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.

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 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.

Autophagy in practice: stevia and leucine.

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

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.

Chronic treatment with pioglitazone does not protect obese patients with diabetes mellitus type II from free fatty acid-induced insulin resistance.

CONTEXT: Thiazolidinediones increase peripheral insulin sensitivity and decrease plasma free fatty acids (FFA). However, their exact mechanism of action has not been fully elucidated. OBJECTIVE: We studied the protective effect of pioglitazone on FFA-induced insulin resistance and the effects on intramyocellular glycosphingolipids. DESIGN: We studied glucose metabolism in the basal state and during a hyperinsulinemic euglycemic clamp by using stable isotopes. Studies were performed at baseline and after 4 months of treatment with pioglitazone. Patients were then studied on a third occasion during infusion of a lipid emulsion to increase plasma FFA to pretreatment levels. All studies were combined with muscle biopsies to measure intramyocellular ceramide and glycosphingolipids. PATIENTS: Patients were obese with poorly controlled type 2 diabetes mellitus. INTERVENTION: Patients were treated with 30 mg pioglitazone once daily. MAIN OUTCOME MEASURE: The change in peripheral insulin sensitivity after treatment with pioglitazone and during the infusion of the lipid emulsion was the main outcome measure. RESULTS: Peripheral glucose uptake (Rd) increased significantly, but returned to baseline levels after increasing plasma FFA to pretreatment levels. Insulin-mediated suppression of FFA was increased significantly. Intramyocellular ceramide concentrations were higher during the hyperinsulinemic clamp after treatment with pioglitazone, but not in the basal state. The intramyocellular content of glycosphingolipids and plasma concentrations of ceramide and glycosphingolipids did not change. CONCLUSIONS: Pioglitazone increases Rd and insulin-mediated suppression of plasma FFA, but does not protect patients with type 2 diabetes mellitus from FFA-induced insulin resistance. This effect of pioglitazone is not attained via a decrease in intramyocellular concentrations of ceramide or glycosphingolipids.

Signalling and autophagy regulation in health, aging and disease.

It has become clear in recent years that autophagy not only serves to produce amino acids for ongoing protein synthesis and to produce substrates for energy production when cells become starved but autophagy is also able to eliminate defective cell structures and for this reason the process may be implicated in several diseased states. Autophagy is controlled by complex signalling pathways, including that used by insulin. In these pathways, phosphatidylinositol 3-kinases and the protein kinase mTOR play important roles.

Regulation and role of autophagy in mammalian cells.

The recent period has witnessed progress in the understanding of the lysosomal autophagic pathway. The discovery of a family of genes conserved from yeast to humans, and involved in the formation of autophagosomes, has unraveled new protein-conjugation systems and has shed light on the importance of autophagy in physiology and pathophysiology. The elucidation of the molecular control of autophagy will also lead to a better understanding of the role of autophagy during cell death. As a great number of extracellular stimuli (starvation, hormonal or therapeutic treatment) as well as intracellular stimuli (accumulation of misfolded proteins, invasion of microorganisms) is able to modulate the autophagic response, it is not surprising that several signaling pathways are involved in the control of autophagy. The mammalian Target of Rapamycin (mTOR) signaling pathway plays a major role in transmitting autophagic stimuli because of its ability to sense nutrient, metabolic and hormonal signals. In addition, autophagy, which is characterized by a flux of membrane from the formation of the autophagosome to the fusion with the lysosome, is regulated by GTPases, similarly to the vesicular transport along the exocytic/endocytic pathway. The aim of the present review is to give an overview of autophagy and to discuss its regulation by activators and effectors of mTOR and GTPases.

A low-carbohydrate/high-fat diet improves glucoregulation in type 2 diabetes mellitus by reducing postabsorptive glycogenolysis.

The aim of this study was to examine the mechanisms by which dietary carbohydrate and fat modulate fasting glycemia. We compared the effects of an eucaloric high-carbohydrate (89% carbohydrate) and high-fat (89% fat) diet on fasting glucose metabolism and insulin sensitivity in seven obese patients with type 2 diabetes using stable isotopes and euglycemic hyperinsulinemic clamps. At basal insulin levels glucose concentrations were 148 +/- 11 and 123 +/- 11 mg/dl (8.2 +/- 0.6 and 6.8 +/- 0.6 mmol/liter) on the high-carbohydrate and high-fat diet, respectively (P < 0.001), with insulin concentrations of 12 +/- 2 and 10 +/- 1 microIU/ml (82 +/- 11 and 66 +/- 10 pmol/liter) (P = 0.08). Glucose production was higher on the high-carbohydrate diet (1.88 +/- 0.06 vs. 1.55 +/- 0.05 mg/kg.min (10.44 +/- 0.33 vs. 8.61 +/- 0.28 micromol/kg.min) (P < 0.001) because of higher glycogenolysis. Gluconeogenic rates were not different between the diets. During the use of hyperinsulinemic euglycemic clamps, insulin-mediated suppression of glucose production and stimulation of glucose disposal were not different between the diets. Free fatty concentrations were suppressed by 89 and 62% (P < 0.0001) on the high-carbohydrate and high-fat diet, respectively. We conclude that short-term variations in dietary carbohydrate to fat ratios affect basal glucose metabolism in people with type 2 diabetes merely through modulation of the rate of glycogenolysis, without affecting insulin sensitivity of glucose metabolism.

Hepatic amino acid-dependent signaling is under the control of AMP-dependent protein kinase.

It has become increasingly clear in recent years that amino acids can stimulate a signal transduction pathway resulting in the phosphorylation of mammalian target of rapamycin downstream targets. We have now found that amino acid-dependent phosphorylation of p70S6 kinase and of S6 in hepatocytes is prevented when AMP-dependent protein kinase (AMPK) is activated by either the purine ribonucleoside analogue AICAriboside, fructose or glycerol. Insulin-dependent phosphorylation of protein kinase B is not affected by AMPK activation. Protein synthesis is strongly inhibited when AMPK is activated. It is concluded that amino acid-dependent signaling, a protein-anabolic signal, can be effectively antagonized by activation of AMPK.

Low-fat, high-carbohydrate and high-fat, low-carbohydrate diets decrease primary bile acid synthesis in humans.

BACKGROUND: Dietary fat content influences bile salt metabolism, but quantitative data from controlled studies in humans are scarce. OBJECTIVE: The objective of the study was to establish the effect of dietary fat content on the metabolism of primary bile salts. DESIGN: The effects of eucaloric extremely low-fat (0%), intermediate-fat (41%; control diet), and extremely high-fat (83%) diets on kinetic values of cholate and chenodeoxycholate metabolism were determined after 11 d by using stable isotope dilution in 6 healthy men. All diets contained identical amounts of cholesterol. RESULTS: The total primary bile salt pool size was not significantly affected by dietary fat content, although the chenodeoxycholate pool was significantly higher during the low-fat diet. Fractional turnover rates of both primary bile salts were 30-50% lower during the low- and high-fat diets than during the control diet. Total hepatic bile salt synthesis was approximately 30% lower during both the high- and low-fat diets, but synthesis rates of the 2 primary bile salts were differentially affected. The molar ratio of cholate to total bile salt synthesis increased from 0.50 +/- 0.05 ( +/- SD) to 0.59 +/- 0.05 and 0.66 +/- 0.04 with increasing fat intake, whereas the molar ratio of chenodeoxycholate to total bile salt synthesis decreased from 0.50 +/- 0.05 to 0.41 +/- 0.05 and 0.34 +/- 0.04. The relative concentration of deoxycholate in plasma increased during the low-fat period, which indicated increased absorption from the colon. CONCLUSIONS: Both low- and high-fat diets reduce the synthesis and turnover rates of primary bile salts in humans, although probably through different mechanisms, and consequently they affect the removal of cholesterol from the body.

Overexpression of arginase I in enterocytes of transgenic mice elicits a selective arginine deficiency and affects skin, muscle, and lymphoid development.

BACKGROUND: Arginine is required for the detoxification of ammonia and the synthesis of proteins, nitric oxide, agmatine, creatine, and polyamines, and it may promote lymphocyte function. In suckling mammals, arginine is synthesized in the enterocytes of the small intestine, but this capacity is lost after weaning. OBJECTIVE: We investigated the significance of intestinal arginine production for neonatal development in a murine model of chronic arginine deficiency. DESIGN: Two lines of transgenic mice that express different levels of arginase I in their enterocytes were analyzed. RESULTS: Both lines suffer from a selective but quantitatively different reduction in circulating arginine concentration. The degree of arginine deficiency correlated with the degree of retardation of hair and muscle growth and with the development of the lymphoid tissue, in particular Peyer's patches. Expression of arginase in all enterocytes was necessary to elicit this phenotype. Phenotypic abnormalities were reversed by daily injections of arginine but not of creatine. The expression level of the very arginine-rich skin protein trichohyalin was not affected in transgenic mice. Finally, nitric oxide synthase-deficient mice did not show any of the features of arginine deficiency. CONCLUSIONS: Enterocytes are important for maintaining arginine homeostasis in neonatal mice. Graded arginine deficiency causes graded impairment of skin, muscle, and lymphoid development. The effects of arginine deficiency are not mediated by impaired synthesis of creatine or by incomplete charging of arginyl-transfer RNA.

In vivo measurement of nitric oxide production in porcine gut, liver and muscle during hyperdynamic endotoxaemia.

1. During prolonged endotoxaemia, an increase in arginine catabolism may result in limiting substrate availability for nitric oxide (NO) production. These effects were quantitated in a chronically instrumented porcine endotoxaemia model. 2. Ten days prior to the beginning of the experiments, pigs were catheterized. On day 0, pigs received a continuous infusion of endotoxin (3 microg kg(-1) h(-1)) over 24 h and were saline resuscitated. Blood was drawn from the catheters at 0 and 24 h during primed-infusion of (15)N(2)-arginine and P-aminohippurate to assess (15)N(2)-arginine to (15)N-citrulline conversion and plasma flow rates, respectively, across the portal-drained viscera, liver and hindquarter. 3. During endotoxin infusion a hyperdynamic circulation with elevated heart rate, cardiac index and decreased mean arterial pressure was achieved, characteristic of the human septic condition. 4. Endotoxin induced NO production by the portal-drained viscera and the liver. The increased NO production was quantitatively matched by an increase in arginine disposal. Nitrite/nitrate levels remained unchanged during endotoxaemia. 5. Despite an increased arginine production from the hindquarter and an increased whole-body arginine appearance rate during endotoxin infusion, the plasma arginine concentration was lower in endotoxin-treated animals than in controls. 6 On a whole-body level, the muscle was found to serve as a major arginine supplier and, considering the lowered arginine plasma levels, seems critical in providing arginine as precursor for NO synthesis in the splanchnic region.

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.

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: 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.

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.