Downregulation of diacylglycerol kinase delta contributes to hyperglycemia-induced insulin resistance.

Type 2 (non-insulin-dependent) diabetes mellitus is a progressive metabolic disorder arising from genetic and environmental factors that impair beta cell function and insulin action in peripheral tissues. We identified reduced diacylglycerol kinase delta (DGKdelta) expression and DGK activity in skeletal muscle from type 2 diabetic patients. In diabetic animals, reduced DGKdelta protein and DGK kinase activity were restored upon correction of glycemia. DGKdelta haploinsufficiency increased diacylglycerol content, reduced peripheral insulin sensitivity, insulin signaling, and glucose transport, and led to age-dependent obesity. Metabolic flexibility, evident by the transition between lipid and carbohydrate utilization during fasted and fed conditions, was impaired in DGKdelta haploinsufficient mice. We reveal a previously unrecognized role for DGKdelta in contributing to hyperglycemia-induced peripheral insulin resistance and thereby exacerbating the severity of type 2 diabetes. DGKdelta deficiency causes peripheral insulin resistance and metabolic inflexibility. These defects in glucose and energy homeostasis contribute to mild obesity later in life.

SAHA and cisplatin sensitize gastric cancer cells to doxorubicin by induction of DNA damage, apoptosis and perturbation of AMPK-mTOR signalling.

Chemotherapy remains the most prescribed anti-cancer therapy, despite patients suffering severe side effects and frequently developing chemoresistance. These complications can be partially overcome by combining different chemotherapeutic agents that target multiple biological pathways. However, selecting efficacious drug combinations remains challenging. We previously used fission yeast Schizosaccharomycespombe as a surrogate model to predict drug combinations, and showed that suberoylanilide hydroxamic acid (SAHA) and cisplatin can sensitise gastric adenocarcinoma cells toward the cytotoxic effects of doxorubicin. Yet, how this combination undermines cell viability is unknown. Here, we show that SAHA and doxorubicin markedly enhance the cleavage of two apoptosis markers, caspase 3 and poly-ADP ribose polymerase (PARP-1), and increase the phosphorylation of γH2AX, a marker of DNA damage. Further, we found a prominent reduction in Ser485 phosphorylation of AMP-dependent protein kinase (AMPK), and reductions in its target mTOR and downstream ribosomal protein S6 phosphorylation. We show that SAHA contributes most of the effect, as confirmed using another histone deacetylase inhibitor, trichostatin A. Overall, our results show that the combination of SAHA and doxorubicin can induce apoptosis in gastric adenocarcinoma in a synthetically lethal manner, and that fission yeast offers an efficient tool for identifying potent drug combinations against human cancer cells.

Mechanistic target of rapamycin complex 1 is an essential mediator of metabolic and mitogenic effects of fibroblast growth factor 19 in hepatoma cells.

UNLABELLED: Fibroblast growth factor 19 (FGF19) is an important postprandial enterokine which regulates liver metabolism and hepatocyte proliferation. However, the precise mechanism by which FGF19 regulates these cellular effects is poorly understood. Given that mechanistic target of rapamycin complex 1 (mTORC1) regulates numerous postprandial adaptations, we investigated the potential role of mTORC1 in FGF19 action. We found that FGF19 activated mTORC1 in HepG2 and HuH7 human hepatoma cells, differentiated 3T3-L1 adipocytes and mouse liver. FGF19 activates the mTORC1-p70S6K and extracellular signal-regulated kinase (Erk)-p90RSK pathways independently to regulate S6 in an additive manner in hepatoma cells, but it uses mTORC1 as the primary pathway to regulate S6 in 3T3-L1 adipocytes. Thus, mTORC1 is a novel mediator of FGF19 signaling, which can act in parallel with Erk or function as the primary pathway to regulate S6. The FGF19-induced mTORC1 pathway requires amino acids for efficient signaling; thus, involvement of mTORC1 confers amino acid sensitivity to FGF19 signaling. Although Akt and Erk are known to activate mTORC1, we found that FGF19 signals to mTORC1 through a third recently identified mTORC1 regulator, Ras-like (Ral) protein. Pharmacological or genetic inhibition of RalA or RalB abolished FGF19-induced mTORC1 activation, demonstrating that Ral proteins are required for FGF19 to activate mTORC1. FGF19 induced metabolic gene expression, fatty acid oxidation, cell growth, and proliferation in HepG2 cells; and these effects were abolished by mTORC1 inhibition, demonstrating an essential role of mTORC1 in FGF19 action. CONCLUSION: mTORC1 is a novel and essential mediator of FGF19 action on metabolic and mitogenic programs; thus, the involvement of mTORC1 in FGF19 signaling is an important factor to consider when targeting the pathway for cancer or diabetes therapy. (Hepatology 2016;64:1289-1301).

Autophagy modulates amino acid signaling network in myotubes: differential effects on mTORC1 pathway and the integrated stress response.

Induction of autophagy and the integrated stress response is important for amino acid homeostasis. It remains unknown whether the autophagy coregulates both mechanistic target of rapamycin complex 1 (mTORC1) signaling and the integrated stress response. In mouse C2C12 myotubes, we found that amino acid limitation induced autophagy and that the subsequent release of amino acid is required to sustain mTORC1 signaling. Inhibition of autophagy by bafilomycin A1 or chloroquine treatment during amino acid scarcity abolished mTORC1 signaling, an effect that could be rescued by inhibiting protein synthesis or amino acid supplementation, respectively. Autophagy is required to sustain the balance of both essential and nonessential amino acids during amino acid starvation, and it has a predominant role over the ubiquitin-proteasome system in the regulation of mTORC1. Inhibition of autophagy was found to activate the integrated stress response, as well as eukaryotic initiation factor 2α (eIF2α) and its target genes independent of amino acid availability. Conversely, autophagy induction via mTOR inhibition is sufficient to reduce eIF2α phosphorylation. Thus, autophagy protects the eIF2α-mediated stress response independent of amino acid supply in cultured myotubes. Our results showed that autophagy uniquely modulates mTORC1 and the integrated stress response in an amino acid-dependent and -independent manner, respectively.

The biochemistry and cell biology of aging: metabolic regulation through mitochondrial signaling.

Cellular and organ metabolism affects organismal lifespan. Aging is characterized by increased risks for metabolic disorders, with age-associated degenerative diseases exhibiting varying degrees of mitochondrial dysfunction. The traditional view of the role of mitochondria generated reactive oxygen species (ROS) in cellular aging, assumed to be causative and simply detrimental for a long time now, is in need of reassessment. While there is little doubt that high levels of ROS are detrimental, mounting evidence points toward a lifespan extension effect exerted by mild to moderate ROS elevation. Dietary caloric restriction, inhibition of insulin-like growth factor-I signaling, and inhibition of the nutrient-sensing mechanistic target of rapamycin are robust longevity-promoting interventions. All of these appear to elicit mitochondrial retrograde signaling processes (defined as signaling from the mitochondria to the rest of the cell, for example, the mitochondrial unfolded protein response, or UPR(mt)). The effects of mitochondrial retrograde signaling may even spread to other cells/tissues in a noncell autonomous manner by yet unidentified signaling mediators. Multiple recent publications support the notion that an evolutionarily conserved, mitochondria-initiated signaling is central to the genetic and epigenetic regulation of cellular aging and organismal lifespan.

Hormone-like fibroblast growth factors and metabolic regulation.

The family of fibroblast growth factors (FGFs) consisting now of 22 members is generally considered to control a wide range of biological functions such as development, differentiation and survival. However, research during the past decade provided substantial evidence that a so called "hormone-like" subgroup of FGFs, comprised of FGF19, FGF21 and FGF23, is involved in the regulation of diverse metabolic pathways to control glucose, lipid, bile acid, phosphate and vitamin D metabolism. The unique properties of these FGFs include predominant production of the factors in selective tissues, their abundance in the blood due to the lack of extracellular heparin-mediated sequestration, and highly specific tissue-targeted action via engagement of their respective co-receptors. The important metabolic context of FGF19, FGF21, and FGF23 actions has revealed important novel roles for FGFs and provided significant means to explore an opportunity for therapeutic targeting of these factors and their corresponding pathways.

Npac Is A Co-factor of Histone H3K36me3 and Regulates Transcriptional Elongation in Mouse Embryonic Stem Cells.

Chromatin modification contributes to pluripotency maintenance in embryonic stem cells (ESCs). However, the related mechanisms remain obscure. Here, we show that Npac, a "reader" of histone H3 lysine 36 trimethylation (H3K36me3), is required to maintain mouse ESC (mESC) pluripotency since knockdown of Npac causes mESC differentiation. Depletion of Npac in mouse embryonic fibroblasts (MEFs) inhibits reprogramming efficiency. Furthermore, our chromatin immunoprecipitation followed by sequencing (ChIP-seq) results of Npac reveal that Npac co-localizes with histone H3K36me3 in gene bodies of actively transcribed genes in mESCs. Interestingly, we find that Npac interacts with positive transcription elongation factor b (p-TEFb), Ser2-phosphorylated RNA Pol II (RNA Pol II Ser2P), and Ser5-phosphorylated RNA Pol II (RNA Pol II Ser5P). Furthermore, depletion of Npac disrupts transcriptional elongation of the pluripotency genes Nanog and Rif1. Taken together, we propose that Npac is essential for the transcriptional elongation of pluripotency genes by recruiting p-TEFb and interacting with RNA Pol II Ser2P and Ser5P.

PATZ1 (MAZR) Co-occupies Genomic Sites With p53 and Inhibits Liver Cancer Cell Proliferation via Regulating p27.

Liver cancer is the third most common cause of cancer death in the world. POZ/BTB and AT-hook-containing zinc finger protein 1 (PATZ1/MAZR) is a transcription factor associated with various cancers. However, the role of PATZ1 in cancer progression remains controversial largely due to lack of genome-wide studies. Here we report that PATZ1 regulates cell proliferation by directly regulating CDKN1B (p27) in hepatocellular carcinoma cells. Our PATZ1 ChIP-seq and gene expression microarray analyses revealed that PATZ1 is strongly related to cancer signatures and cellular proliferation. We further discovered that PATZ1 depletion led to an increased rate of colony formation, elevated Ki-67 expression and greater S phase entry. Importantly, the increased cancer cell proliferation was accompanied with suppressed expression of the cyclin-dependent kinase inhibitor CDKN1B. Consistently, we found that PATZ1 binds to the genomic loci flanking the transcriptional start site of CDKN1B and positively regulates its transcription. Notably, we demonstrated that PATZ1 is a p53 partner and p53 is essential for CDKN1B regulation. In conclusion, our study provides novel mechanistic insights into the inhibitory role of PATZ1 in liver cancer progression, thereby yielding a promising therapeutic intervention to alleviate tumor burden.

Cysteine Deprivation Targets Ovarian Clear Cell Carcinoma Via Oxidative Stress and Iron-Sulfur Cluster Biogenesis Deficit.

Aims: Current treatment options for ovarian clear cell carcinoma (OCCC) are limited to combination of platinum-based and other cytotoxic agents to which patients respond poorly due to intrinsic chemoresistance. There is therefore an urgent need to develop alternative therapeutic strategies for OCCC. Results: Cysteine deprivation suppresses OCCC growth in vitro and in vivo with no apparent toxicity. Modes of cell death induced by cysteine deprivation in OCCC are determined by their innate metabolic profiles. Cysteine-deprived glycolytic OCCC is abolished primarily by oxidative stress-dependent necrosis and ferroptosis, which can otherwise be prevented by pretreatment with antioxidative agents. Meanwhile, OCCC that relies on mitochondria respiration for its bioenergetics is suppressed through apoptosis, which can otherwise be averted by pretreatment with cysteine precursor alone, but not with antioxidative agents. Cysteine deprivation induces apoptosis in respiring OCCC by limiting iron-sulfur (Fe-S) cluster synthesis in the mitochondria, without which electron transport chain may be disrupted. Respiring OCCC responds to Fe-S cluster deficit by increasing iron influx into the mitochondria, which leads to iron overload, mitochondria damage, and eventual cell death. Innovation/Conclusion: This study demonstrates the importance of cysteine availability in OCCC that is for its antioxidative property and its less appreciated role in mitochondria respiration. Regardless of OCCC metabolic profiles, cysteine deprivation abolishes both glycolytic and respiring OCCC growth in vitro and in vivo. Conclusion: This study highlights the therapeutic potential of cysteine deprivation for OCCC.

AMP-activated protein kinase signaling in metabolic regulation.

AMP-activated protein kinase (AMPK) is an energy sensor that regulates cellular metabolism. When activated by a deficit in nutrient status, AMPK stimulates glucose uptake and lipid oxidation to produce energy, while turning off energy-consuming processes including glucose and lipid production to restore energy balance. AMPK controls whole-body glucose homeostasis by regulating metabolism in multiple peripheral tissues, such as skeletal muscle, liver, adipose tissues, and pancreatic beta cells--key tissues in the pathogenesis of type 2 diabetes. By responding to diverse hormonal signals including leptin and adiponectin, AMPK serves as an intertissue signal integrator among peripheral tissues, as well as the hypothalamus, in the control of whole-body energy balance.

Role of adenosine 5'-monophosphate-activated protein kinase subunits in skeletal muscle mammalian target of rapamycin signaling.

AMP-activated protein kinase (AMPK) is an important energy-sensing protein in skeletal muscle. Mammalian target of rapamycin (mTOR) mediates translation initiation and protein synthesis through ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). AMPK activation reduces muscle protein synthesis by down-regulating mTOR signaling, whereas insulin mediates mTOR signaling via Akt activation. We hypothesized that AMPK-mediated inhibitory effects on mTOR signaling depend on catalytic alpha2 and regulatory gamma3 subunits. Extensor digitorum longus muscle from AMPK alpha2 knockout (KO), AMPK gamma3 KO, and respective wild-type (WT) littermates (C57BL/6) were incubated in the presence of 5-aminoimidazole-4-carboxamide-1-beta-d-ribonucleoside (AICAR), insulin, or AICAR plus insulin. Phosphorylation of AMPK, Akt, and mTOR-associated signaling proteins were assessed. Insulin increased Akt Ser473 phosphorylation (P < 0.01), irrespective of genotype or presence of AICAR. AICAR increased phosphorylation of AMPK Thr172 (P < 0.01) in WT but not KO mice. Insulin stimulation increased phosphorylation of S6K1 (Thr389), ribosomal protein S6 (Ser235/236), and 4E-BP1 (Thr37/46) (P < 0.01) in WT, AMPK alpha2 KO, and AMPK gamma3 KO mice. However, in WT mice, preincubation with AICAR completely inhibited insulin-induced phosphorylation of mTOR targets, suggesting mTOR signaling is blocked by prior AMPK activation. The AICAR-induced inhibition was partly rescued in extensor digitorum longus muscle from either alpha2 or gamma3 AMPK KO mice, indicating functional alpha2 and gamma3 subunits of AMPK are required for the reduction in mTOR signaling. AICAR alone was without effect on basal phosphorylation of S6K1 (Thr389), ribosomal protein S6 (Ser235/236), and 4E-BP1 (Thr37/46). In conclusion, functional alpha2 and gamma3 AMPK subunits are required for AICAR-induced inhibitory effects on mTOR signaling.

AMPK-mediated AS160 phosphorylation in skeletal muscle is dependent on AMPK catalytic and regulatory subunits.

AMP-activated protein kinase (AMPK) is a heterotrimeric protein that regulates glucose transport mediated by cellular stress or pharmacological agonists such as 5-aminoimidazole-4-carboxamide 1 beta-d-ribonucleoside (AICAR). AS160, a Rab GTPase-activating protein, provides a mechanism linking AMPK signaling to glucose uptake. We show that AICAR increases AMPK, acetyl-CoA carboxylase, and AS160 phosphorylation by insulin-independent mechanisms in isolated skeletal muscle. Recombinant AMPK heterotrimeric complexes (alpha1beta1gamma1 and alpha2beta2gamma1) phosphorylate AS160 in a cell-free assay. In mice deficient in AMPK signaling (alpha2 AMPK knockout [KO], alpha2 AMPK kinase dead [KD], and gamma3 AMPK KO), AICAR effects on AS160 phosphorylation were severely blunted, highlighting that complexes containing alpha2 and gamma3 are necessary for AICAR-stimulated AS160 phosphorylation in intact skeletal muscle. Contraction-mediated AS160 phosphorylation was also impaired in alpha2 AMPK KO and KD but not gamma3 AMPK KO mice. Our results implicate AS160 as a downstream target of AMPK.

Glutamine metabolism regulates autophagy-dependent mTORC1 reactivation during amino acid starvation.

Activation of autophagy and elevation of glutamine synthesis represent key adaptations to maintain amino acid balance during starvation. In this study, we investigate the role of autophagy and glutamine on the regulation of mTORC1, a critical kinase that regulates cell growth and proliferation. We report that supplementation of glutamine alone is sufficient to restore mTORC1 activity during prolonged amino acid starvation. Inhibition of autophagy abolishes the restorative effect of glutamine, suggesting that reactivation of mTORC1 is autophagy-dependent. Inhibition of glutaminolysis or transamination impairs glutamine-mediated mTORC1 reactivation, suggesting glutamine reactivates mTORC1 specifically through its conversion to glutamate and restoration of non-essential amino acid pool. Despite a persistent drop in essential amino acid pool during amino acid starvation, crosstalk between glutamine and autophagy is sufficient to restore insulin sensitivity of mTORC1. Thus, glutamine metabolism and autophagy constitute a specific metabolic program which restores mTORC1 activity during amino acid starvation.mTORC1 is a critical kinase that regulates cell growth and proliferation. Here the authors show that glutamine metabolism is sufficient to restore mTORC1 activity during prolonged amino acid starvation in an autophagy-dependent manner.

Changes in exercise-induced gene expression in 5'-AMP-activated protein kinase gamma3-null and gamma3 R225Q transgenic mice.

5'-AMP-activated protein kinase (AMPK) is important for metabolic sensing. We used AMPKgamma3 mutant-overexpressing Tg-Prkag3(225Q) and AMPKgamma3-knockout Prkag3-/- mice to determine the role of the AMPKgamma3 isoform in exercise-induced metabolic and gene regulatory responses in skeletal muscle. Mice were studied after 2 h swimming or 2.5 h recovery. Exercise increased basal and insulin-stimulated glucose transport, with similar responses among genotypes. In Tg-Prkag3(225Q) mice, acetyl-CoA carboxylase (ACC) phosphorylation was increased and triglyceride content was reduced after exercise, suggesting that this mutation promotes greater reliance on lipid oxidation. In contrast, ACC phosphorylation and triglyceride content was similar between wild-type and Prkag3-/- mice. Expression of genes involved in lipid and glucose metabolism was altered by genetic modification of AMPKgamma3. Expression of lipoprotein lipase 1, carnitine palmitoyl transferase 1b, and 3-hydroxyacyl-CoA dehydrogenase was increased in Tg-Prkag3(225Q) mice, with opposing effects in Prkag3-/- mice after exercise. GLUT4, hexokinase II (HKII), and glycogen synthase mRNA expression was increased in Tg-Prkag3(225Q) mice after exercise. GLUT4 and HKII mRNA expression was increased in wild-type mice and blunted in Prkag3-/- mice after recovery. In conclusion, the Prkag3(225Q) mutation, rather than presence of a functional AMPKgamma3 isoform, directly promotes metabolic and gene regulatory responses along lipid oxidative pathways in skeletal muscle after endurance exercise.

5'-AMP-activated protein kinase regulates skeletal muscle glycogen content and ergogenics.

5'-AMP-activated protein kinase (AMPK) activity is increased during exercise in an intensity- and glycogen-dependent manner. We previously reported that a mutation in the AMPK3 subunit (Prkag3225Q) increases AMPK activity and skeletal muscle glycogen content. Transfection experiments revealed the R225Q mutation is associated with high basal AMPK activity and diminished AMP dependence. Thus, the R225Q mutation can be considered a loss-of-function mutation that abolished allosteric regulation by AMP/ATP, causing increased basal AMPK activity. We used AMPK3 transgenic (Tg-Prkag3225Q) and knockout (Prkag3-/-) mice to determine the relationship between AMPK activity, glycogen content, and ergogenics (ability to perform work) in isolated extensor digitorum longus skeletal muscle after contractions induced by electrical stimulation. Contraction-induced AMPK activity was inversely coupled to glycogen content in wild-type and Tg-Prkag3225Q mice, but not in Prkag3-/- mice, highlighting a partial feedback control of glycogen on contraction-induced AMPK activity in the presence of a functional AMPK3 isoform. Skeletal muscle glycogen content was positively correlated to work performance, regardless of genotype. Thus, chronic activation of AMPK by the Prkag3225Q mutation directly influences skeletal muscle ergogenics by enhancing glycogen content. In conclusion, functional studies of the AMPK3 isoform further support the close connection between glycogen content and exercise performance in skeletal muscle.

Crosstalk between cystine and glutathione is critical for the regulation of amino acid signaling pathways and ferroptosis.

Although essential amino acids regulate mechanistic target of rapamycin complex 1 (mTORC1) and the integrated stress response (ISR), the role of cysteine is unknown. We found that in hepatoma HepG2 cells, cystine (oxidized form of cysteine) activated mTORC1 and suppressed the ISR. Cystine deprivation induced GSH efflux and extracellular degradation, which aimed to restore cellular cysteine. Inhibition of γ-glutamyl transpeptidase (GGT) impaired the ability of GSH or cell-permeable GSH to restore mTORC1 signaling and the ISR, suggesting that the capacity of GSH to release cysteine, but not GSH per se, regulated the signaling networks. Inhibition of protein translation restored both mTORC1 signaling and the ISR during cystine starvation, suggesting the bulk of cellular cysteine was committed to the biosynthetic process. Cellular cysteine and GSH displayed overlapping protective roles in the suppression of ferroptosis, further supporting their cooperation in the regulation of cell signaling. Thus, cellular cysteine and its derivative GSH cooperate to regulate mTORC1 pathway, the ISR and ferroptosis.

FoxO1 interacts with transcription factor EB and differentially regulates mitochondrial uncoupling proteins via autophagy in adipocytes.

Mitochondrial uncoupling proteins (UCPs) are inducible and play an important role in metabolic and redox homeostasis. Recent studies have suggested that FoxO1 controls mitochondrial biogenesis and morphology, but it remains largely unknown how FoxO1 may regulate mitochondrial UCPs. Here we show that FoxO1 interacted with transcription factor EB (Tfeb), a key regulator of autophagosome and lysosome, and mediated the expression of UCP1, UCP2 and UCP3 differentially via autophagy in adipocytes. UCP1 was down-regulated but UCP2 and UCP3 were upregulated during adipocyte differentiation, which was associated with increased Tfeb and autophagy activity. However, inhibition of FoxO1 suppressed Tfeb and autophagy, attenuating UCP2 and UCP3 but increasing UCP1 expression. Pharmacological blockade of autophagy recapitulated the effects of FoxO1 inhibition on UCPs. Chromatin immunoprecipitation assay demonstrated that FoxO1 interacted with Tfeb by directly binding to its promoter, and silencing FoxO1 led to drastic decrease in Tfeb transcript and protein levels. These data provide the first line of evidence that FoxO1 interacts with Tfeb to regulate autophagy and UCP expression in adipocytes. Dysregulation of FoxO1→autophagy→UCP pathway may account for metabolic changes in obesity.

FoxO1 antagonist suppresses autophagy and lipid droplet growth in adipocytes.

Obesity and related metabolic disorders constitute one of the most pressing heath concerns worldwide. Increased adiposity is linked to autophagy upregulation in adipose tissues. However, it is unknown how autophagy is upregulated and contributes to aberrant adiposity. Here we show a FoxO1-autophagy-FSP27 axis that regulates adipogenesis and lipid droplet (LD) growth in adipocytes. Adipocyte differentiation was associated with upregulation of autophagy and fat specific protein 27 (FSP27), a key regulator of adipocyte maturation and expansion by promoting LD formation and growth. However, FoxO1 specific inhibitor AS1842856 potently suppressed autophagy, FSP27 expression, and adipocyte differentiation. In terminally differentiated adipocytes, AS1842856 significantly reduced FSP27 level and LD size, which was recapitulated by autophagy inhibitors (bafilomycin-A1 and leupeptin, BL). Similarly, AS1842856 and BL dampened autophagy activity and FSP27 expression in explant cultures of white adipose tissue. To our knowledge, this is the first study addressing FoxO1 in the regulation of adipose autophagy, shedding light on the mechanism of increased autophagy and adiposity in obese individuals. Given that adipogenesis and adipocyte expansion contribute to aberrant adiposity, targeting the FoxO1-autophagy-FSP27 axis may lead to new anti-obesity options.

Differential regulatory functions of three classes of phosphatidylinositol and phosphoinositide 3-kinases in autophagy.

Autophagy is an evolutionarily conserved and exquisitely regulated self-eating cellular process with important biological functions. Phosphatidylinositol 3-kinases (PtdIns3Ks) and phosphoinositide 3-kinases (PI3Ks) are involved in the autophagic process. Here we aim to recapitulate how 3 classes of these lipid kinases differentially regulate autophagy. Generally, activation of the class I PI3K suppresses autophagy, via the well-established PI3K-AKT-MTOR (mechanistic target of rapamycin) complex 1 (MTORC1) pathway. In contrast, the class III PtdIns3K catalytic subunit PIK3C3/Vps34 forms a protein complex with BECN1 and PIK3R4 and produces phosphatidylinositol 3-phosphate (PtdIns3P), which is required for the initiation and progression of autophagy. The class II enzyme emerged only recently as an alternative source of PtdIns3P and autophagic initiator. However, the orthodox paradigm is challenged by findings that the PIK3CB catalytic subunit of class I PI3K acts as a positive regulator of autophagy, and PIK3C3 was thought to be an amino acid sensor for MTOR, which curbs autophagy. At present, a number of PtdIns3K and PI3K inhibitors, including specific PIK3C3 inhibitors, have been developed for suppression of autophagy and for clinical applications in autophagy-related human diseases.