ABSTRACT
Metformin, the most prescribed antidiabetic medicine, has shown other benefits such as anti-ageing and anticancer effects1-4. For clinical doses of metformin, AMP-activated protein kinase (AMPK) has a major role in its mechanism of action4,5; however, the direct molecular target of metformin remains unknown. Here we show that clinically relevant concentrations of metformin inhibit the lysosomal proton pump v-ATPase, which is a central node for AMPK activation following glucose starvation6. We synthesize a photoactive metformin probe and identify PEN2, a subunit of γ-secretase7, as a binding partner of metformin with a dissociation constant at micromolar levels. Metformin-bound PEN2 forms a complex with ATP6AP1, a subunit of the v-ATPase8, which leads to the inhibition of v-ATPase and the activation of AMPK without effects on cellular AMP levels. Knockout of PEN2 or re-introduction of a PEN2 mutant that does not bind ATP6AP1 blunts AMPK activation. In vivo, liver-specific knockout of Pen2 abolishes metformin-mediated reduction of hepatic fat content, whereas intestine-specific knockout of Pen2 impairs its glucose-lowering effects. Furthermore, knockdown of pen-2 in Caenorhabditis elegans abrogates metformin-induced extension of lifespan. Together, these findings reveal that metformin binds PEN2 and initiates a signalling route that intersects, through ATP6AP1, the lysosomal glucose-sensing pathway for AMPK activation. This ensures that metformin exerts its therapeutic benefits in patients without substantial adverse effects.
Subject(s)
Hypoglycemic Agents , Metformin , Vacuolar Proton-Translocating ATPases , AMP-Activated Protein Kinases/metabolism , Adenosine Triphosphatases/metabolism , Amyloid Precursor Protein Secretases , Animals , Caenorhabditis elegans/metabolism , Diabetes Mellitus/drug therapy , Glucose/metabolism , Humans , Hypoglycemic Agents/administration & dosage , Hypoglycemic Agents/metabolism , Hypoglycemic Agents/pharmacology , Lysosomes/metabolism , Membrane Proteins , Metformin/agonists , Metformin/metabolism , Metformin/pharmacology , Vacuolar Proton-Translocating ATPases/metabolismABSTRACT
Metabolic reprogramming is fundamental to biological homeostasis, enabling cells to adjust metabolic routes after sensing altered availability of fuels and growth factors. ULK1 and ULK2 represent key integrators that relay metabolic stress signals to the autophagy machinery. Here, we demonstrate that, during deprivation of amino acid and growth factors, ULK1/2 directly phosphorylate key glycolytic enzymes including hexokinase (HK), phosphofructokinase 1 (PFK1), enolase 1 (ENO1), and the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBP1). Phosphorylation of these enzymes leads to enhanced HK activity to sustain glucose uptake but reduced activity of FBP1 to block the gluconeogenic route and reduced activity of PFK1 and ENO1 to moderate drop of glucose-6-phosphate and to repartition more carbon flux to pentose phosphate pathway (PPP), maintaining cellular energy and redox homeostasis at cellular and organismal levels. These results identify ULK1/2 as a bifurcate-signaling node that sustains glucose metabolic fluxes besides initiation of autophagy in response to nutritional deprivation.
Subject(s)
Autophagy-Related Protein-1 Homolog/metabolism , Autophagy , Glucose/metabolism , Glycolysis , Intracellular Signaling Peptides and Proteins/metabolism , Pentose Phosphate Pathway , Protein Serine-Threonine Kinases/metabolism , Stress, Physiological , Amino Acids/deficiency , Amino Acids/metabolism , Animals , Autophagy-Related Protein-1 Homolog/deficiency , Autophagy-Related Protein-1 Homolog/genetics , Biomarkers, Tumor/metabolism , Cell Death , DNA-Binding Proteins/metabolism , Female , Fructose-Bisphosphatase/metabolism , Genotype , HCT116 Cells , Hexokinase/metabolism , Humans , Intracellular Signaling Peptides and Proteins/genetics , MCF-7 Cells , Male , Mice, Knockout , Phenotype , Phosphofructokinase-1/metabolism , Phosphopyruvate Hydratase/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/deficiency , Protein Serine-Threonine Kinases/genetics , RNA Interference , Reactive Oxygen Species/metabolism , Signal Transduction , Time Factors , Transfection , Tumor Suppressor Proteins/metabolismABSTRACT
The major energy source for most cells is glucose, from which ATP is generated via glycolysis and/or oxidative metabolism. Glucose deprivation activates AMP-activated protein kinase (AMPK), but it is unclear whether this activation occurs solely via changes in AMP or ADP, the classical activators of AMPK. Here, we describe an AMP/ADP-independent mechanism that triggers AMPK activation by sensing the absence of fructose-1,6-bisphosphate (FBP), with AMPK being progressively activated as extracellular glucose and intracellular FBP decrease. When unoccupied by FBP, aldolases promote the formation of a lysosomal complex containing at least v-ATPase, ragulator, axin, liver kinase B1 (LKB1) and AMPK, which has previously been shown to be required for AMPK activation. Knockdown of aldolases activates AMPK even in cells with abundant glucose, whereas the catalysis-defective D34S aldolase mutant, which still binds FBP, blocks AMPK activation. Cell-free reconstitution assays show that addition of FBP disrupts the association of axin and LKB1 with v-ATPase and ragulator. Importantly, in some cell types AMP/ATP and ADP/ATP ratios remain unchanged during acute glucose starvation, and intact AMP-binding sites on AMPK are not required for AMPK activation. These results establish that aldolase, as well as being a glycolytic enzyme, is a sensor of glucose availability that regulates AMPK.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Fructose-Bisphosphate Aldolase/metabolism , Fructosediphosphates/metabolism , Glucose/metabolism , AMP-Activated Protein Kinase Kinases , Adenosine Diphosphate/metabolism , Adenosine Monophosphate/metabolism , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Animals , Axin Protein/metabolism , Binding Sites , Enzyme Activation , Fibroblasts , Fructose-Bisphosphate Aldolase/genetics , Glucose/deficiency , Humans , Male , Mice , Phosphorylation , Protein Serine-Threonine Kinases/metabolismABSTRACT
unc-51-like autophagy activating kinase 1 and 2 (Ulk1/2) regulate autophagy initiation under various stress conditions. However, the physiological functions of these Ser/Thr kinases are not well characterized. Here, we show that mice with liver-specific double knockout (LDKO) of Ulk1 and Ulk2 (Ulk1/2 LDKO) are viable, but exhibit overt hepatomegaly phenotype. Surprisingly, Ulk1/2 LDKO mice display normal autophagic activity in hepatocytes upon overnight fasting, but are strongly resistant to acetaminophen (APAP)-induced liver injury. Further studies revealed that Ulk1/2 are also dispensable for APAP-induced autophagy process, but are essential for the maximum activation of c-Jun N-terminal kinase (JNK) signaling both in vivo and in isolated primary hepatocytes during APAP treatment. Mechanistically, APAP-induced inhibition of mechanistic target of rapamycin complex 1 releases Ulk1 from an inactive state. Activated Ulk1 then directly phosphorylates and increases the kinase activity of mitogen-activated protein kinase kinase 4 and 7 (MKK4/7), the upstream kinases and activator of JNK, and mediates APAP-induced liver injury. Ulk1-dependent phosphorylation of MKK7 was further confirmed by a context-dependent phosphorylation antibody. Moreover, activation of JNK and APAP-induced cell death was markedly attenuated in Mkk4/7 double knockdown hepatocytes reconstituted with an Ulk1-unphosphorylatable mutant of MKK7 compared to those in cells rescued with wild-type MKK7. CONCLUSION: Together, these findings reveal an important role of Ulk1/2 for APAP-induced JNK activation and liver injury, and understanding of this regulatory mechanism may offer us new strategies for prevention and treatment of human APAP hepatotoxicity. (Hepatology 2018;67:2397-2413).
Subject(s)
Acetaminophen/adverse effects , Analgesics, Non-Narcotic/adverse effects , Antipyretics/adverse effects , Autophagy-Related Protein-1 Homolog/deficiency , Chemical and Drug Induced Liver Injury/enzymology , Chemical and Drug Induced Liver Injury/etiology , Liver/enzymology , Protein Serine-Threonine Kinases/deficiency , Animals , Male , MiceABSTRACT
Growth factors, typically defined as natural substances capable of stimulating cell growth and differentiation, are vital regulators for the survival of metazoan cells. In this review, we will focus on growth factor signaling pathways that are closely related to autophagy induction and discuss the critical roles of this fascinating cellular process in intracellular energy homeostasis, cell fate determination, and pathophysiological regulation.
Subject(s)
Autophagy , Intercellular Signaling Peptides and Proteins/metabolism , Signal Transduction , Animals , Apoptosis , Homeostasis , Humans , Proto-Oncogene Proteins c-akt/metabolismABSTRACT
Probucol has been utilized as a cholesterol-lowering drug with antioxidative properties. However, the impact and fundamental mechanisms of probucol in obesity-related cognitive decline are unclear. In this study, male C57BL/6J mice were allocated to a normal chow diet (NCD) group or a high-fat diet (HFD) group, followed by administration of probucol to half of the mice on the HFD regimen. Subsequently, the mice were subjected to a series of behavioral assessments, alongside the measurement of metabolic and redox parameters. Notably, probucol treatment effectively alleviates cognitive and social impairments induced by HFD in mice, while exhibiting no discernible influence on mood-related behaviors. Notably, the beneficial effects of probucol arise independently of rectifying obesity or restoring systemic glucose and lipid homeostasis, as evidenced by the lack of changes in body weight, serum cholesterol levels, blood glucose, hyperinsulinemia, systemic insulin resistance, and oxidative stress. Instead, probucol could regulate the levels of nitric oxide and superoxide-generating proteins, and it could specifically alleviate HFD-induced hippocampal insulin resistance. These findings shed light on the potential role of probucol in modulating obesity-related cognitive decline and urge reevaluation of the underlying mechanisms by which probucol exerts its beneficial effects.
ABSTRACT
The shift of carbon utilization from primarily glucose to other nutrients is a fundamental metabolic adaptation to cope with decreased blood glucose levels and the consequent decline in glucose oxidation. AMP-activated protein kinase (AMPK) plays crucial roles in this metabolic adaptation. However, the underlying mechanism is not fully understood. Here, we show that PDZ domain containing 8 (PDZD8), which we identify as a new substrate of AMPK activated in low glucose, is required for the low glucose-promoted glutaminolysis. AMPK phosphorylates PDZD8 at threonine 527 (T527) and promotes the interaction of PDZD8 with and activation of glutaminase 1 (GLS1), a rate-limiting enzyme of glutaminolysis. In vivo, the AMPK-PDZD8-GLS1 axis is required for the enhancement of glutaminolysis as tested in the skeletal muscle tissues, which occurs earlier than the increase in fatty acid utilization during fasting. The enhanced glutaminolysis is also observed in macrophages in low glucose or under acute lipopolysaccharide (LPS) treatment. Consistent with a requirement of heightened glutaminolysis, the PDZD8-T527A mutation dampens the secretion of pro-inflammatory cytokines in macrophages in mice treated with LPS. Together, we have revealed an AMPK-PDZD8-GLS1 axis that promotes glutaminolysis ahead of increased fatty acid utilization under glucose shortage.
ABSTRACT
Glycolytic intermediary metabolites such as fructose-1,6-bisphosphate can serve as signals, controlling metabolic states beyond energy metabolism. However, whether glycolytic metabolites also play a role in controlling cell fate remains unexplored. Here, we find that low levels of glycolytic metabolite 3-phosphoglycerate (3-PGA) can switch phosphoglycerate dehydrogenase (PHGDH) from cataplerosis serine synthesis to pro-apoptotic activation of p53. PHGDH is a p53-binding protein, and when unoccupied by 3-PGA interacts with the scaffold protein AXIN in complex with the kinase HIPK2, both of which are also p53-binding proteins. This leads to the formation of a multivalent p53-binding complex that allows HIPK2 to specifically phosphorylate p53-Ser46 and thereby promote apoptosis. Furthermore, we show that PHGDH mutants (R135W and V261M) that are constitutively bound to 3-PGA abolish p53 activation even under low glucose conditions, while the mutants (T57A and T78A) unable to bind 3-PGA cause constitutive p53 activation and apoptosis in hepatocellular carcinoma (HCC) cells, even in the presence of high glucose. In vivo, PHGDH-T57A induces apoptosis and inhibits the growth of diethylnitrosamine-induced mouse HCC, whereas PHGDH-R135W prevents apoptosis and promotes HCC growth, and knockout of Trp53 abolishes these effects above. Importantly, caloric restriction that lowers whole-body glucose levels can impede HCC growth dependent on PHGDH. Together, these results unveil a mechanism by which glucose availability autonomously controls p53 activity, providing a new paradigm of cell fate control by metabolic substrate availability.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Animals , Mice , Phosphoglycerate Dehydrogenase/genetics , Phosphoglycerate Dehydrogenase/metabolism , Tumor Suppressor Protein p53/metabolism , Serine/metabolism , Cell Line, TumorABSTRACT
The activity of 5'-adenosine monophosphate-activated protein kinase (AMPK) is inversely correlated with the cellular availability of glucose. When glucose levels are low, the glycolytic enzyme aldolase is not bound to fructose-1,6-bisphosphate (FBP) and, instead, signals to activate lysosomal AMPK. Here, we show that blocking FBP binding to aldolase with the small molecule aldometanib selectively activates the lysosomal pool of AMPK and has beneficial metabolic effects in rodents. We identify aldometanib in a screen for aldolase inhibitors and show that it prevents FBP from binding to v-ATPase-associated aldolase and activates lysosomal AMPK, thereby mimicking a cellular state of glucose starvation. In male mice, aldometanib elicits an insulin-independent glucose-lowering effect, without causing hypoglycaemia. Aldometanib also alleviates fatty liver and nonalcoholic steatohepatitis in obese male rodents. Moreover, aldometanib extends lifespan and healthspan in both Caenorhabditis elegans and mice. Taken together, aldometanib mimics and adopts the lysosomal AMPK activation pathway associated with glucose starvation to exert physiological roles, and might have potential as a therapeutic for metabolic disorders in humans.
Subject(s)
Insulins , Starvation , Humans , Male , Mice , Animals , AMP-Activated Protein Kinases/metabolism , Glucose/metabolism , Fructose-Bisphosphate Aldolase/metabolism , Lysosomes/metabolism , Starvation/metabolism , Adenosine Triphosphatases/metabolism , Caenorhabditis elegans , Adenosine Monophosphate/metabolism , Fructose/metabolism , Insulins/metabolismABSTRACT
Remodeling of lipid metabolism has been implicated in cancers; however, it remains obscure how the lipid metabolic pathways are altered by oncogenic signaling to affect tumor development. We have recently shown that proto-oncogene tyrosine-protein kinase Src interacts with and phosphorylates the lipogenesis enzyme phosphatidate phosphatase LPIN1 to promote breast cancer development.
ABSTRACT
The sympathetic nervous system-catecholamine-uncoupling protein 1 (UCP1) axis plays an essential role in non-shivering adaptive thermogenesis. However, whether there exists a direct effector that physically connects catecholamine signalling to UCP1 in response to acute cold is unknown. Here we report that outer mitochondrial membrane-located AIDA is phosphorylated at S161 by the catecholamine-activated protein kinase A (PKA). Phosphorylated AIDA translocates to the intermembrane space, where it binds to and activates the uncoupling activity of UCP1 by promoting cysteine oxidation of UCP1. Adipocyte-specific depletion of AIDA abrogates UCP1-dependent thermogenesis, resulting in hypothermia during acute cold exposure. Re-expression of S161A-AIDA, unlike wild-type AIDA, fails to restore the acute cold response in Aida-knockout mice. The PKA-AIDA-UCP1 axis is highly conserved in mammals, including hibernators. Denervation of the sympathetic postganglionic fibres abolishes cold-induced AIDA-dependent thermogenesis. These findings uncover a direct mechanistic link between sympathetic input and UCP1-mediated adaptive thermogenesis.
Subject(s)
Adipocytes, Brown/metabolism , Adipose Tissue, Brown/innervation , Phospholipid Transfer Proteins/metabolism , Sympathetic Nervous System/physiology , Thermogenesis , Uncoupling Protein 1/metabolism , Adiponectin/genetics , Adiponectin/metabolism , Animals , Cells, Cultured , Cyclic AMP-Dependent Protein Kinases/metabolism , Energy Metabolism , Male , Mice, Inbred C57BL , Mice, Knockout , Oxidation-Reduction , Phospholipid Transfer Proteins/deficiency , Phospholipid Transfer Proteins/genetics , Phosphorylation , Signal Transduction , Uncoupling Protein 1/deficiency , Uncoupling Protein 1/geneticsABSTRACT
Increased lipogenesis has been linked to an increased cancer risk and poor prognosis; however, the underlying mechanisms remain obscure. Here we show that phosphatidic acid phosphatase (PAP) lipin-1, which generates diglyceride precursors necessary for the synthesis of glycerolipids, interacts with and is a direct substrate of the Src proto-oncogenic tyrosine kinase. Obesity-associated microenvironmental factors and other Src-activating growth factors, including the epidermal growth factor, activate Src and promote Src-mediated lipin-1 phosphorylation on Tyr398, Tyr413 and Tyr795 residues. The tyrosine phosphorylation of lipin-1 markedly increases its PAP activity, accelerating the synthesis of glycerophospholipids and triglyceride. Alteration of the three tyrosine residues to phenylalanine (3YF-lipin-1) disables lipin-1 from mediating Src-enhanced glycerolipid synthesis, cell proliferation and xenograft growth. Re-expression of 3YF-lipin-1 in PyVT;Lpin1-/- mice fails to promote progression and metastasis of mammary tumours. Human breast tumours exhibit increased p-Tyr-lipin-1 levels compared to the adjacent tissues. Importantly, statistical analyses show that levels of p-Tyr-lipin-1 correlate with tumour sizes, lymph node metastasis, time to recurrence and survival of the patients. These results illustrate a direct lipogenesis-promoting role of the pro-oncogenic Src, providing a mechanistic link between obesity-associated mitogenic signaling and breast cancer malignancy.
Subject(s)
Breast Neoplasms/pathology , CSK Tyrosine-Protein Kinase/genetics , Phosphatidate Phosphatase/metabolism , Animals , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/mortality , CSK Tyrosine-Protein Kinase/metabolism , Cell Line, Tumor , Cell Proliferation , Female , Humans , Lipogenesis/physiology , Male , Mammary Neoplasms, Animal/genetics , Mammary Neoplasms, Animal/pathology , Mice, Mutant Strains , Mice, Transgenic , Phosphatidate Phosphatase/genetics , Phosphorylation , Proto-Oncogene Mas , Tyrosine/metabolism , Xenograft Model Antitumor AssaysABSTRACT
AMPK, a master regulator of metabolic homeostasis, is activated by both AMP-dependent and AMP-independent mechanisms. The conditions under which these different mechanisms operate, and their biological implications are unclear. Here, we show that, depending on the degree of elevation of cellular AMP, distinct compartmentalized pools of AMPK are activated, phosphorylating different sets of targets. Low glucose activates AMPK exclusively through the AMP-independent, AXIN-based pathway in lysosomes to phosphorylate targets such as ACC1 and SREBP1c, exerting early anti-anabolic and pro-catabolic roles. Moderate increases in AMP expand this to activate cytosolic AMPK also in an AXIN-dependent manner. In contrast, high concentrations of AMP, arising from severe nutrient stress, activate all pools of AMPK independently of AXIN. Surprisingly, mitochondrion-localized AMPK is activated to phosphorylate ACC2 and mitochondrial fission factor (MFF) only during severe nutrient stress. Our findings reveal a spatiotemporal basis for hierarchical activation of different pools of AMPK during differing degrees of stress severity.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Energy Metabolism , Nutrients/metabolism , AMP-Activated Protein Kinases/biosynthesis , Animals , CRISPR-Cas Systems , Cells, Cultured , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Microscopy, Fluorescence , PhosphorylationABSTRACT
Fructose-1,6-bisphosphate (FBP) aldolase links sensing of declining glucose availability to AMPK activation via the lysosomal pathway. However, how aldolase transmits lack of occupancy by FBP to AMPK activation remains unclear. Here, we show that FBP-unoccupied aldolase interacts with and inhibits endoplasmic reticulum (ER)-localized transient receptor potential channel subfamily V, inhibiting calcium release in low glucose. The decrease of calcium at contact sites between ER and lysosome renders the inhibited TRPV accessible to bind the lysosomal v-ATPase that then recruits AXIN:LKB1 to activate AMPK independently of AMP. Genetic depletion of TRPVs blocks glucose starvation-induced AMPK activation in cells and liver of mice, and in nematodes, indicative of physical requirement of TRPVs. Pharmacological inhibition of TRPVs activates AMPK and elevates NAD+ levels in aged muscles, rejuvenating the animals' running capacity. Our study elucidates that TRPVs relay the FBP-free status of aldolase to the reconfiguration of v-ATPase, leading to AMPK activation in low glucose.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Fructose-Bisphosphate Aldolase/metabolism , Glucose/metabolism , TRPV Cation Channels/metabolism , Acrylamides/pharmacology , Adenosine Triphosphatases/metabolism , Animals , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Caenorhabditis elegans/metabolism , Calcium/metabolism , Calcium Channels/metabolism , Endoplasmic Reticulum/metabolism , Enzyme Activation/drug effects , Enzyme Activation/genetics , Gene Knockout Techniques , HEK293 Cells , Humans , Lysosomes/metabolism , Male , Mice , Signal Transduction/drug effects , Signal Transduction/genetics , TRPV Cation Channels/antagonists & inhibitors , TRPV Cation Channels/genetics , TransfectionABSTRACT
Dietary carbohydrates have been demonized for presumed negative effects on health. However, Liu et al. (2018) identify new pathways for conversion of glucose into acetate that consume reactive oxygen species. A study relating human carbohydrate consumption to all-cause mortality also suggests that moderately high-carbohydrate diets can be beneficial.
Subject(s)
Dietary Carbohydrates , Glucose , Acetates , Animals , Humans , MammalsABSTRACT
Obesity is characterized by excessive fatty acid conversion to triacylglycerols (TAGs) in adipose tissues. However, how signaling networks sense fatty acids and connect to the stimulation of lipid synthesis remains elusive. Here, we show that homozygous knock-in mice carrying a point mutation at the Ser86 phosphorylation site of acetyltransferase Tip60 (Tip60 SA/SA ) display remarkably reduced body fat mass, and Tip60 SA/SA females fail to nurture pups to adulthood due to severely reduced milk TAGs. Mechanistically, fatty acids stimulate Tip60-dependent acetylation and endoplasmic reticulum translocation of phosphatidic acid phosphatase lipin 1 to generate diacylglycerol for TAG synthesis, which is repressed by deacetylase Sirt1. Inhibition of Tip60 activity strongly blocks fatty acid-induced TAG synthesis while Sirt1 suppression leads to increased adiposity. Genetic analysis of loss-of-function mutants in Saccharomyces cerevisiae reveals a requirement of ESA1, yeast ortholog of Tip60, in TAG accumulation. These findings uncover a conserved mechanism linking fatty acid sensing to fat synthesis.
Subject(s)
Endoplasmic Reticulum/enzymology , Lysine Acetyltransferase 5/metabolism , Nuclear Proteins/metabolism , Phosphatidate Phosphatase/metabolism , Trans-Activators/metabolism , Triglycerides/biosynthesis , Acetylation , Animals , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/metabolism , Fatty Acids/metabolism , Female , Histone Acetyltransferases/genetics , Histone Acetyltransferases/metabolism , Kinetics , Lysine Acetyltransferase 5/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Nuclear Proteins/genetics , Phosphatidate Phosphatase/genetics , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sirtuin 1/genetics , Sirtuin 1/metabolism , Trans-Activators/genetics , Triglycerides/chemistryABSTRACT
The efficiency of intestinal absorption of dietary fat constitutes a primary determinant accounting for individual vulnerability to obesity. However, how fat absorption is controlled and contributes to obesity remains unclear. Here, we show that inhibition of endoplasmic-reticulum-associated degradation (ERAD) increases the abundance of triacylglycerol synthesis enzymes and fat absorption in small intestine. The C2-domain protein AIDA acts as an essential factor for the E3-ligase HRD1 of ERAD to downregulate rate-limiting acyltransferases GPAT3, MOGAT2, and DGAT2. Aida-/- mice, when grown in a thermal-neutral condition or fed high-fat diet, display increased intestinal fatty acid re-esterification, circulating and tissue triacylglycerol, accompanied with severely increased adiposity without enhancement of adipogenesis. Intestine-specific knockout of Aida largely phenocopies its whole-body knockout, strongly indicating that increased intestinal TAG synthesis is a primary impetus to obesity. The AIDA-mediated ERAD system may thus represent an anti-thrifty mechanism impinging on the enzymes for intestinal fat absorption and systemic fat storage.
Subject(s)
Dietary Fats/metabolism , Endoplasmic Reticulum-Associated Degradation , Intestinal Absorption , Intestine, Small/enzymology , Obesity/metabolism , Phospholipid Transfer Proteins/metabolism , Triglycerides/biosynthesis , Animals , Esterification , Mice, Inbred C57BL , Phospholipid Transfer Proteins/geneticsABSTRACT
MUC16/CA125 has been identified as a prominent cancer biomarker, especially for epithelial ovarian cancers, in clinical test for over three decades. Due to its huge mass, limited knowledge of MUC16 was acquired previously. By utilizing a well characterized self-made MUC16 monoclonal antibody, we identified the endogenous interaction between a C-terminal fragment of MUC16 (MUC16C) and ß-catenin for the first time, and further elucidated that trans-activation domain of ß-catenin is required for this interaction. Such interaction could activate the Wnt/ß-catenin signaling pathway by facilitating cytosol-nucleus transportation of ß-catenin, consequently induce cell proliferation and the migration, eventually lead to tumorigenesis and metastasis in nude mice. Consistently, knockdown of MUC16 significantly weakened the capabilities of cells for proliferation and migration. Based on our discovery, we suggest that MUC16 appears as an attractive target for the development of effective anticancer drugs.
Subject(s)
CA-125 Antigen/metabolism , Carcinogenesis/metabolism , Membrane Proteins/metabolism , Wnt Signaling Pathway/physiology , beta Catenin/metabolism , Animals , Cell Line, Tumor , Cell Proliferation/physiology , Heterografts , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Invasiveness , Peptide FragmentsABSTRACT
Hypoxia-inducible factors (HIFs) are master regulators of adaptive responses to low oxygen, and their α-subunits are rapidly degraded through the ubiquitination-dependent proteasomal pathway after hydroxylation. Aberrant accumulation or activation of HIFs is closely linked to many types of cancer. However, how hydroxylation of HIFα and its delivery to the ubiquitination machinery are regulated remains unclear. Here we show that Rho-related BTB domain-containing protein 3 (RHOBTB3) directly interacts with the hydroxylase PHD2 to promote HIFα hydroxylation. RHOBTB3 also directly interacts with the von Hippel-Lindau (VHL) protein, a component of the E3 ubiquitin ligase complex, facilitating ubiquitination of HIFα. Remarkably, RHOBTB3 dimerizes with LIMD1, and constructs a RHOBTB3/LIMD1-PHD2-VHL-HIFα complex to effect the maximal degradation of HIFα. Hypoxia reduces the RHOBTB3-centered complex formation, resulting in an accumulation of HIFα. Importantly, the expression level of RHOBTB3 is greatly reduced in human renal carcinomas, and RHOBTB3 deficiency significantly elevates the Warburg effect and accelerates xenograft growth. Our work thus reveals that RHOBTB3 serves as a scaffold to organize a multi-subunit complex that promotes the hydroxylation, ubiquitination and degradation of HIFα.