Brain Responses to High-Protein Diets

Protein, amino acids, vagus nerve signaling, and the brain

The protein content of the diet has long been investigated for its influence on food behavior. High-protein diets promote satiety and reduce calorie intake, whereas results for low-protein diets are more contradictory and less established. Protein sensing might take place in the oral cavity or in the post-oral gastrointestinal tract, where specific receptors have been found. Protein signaling to the brain may act through the vagal nerve and involve gastric hormones, such as cholecystokinin and peptide YY. Other pathways are post-absorptive signaling and the direct influence of brain levels of amino acids. High-protein diet enhances the activity of brain satiety centers, mainly the nucleus of the solitary tract and arcuate nucleus, although the activity of brain reward centers might also be modified. A better understanding of the role of both homeostatic and hedonic systems is needed to fully describe the influence of protein on food intake.

The mammalian target of rapamycin (mTOR) is a central controller of cell growth and proliferation. mTOR forms two distinct complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 is regulated by multiple signals such as growth factors, amino acids, and cellular energy and regulates numerous essential cellular processes including translation, transcription, and autophagy. The AMP-activated protein kinase (AMPK) is a cellular energy sensor and signal transducer that is regulated by a wide array of metabolic stresses. These two pathways serve as a signaling nexus for regulating cellular metabolism, energy homeostasis, and cell growth, and dysregulation of each pathway may contribute to the development of metabolic disorders such as obesity, type 2 diabetes, and cancer. This review focuses on our current understanding of the relationship between AMPK and mTORC1 signaling and discusses their roles in cellular and organismal energy homeostasis.

Recent advances have demonstrated that the adipose tissue plays a central role in regulating overall energy balance. Obesity results from a chronic deregulation of energy balance, with energy intake exceeding energy expenditure. Recently, new mechanisms that control the obesity phenotype such as the equilibrium between white and brown adipose tissue function has been identified. In this context, it is becoming increasingly clear that in addition to cellular growth, AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) also regulate lipid metabolism and adipogenesis. Here, we review recent advances in the understanding of the molecular mechanisms involved in white and brown differentiation programs focusing on AMPK and mTOR signaling pathways, which may play differential roles in white adipose tissue and brown adipose tissue development. In view of the worldwide epidemic of obesity and its associated metabolic disorders such as insulin resistance and type 2 diabetes, targeting these kinases may represent a potential approach for reducing adiposity and improving obesity-related diseases. © 2013 IUBMB Life, 65(7):572-583, 2013.

The goal of the study is to examine the relationship between the sensor molecules, Hypoxia Inducible Factor-1 (HIF-1), AMP activated Protein Kinase (AMPK) and mammalian Target of Rapamycin (mTOR) in chondrocyte survival and autophagy. We showed that chondrocytes expressed the energy sensor AMPK-1 and that activation increased with maturation. In addition, we showed that thapsigargin treatment activated AMPK and autophagy in a HIF-1-dependent manner. Using serum-starved AMPK-silenced cells, we demonstrated that AMPK was required for the induction of the autophagic response. We also noted a change in chondrocyte sensitivity to apoptogens, due to activation of caspase-8 and cleavage and activation of the pro-apoptotic protein, BID. To test the hypothesis that AMPK signaling directly promoted autophagy, we inhibited AMPK activity in mTOR silenced cells and showed that while mTOR suppression induced autophagy, AMPK inhibition did not block this activity. Based on these findings, it is concluded that because of the micro-environmental changes experienced by the chondrocyte, autophagy is activated by AMPK in a HIF-1-dependent manner.

Weight loss of ≥10% improves glucose control and may remit type 2 diabetes (T2D). High-protein (HP) diets are commonly used for weight loss, but whether protein sources, especially red meat, impact weight loss-induced T2D management is unknown. This trial compared an HP diet including beef and a normal-protein (NP) diet without red meat for weight loss, body composition changes, and glucose control in individuals with T2D. A total of 106 adults (80 female) with T2D consumed an HP (40% protein) diet with ≥4 weekly servings of lean beef or an NP (21% protein) diet excluding red meat during a 52-week weight loss intervention. Body weight, body composition, and cardiometabolic parameters were measured before and after intervention. Weight loss was not different between the HP (-10.2 ± 1.6 kg) and NP (-12.7 ± 4.8 kg, p = 0.336) groups. Both groups reduced fat mass and increased fat-free mass percent. Hemoglobin A1c, glucose, insulin, insulin resistance, blood pressure, and triglycerides improved, with no differences between groups. The lack of observed effects of dietary protein and red meat consumption on weight loss and improved cardiometabolic health suggests that achieved weight loss, rather than diet composition, should be the principal target of dietary interventions for T2D management.

Resistin has been suggested to be involved in the development of diabetes and insulin resistance. We recently reported that resistin is expressed in diabetic hearts and promotes cardiac hypertrophy; however, the mechanisms underlying this process are currently unknown. Therefore, we wanted to elucidate the mechanisms associated with resistin-induced cardiac hypertrophy and myocardial insulin resistance. Overexpression of resistin using adenoviral vector in neonatal rat ventricular myocytes was associated with inhibition of AMP-activated protein kinase (AMPK) activity, activation of tuberous sclerosis complex 2/mammalian target of rapamycin (mTOR) pathway, and increased cell size, [(3)H]leucine incorporation (i.e. protein synthesis) and mRNA expression of the hypertrophic marker genes, atrial natriuretic factor, brain natriuretic peptide, and β-myosin heavy chain. Activation of AMPK with 5-aminoimidazole-4-carbozamide-1-β-D-ribifuranoside or inhibition of mTOR with rapamycin or mTOR siRNA attenuated these resistin-induced changes. Furthermore, resistin increased serine phosphorylation of insulin receptor substrate (IRS1) through the activation of the apoptosis signal-regulating kinase 1/c-Jun N-terminal Kinase (JNK) pathway, a module known to stimulate insulin resistance. Inhibition of JNK (with JNK inhibitor SP600125 or using dominant-negative JNK) reduced serine 307 phosphorylation of IRS1. Resistin also stimulated the activation of p70(S6K), a downstream kinase target of mTOR, and increased phosphorylation of the IRS1 serine 636/639 residues, whereas treatment with rapamycin reduced the phosphorylation of these residues. Interestingly, these in vitro signaling pathways were also operative in vivo in ventricular tissues from adult rat hearts overexpressing resistin. These data demonstrate that resistin induces cardiac hypertrophy and myocardial insulin resistance, possibly via the AMPK/mTOR/p70(S6K) and apoptosis signal-regulating kinase 1/JNK/IRS1 pathways.

Sulfated cholecystokinin-8 activates phospho-mTOR immunoreactive neurons of the paraventricular nucleus in rats

Niida et al provide the first conclusive evidence that the DNA damage checkpoint kinases Chk1 and Chk2 are bona fide tumour suppressors, also giving insight into the unique and redundant functions of these two factors in vivo.

Effects of AMP-activated protein kinase (AMPK) signaling and essential amino acids on mammalian target of rapamycin (mTOR) signaling and protein synthesis rates in mammary cells

To investigate the influences of adiponectin (APN) on the liver injury in sepsis rats and to explore whether it exerts a therapeutic effect through the adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway. A rat model of sepsis was established through cecal ligation and puncture (CLP) (CLP group), and APN treatment group (APN group) and control group were also set. The changes in the liver function-related indicators, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), were determined by automatic biochemistry analyzer, and the levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1, and IL-6 were measured via enzyme-linked immunosorbent assay (ELISA). Hematoxylin-eosin (HE) staining was employed to detect liver tissue injury, and the hepatocyte apoptosis and necrosis after intervention with APN were evaluated using in situ fluorescence staining. Moreover, the mRNA expression of APN in liver tissues was detected via quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR), and the expression levels of phosphorylated AMPK and mTOR proteins in liver tissue samples were determined using Western blotting. In terms of changes in liver function-related indicators, the concentrations of ALT and AST were substantially raised in the CLP group, and compared with those in the control group, the concentrations of the two indicators significantly declined in the APN group, showing statistically significant differences (p<0.05). CLP and APN group had evidently higher levels of inflammatory factors than the control group, but their levels in APN group were notably lower than those in the CLP group (p<0.05). It was found through the HE staining that the sepsis rats in CLP group had massive inflammatory cell infiltration, and that the inflammatory cells were remarkably decreased in the APN group after APN treatment. According to the in-situ fluorescence staining detection results, CLP group exhibited a notable increase in the cell apoptosis rate, and APN group had substantially reduced apoptotic cells (p<0.05). The determination results of APN expression revealed that CLP group had a lowered level of APN, and that the level of APN in APN group was markedly higher than that in the control group. Based on the results of Western blotting, the level of phosphorylated AMPK was remarkably elevated, and that of phosphorylated mTOR was lowered in the CLP group compared with those in the control group, while in comparison with CLP group, APN group showed a considerable elevation of phosphorylated AMPK level and a distinct decline in the phosphorylated mTOR level. APN can activate the AMPK/mTOR pathway and reduce hepatocyte apoptosis to alleviate liver injury in sepsis rats.

Hypoxic pulmonary hypertension (HPH) is characterized by sustained elevation of pulmonary artery pressure produced by vasoconstriction and hyperproliferative remodeling of the pulmonary artery and subsequent right ventricular hypertrophy (RVH). The search for therapeutic targets for cardiovascular pathophysiology has extended in many directions. However, studies focused on mitigating high-altitude pulmonary hypertension (HAPH) have been rare. Because AMP-activated protein kinase (AMPK) is involved in cardiovascular and metabolic pathology, AMPK is often studied as a potential therapeutic target. AMPK is best characterized as a sensor of cellular energy that can also restore cellular metabolic homeostasis. However, AMPK has been implicated in other pathways with vasculoprotective effects. Notably, cellular metabolic stress increases the intracellular ADP/ATP or AMP/ATP ratio, and AMPK activation restores ATP levels by activating energy-producing catabolic pathways and inhibiting energy-consuming anabolic pathways, such as cell growth and proliferation pathways, promoting cardiovascular protection. Thus, AMPK activation plays an important role in antiproliferative, antihypertrophic and antioxidant pathways in the pulmonary artery in HPH. However, AMPK plays contradictory roles in promoting HPH development. This review describes the main findings related to AMPK participation in HPH and its potential as a therapeutic target. It also extrapolates known AMPK functions to discuss the less-studied HAPH context.

Episodes of insulin-induced hypoglycemia are frequent in type 1 and advanced type 2 diabetes, but pharmaceutical approaches to prevent these are lacking. Cellular stresses such as low blood sugar activate AMPK, which has emerged as a whole body and cellular energy sensor. Direct delivery of AMPK activator to the ventromedial hypothalamus of rodents leads to increased hepatic glucose-production, observed during hyperinsulinemic-hypoglycemic clamp studies, and genetic activation of AMPK in the pancreatic alpha cell increases glucagon release. It may therefore be suitable to target AMPK for hypoglycemia prevention. Here, R481, a novel metformin-like brain permeable AMPK activator, was used to assess the impact of AMPK pathway activation on the counterregulatory response to hypoglycemia. Hypothalamic glucose sensing GT1-7 cells were treated with R481 and activation of AMPK pathway by phosphorylation assessed using Western Blotting. R481 was administered orally to male Sprague Dawley rats prior to insulin-induced hypoglycemia, following which blood glucose and feeding were measured. A separate cohort of rats underwent a hyperinsulinemic-hypoglycemic clamp study where glucose infusion rates and counterregulatory hormones levels were determined. Nanomolar concentrations of R481 increased AMPK pathway phosphorylation in GT1-7 neurons. Administration of R481 (5-20 mg/kg) to rats attenuated insulin-mediated drop in blood glucose during acute insulin-induced hypoglycemia, without altering fast-induced refeeding. R481 decreased the glucose infusion rate during hyperinsulinemic-hypoglycemic clamps, by amplifying plasma glucagon secretion, without altering epinephrine. Peripheral administration of AMPK activator R481 amplified glucagon release to improve counterregulatory response to hypoglycemia in healthy rats. Disclosure A.M.L. Cruz: None. Y. Malekizadeh: None. J.M. Vlachaki Walker: None. S.J. Shaw: Employee; Self; Rigel Pharmaceuticals. K.L. Ellacott: None. C. Beall: None. Funding JDRF; Diabetes UK

Sensing of energy and nutrients by AMP-activated protein kinase

Nitric oxide (NO) is a key regulator of cardiomyogenesis of embryonic stem (ES) cells. However, signaling pathways involving the energy sensor AMP-activated protein kinase (AMPK) and/or mammalian target of rapamycin (mTOR) resulting in NO generation and stimulation of cardiomyogenesis are currently not known. Herein, the role of AMPK- versus mTOR-regulated signaling pathways and the impact of NO for cardiomyogenesis of mouse ES cells were investigated. Activation of AMPK by 5-amino-4-imidazolecarboxamide riboside (AICAr) or metformin as well as inactivation of AMPK by compound C (Comp C), siRNA ablation of AMPKα2, or exogenous ATP stimulated cardiomyogenesis of ES cells. Inhibition of AMPK by Comp C resulted in phosphorylation of mTOR and generation of NO. NO generation was likewise achieved when AMPK was either activated by AICAr or mTOR was inhibited by rapamycin, suggesting that NO generation occurred by two mutually active parallel signaling pathways, one being AMPK dependent and mTOR independent (AICAr pathway) and the other being AMPK independent and mTOR dependent (Comp C pathway). Consequently, cardiomyogenesis as well as NO generation was completely abrogated when ES cells were cultivated in the presence of rapamycin and Comp C, which inhibit both signaling pathways. The impact of NO for cardiomyogenesis of ES cells was corroborated in experiments showing that the effects of Comp C on cardiomyogenesis of ES cells were abolished by the NO synthase inhibitors NG-monomethyl-l-arginine and N (G)-nitro-l-arginine methyl ester. In summary, our data demonstrate that NO generation downstream of AMPK and mTOR is activated by distinct, interacting signaling pathways that initiate cardiomyogenesis of ES cells.