatocellular carcinoma incidence.

Hepatocellular carcinoma (HCC) is the sixth most common cancer type and the fourth leading cause of cancer-related death. This cancer appears with higher incidence in men and during obesity; however, the specific mechanisms underlying this correlation are unknown. Adipose tissue, a key organ in metabolic syndrome, shows evident gender disparities in the production of adipokines. Levels of the important adipokine adiponectin decrease in men during puberty, as well as in the obese state. Here, we show that this decrease in adiponectin levels is responsible for the increased liver cancer risk in males. We found that testosterone activates the protein JNK in mouse and human adipocytes. JNK-mediated inhibition of adiponectin secretion increases liver cancer cell proliferation, since adiponectin protects against liver cancer development through the activation of AMP-activated protein kinase (AMPK) and p38α. This study provides insight into adipose tissue to liver crosstalk and its gender relation during cancer development, having the potential to guide strategies for new cancer therapeutics.

p38α blocks brown adipose tissue thermogenesis through p38δ inhibition.

Adipose tissue has emerged as an important regulator of whole-body metabolism, and its capacity to dissipate energy in the form of heat has acquired a special relevance in recent years as potential treatment for obesity. In this context, the p38MAPK pathway has arisen as a key player in the thermogenic program because it is required for the activation of brown adipose tissue (BAT) thermogenesis and participates also in the transformation of white adipose tissue (WAT) into BAT-like depot called beige/brite tissue. Here, using mice that are deficient in p38α specifically in adipose tissue (p38αFab-KO), we unexpectedly found that lack of p38α protected against high-fat diet (HFD)-induced obesity. We also showed that p38αFab-KO mice presented higher energy expenditure due to increased BAT thermogenesis. Mechanistically, we found that lack of p38α resulted in the activation of the related protein kinase family member p38δ. Our results showed that p38δ is activated in BAT by cold exposure, and lack of this kinase specifically in adipose tissue (p38δ Fab-KO) resulted in overweight together with reduced energy expenditure and lower body and skin surface temperature in the BAT region. These observations indicate that p38α probably blocks BAT thermogenesis through p38δ inhibition. Consistent with the results obtained in animals, p38α was reduced in visceral and subcutaneous adipose tissue of subjects with obesity and was inversely correlated with body mass index (BMI). Altogether, we have elucidated a mechanism implicated in physiological BAT activation that has potential clinical implications for the treatment of obesity and related diseases such as diabetes.

MKK6 controls T3-mediated browning of white adipose tissue.

Increasing the thermogenic capacity of adipose tissue to enhance organismal energy expenditure is considered a promising therapeutic strategy to combat obesity. Here, we report that expression of the p38 MAPK activator MKK6 is elevated in white adipose tissue of obese individuals. Using knockout animals and shRNA, we show that Mkk6 deletion increases energy expenditure and thermogenic capacity of white adipose tissue, protecting mice against diet-induced obesity and the development of diabetes. Deletion of Mkk6 increases T3-stimulated UCP1 expression in adipocytes, thereby increasing their thermogenic capacity. Mechanistically, we demonstrate that, in white adipose tissue, p38 is activated by an alternative pathway involving AMPK, TAK, and TAB. Our results identify MKK6 in adipocytes as a potential therapeutic target to reduce obesity.Brown and beige adipose tissues dissipate heat via uncoupling protein 1 (UCP1). Here the authors show that the stress activated kinase MKK6 acts as a repressor of UCP1 expression, suggesting that its inhibition promotes adipose tissue browning and increases organismal energy expenditure.

p38γ and p38δ reprogram liver metabolism by modulating neutrophil infiltration.

Non-alcoholic fatty liver disease (NAFLD) is a major health problem and the main cause of liver disease in Western countries. Although NAFLD is strongly associated with obesity and insulin resistance, its pathogenesis remains poorly understood. The disease begins with an excessive accumulation of triglycerides in the liver, which stimulates an inflammatory response. Alternative p38 mitogen-activated kinases (p38γ and p38δ) have been shown to contribute to inflammation in different diseases. Here we demonstrate that p38δ is elevated in livers of obese patients with NAFLD and that mice lacking p38γ/δ in myeloid cells are resistant to diet-induced fatty liver, hepatic triglyceride accumulation and glucose intolerance. This protective effect is due to defective migration of p38γ/δ-deficient neutrophils to the damaged liver. We further show that neutrophil infiltration in wild-type mice contributes to steatosis development by means of inflammation and liver metabolic changes. Therefore, p38γ and p38δ in myeloid cells provide a potential target for NAFLD therapy.

Quantitative analysis of protein-lipid interactions using tryptophan fluorescence.

The fluorescent properties of the amino acid tryptophan make it a useful tool for fluorometric assays. Because tryptophan fluorescence is remarkably sensitive to the polarity of the environment, it can be used to determine the affinity of tryptophan-containing peptides for phospholipid vesicles of varying compositions. Here, we describe a method for using tryptophan fluorescence to determine the binding affinities of peptides derived from the proteins Raf-1 and KSR-1 to small unilamellar vesicles containing phosphatidic acid. The method can be extrapolated to measure the binding of other tryptophan-containing peptides or proteins to lipid vesicles.

Role of phosphatidic acid in the coupling of the ERK cascade.

The production of phosphatidic acid plays a crucial role in the activation of the ERK cascade. This role was linked to the binding of phosphatidate to a specific polybasic site within the kinase domain of Raf-1. Here we show that phosphatidate promotes ERK phosphorylation in intact cells but does not activate Raf in vitro. The kinase suppressor of Ras (KSR) contains a sequence homologous to the phosphatidate binding site of Raf-1. Direct binding of phosphatidate to synthetic peptides derived from the sequences of the binding domains of Raf-1 and KSR was demonstrated by spectroscopic techniques. The specificity of these interactions was confirmed using synthetic lipids and mutated peptides in which the core of the phosphatidic acid binding domain was disrupted. Insulin and exogenous dioleoyl phosphatidate induced a rapid translocation of a mouse KSR1-EGFP construct to the plasma membrane of HIRcB cells. Mutation of two arginines located in the core of the putative phosphatidate binding site abolished dioleoyl phosphatidate- and insulin-induced translocation of KSR1. Overexpression of the mutant KSR1 in HIRcB cells inhibited insulin-dependent MEK and ERK phosphorylation. The addition of dioleoyl phosphatidate or insulin increased the co-localization of KSR1 and H-Ras and promoted the formation of plasma membrane patches enriched in both proteins and phosphatidic acid. These results, in conjunction with our previous work, suggest the formation of phosphatidate-enriched membrane microdomains that contain all components of the ERK cascade. We propose that these domains act as molecular scaffolds in the coupling of signaling events.