Autophagy in practice: stevia and leucine.

Beginning with this issue, we present answers to practical questions regarding autophagy from the lay public.

Regulation of autophagy by amino acids and MTOR-dependent signal transduction.

Amino acids not only participate in intermediary metabolism but also stimulate insulin-mechanistic target of rapamycin (MTOR)-mediated signal transduction which controls the major metabolic pathways. Among these is the pathway of autophagy which takes care of the degradation of long-lived proteins and of the elimination of damaged or functionally redundant organelles. Proper functioning of this process is essential for cell survival. Dysregulation of autophagy has been implicated in the etiology of several pathologies. The history of the studies on the interrelationship between amino acids, MTOR signaling and autophagy is the subject of this review. The mechanisms responsible for the stimulation of MTOR-mediated signaling, and the inhibition of autophagy, by amino acids have been studied intensively in the past but are still not completely clarified. Recent developments in this field are discussed.

Glutamate dehydrogenase contributes to leucine sensing in the regulation of autophagy.

Amino acids, leucine in particular, are known to inhibit autophagy, at least in part by their ability to stimulate MTOR-mediated signaling. Evidence is presented showing that glutamate dehydrogenase, the central enzyme in amino acid catabolism, contributes to leucine sensing in the regulation of autophagy. The data suggest a dual mechanism by which glutamate dehydrogenase activity modulates autophagy, i.e., by activating MTORC1 and by limiting the formation of reactive oxygen species.

Autophagy, signaling and obesity.

Autophagy is a cellular pathway crucial for development, differentiation, survival and homeostasis. Autophagy can provide protection against aging and a number of pathologies such as cancer, neurodegeneration, cardiac disease and infection. Recent studies have reported new functions of autophagy in the regulation of cellular processes such as lipid metabolism and insulin sensitivity. Important links between the regulation of autophagy and obesity including food intake, adipose tissue development, ß cell function, insulin sensitivity and hepatic steatosis exist. This review will provide insight into the current understanding of autophagy, its regulation, and its role in the complications associated with obesity.

Mammalian NADH:ubiquinone oxidoreductase (Complex I) and nicotinamide nucleotide transhydrogenase (Nnt) together regulate the mitochondrial production of H2O2--implications for their role in disease, especially cancer.

Mammalian NADH:ubiquinone oxidoreductase (Complex I) in the mitochondrial inner membrane catalyzes the oxidation of NADH in the matrix. Excess NADH reduces nine of the ten prosthetic groups of the enzyme in bovine-heart submitochondrial particles with a rate of at least 3,300 s⁻¹. This results in an overall NADH→O2 rate of ca. 150 s⁻¹. It has long been known that the bovine enzyme also has a specific reaction site for NADPH. At neutral pH excess NADPH reduces only three to four of the prosthetic groups in Complex I with a rate of 40 s⁻¹ at 22 °C. The reducing equivalents remain essentially locked in the enzyme because the overall NADPH→O2 rate (1.4 s⁻¹) is negligible. The physiological significance of the reaction with NADPH is still unclear. A number of recent developments has revived our thinking about this enigma. We hypothesize that Complex I and the Δp-driven nicotinamide nucleotide transhydrogenase (Nnt) co-operate in an energy-dependent attenuation of the hydrogen-peroxide generation by Complex I. This co-operation is thought to be mediated by the NADPH/NADP⁺ ratio in the vicinity of the NADPH site of Complex I. It is proposed that the specific H2O2 production by Complex I, and the attenuation of it, is of importance for apoptosis, autophagy and the survival mechanism of a number of cancers. Verification of this hypothesis may contribute to a better understanding of the regulation of these processes.

Autophagy: regulation by energy sensing.

Autophagy is inhibited by the mTOR signaling pathway, which is stimulated by increased amino acid levels. When cellular energy production is compromised, AMP-activated protein kinase is activated, mTOR is inhibited and autophagy is stimulated. Two recent studies have shed light on the molecular mechanism by which AMPK controls autophagic flux.

From the urea cycle to autophagy: Alfred J. Meijer.

Now that many of the components of the autophagy machinery have been identified, in particular the autophagy-related (Atg) proteins, increasing focus is being directed toward the role of autophagy in health and disease. Accordingly, it is of ever-greater importance to understand the central role of autophagy in cellular metabolism, a point with which many people will likely agree. However, in our rush to understand autophagy's function in metabolism, we tend to overlook the role of metabolism in regulating autophagy, even though substantial work has been done on this topic. One of the pioneers in this area is Alfred "Fred" J. Meijer.

Autophagy: a potential link between obesity and insulin resistance.

Dysregulation of autophagy contributes to aging and to diseases such as neurodegeneration, cardiomyopathy, and cancer. The paper by Yang et al. (2010) in this issue of Cell Metabolism indicates that defective autophagy may also underlie impaired insulin sensitivity in obesity and that upregulating autophagy can combat insulin resistance.

Dual-action lipophilic iminosugar improves glycemic control in obese rodents by reduction of visceral glycosphingolipids and buffering of carbohydrate assimilation.

The lipophilic iminosugar N-[5-(adamantan-1-ylmethoxy)pentyl]-1-deoxynojirimycin (2, AMP-DNM) potently controls hyperglycemia in obese rodent models of insulin resistance. The reduction of visceral glycosphingolipids by 2 is thought to underlie its beneficial action. It cannot, however, be excluded that concomitant inhibition of intestinal glycosidases and associated buffering of carbohydrate assimilation add to this. To firmly establish the mode of action of 2, we developed a panel of lipophilic iminosugars varying in configuration at C-4/C-5 and N-substitution of the iminosugar. From these we identified the l-ido derivative of 2, l-ido-AMP-DNM (4), as a selective inhibitor of glycosphingolipid synthesis. Compound 4 lowered visceral glycosphingolipids in ob/ob mice and ZDF rats on a par with 2. In contrast to 2, 4 did not inhibit sucrase activity or sucrose assimilation. Treatment with 4 was significantly less effective in reducing blood glucose and HbA1c. We conclude that the combination of reduction of glycosphingolipids in tissue and buffering of carbohydrate assimilation by 2 produces a superior glucose homeostasis.

Autophagy: regulation and role in disease.

Autophagy, a lysosomal process involved in the maintenance of cellular homeostasis, is responsible for the turnover of long-lived proteins and organelles that are either damaged or functionally redundant. The process is tightly controlled by the insulin-amino acid-mammalian target of the rapamycin-dependent signal-transduction pathway. Research in the last decade has indicated not only that autophagy provides cells with oxidizable substrate when nutrients become scarce but also that it can provide protection against aging and a number of pathologies such as cancer, neurodegeneration, cardiac disease, diabetes, and infections.

Assaying of autophagic protein degradation.

Macroautophagy is a three-step process: (1) autophagosomes form and mature, (2) the autophagosomes fuse with lysosomes, and (3) the autophagic cargo is degraded in the lysosomes. It is this lysosomal degradation of the autophagic cargo that constitutes the autophagic flux. As in the case of metabolic pathways, the steady-state concentration of the intermediary autophagic structures alone is insufficient for investigating the flux. Assaying the degradation of long-lived proteins as described in this chapter is one of the methods that can be used to measure autophagic flux.

Autophagy research: lessons from metabolism.

Autophagy research continues to expand exponentially. Clearly autophagy and metabolism are intimately connected; however, the rapid expansion of research into this topic inevitably brings the risk that important basic knowledge of metabolism will be overlooked when considering experimental data. Unfortunately, unawareness of possible metabolic complications may sometimes lead to misinterpretation of the data obtained.

Regulation of adiponectin secretion by insulin and amino acids in 3T3-L1 adipocytes.

Adiponectin is a fat cell-derived hormone with insulin-sensitizing properties. Low plasma adiponectin levels are associated with insulin resistance as found in obesity. One of the mechanisms for this finding is hampered insulin signaling via phosphatidylinositol 3-kinase (PI3K) with concomitant decreased adiponectin secretion. Because insulin can also stimulate signaling at the level of mammalian target of rapamycin (mTOR) by a mechanism that is dependent on the presence of amino acids, the role of mTOR signaling in adiponectin secretion was studied. In view of the vesicular nature of adiponectin secretion, the role of lysosomes was explored as well. In 3T3-L1 adipocytes, both insulin and amino acids stimulated adiponectin secretion. The stimulation by insulin was PI3K dependent but mTOR independent. The stimulation by amino acids was independent of both PI3K and mTOR. Whereas the effect of insulin via PI3K was mainly on adiponectin secretion from adipocytes, the effect of amino acids was predominantly due to their role as substrates for adiponectin synthesis. The acidotropic agents ammonia and methylamine, but not the lysosomal protease inhibitor leupeptin and the autophagy inhibitor 3-methyladenine, strongly inhibited adiponectin secretion and increased the intracellular adiponectin pool. In conclusion, adiponectin production is substrate driven. Phosphatidylinositol 3-kinase and an acidic lysosomal pH, but not amino acid-mediated mTOR signaling or lysosomal breakdown, are involved in adiponectin secretion.

Autophagy: a sweet process in diabetes.

Autophagy is inhibited by the insulin-amino acid-mTOR signaling pathway. Two papers in this issue of Cell Metabolism (Ebato et al., 2008; Jung et al., 2008) provide evidence that basal autophagy is necessary to maintain the architecture and function of pancreatic beta cells and that its induction in diabetic mice protects beta cells against damage by oxidative stress.

Amino acid regulation of autophagosome formation.

Amino acids are not only substrates for various metabolic pathways, but can also serve as signaling molecules controlling signal transduction pathways. One of these signaling pathways is mTOR-dependent and is activated by amino acids (leucine in particular) in synergy with insulin. Activation of this pathway inhibits autophagy. Because activation of mTOR-mediated signaling also stimulates protein synthesis, it appears that protein synthesis and autophagic protein degradation are reciprocally controlled by the same signaling pathway. Recent developments indicate that amino acid-stimulated mTOR-dependent signaling is subject to complex regulation. The mechanism by which amino acids stimulate mTORdependent signaling (and other signaling pathways), and its molecular connection with the autophagic machinery, is still unknown.

Macroautophagy: protector in the diabetes drama?

Macroautophagy ("autophagy") is regulated by the same insulin-amino acid-mTOR signaling pathway that controls protein synthesis. Although the literature does not so far include any direct studies confirming this, we expect autophagy to increase during insulin resistance. We discuss the possibility that this may be a useful mechanism for eliminating damaged mitochondria and other cell structures to prevent cell death.

AMP-activated protein kinase and autophagy.

Autophagy is inhibited by TOR-dependent signaling. Interruption of signalling by rapamycin is known to stimulate autophagy, both in mammalian cells and in yeast. However, inactivation of TOR by AMPK has yielded controversial results in the literature with regard to its effect on autophagy: activation of autophagy in yeast but inhibition in hepatocytes. In a recent study, carried out with hepatocytes, HT-29 cells, and HeLa cells, the possible role of AMPK in the control of mammalian autophagy was reexamined. The data suggest that in mammalian cells, as in yeast, AMPK is required for autophagy.