抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

Autophagy is a process by which components of the cell are degraded to maintain essential activity and viability in response to nutrient limitation. Extensive genetic studies have shown that the yeast ATG1 kinase has an essential role in autophagy induction. Furthermore, autophagy is promoted by AMP activated protein kinase (AMPK), which is a key energy sensor and regulates cellular metabolism to maintain energy homeostasis. Conversely, autophagy is inhibited by the mammalian target of rapamycin (mTOR), a central cell-growth regulator that integrates growth factor and nutrient signals. Here we demonstrate a molecular mechanism for regulation of the mammalian autophagy-initiating kinase Ulk1, a homologue of yeast ATG1. Under glucose starvation, AMPK promotes autophagy by directly activating Ulk1 through phosphorylation of Ser 317 and Ser 777. Under nutrient sufficiency, high mTOR activity prevents Ulk1 activation by phosphorylating Ulk1 Ser 757 and disrupting the interaction between Ulk1 and AMPK. This coordinated phosphorylation is important for Ulk1 in autophagy induction. Our study has revealed a signalling mechanism for Ulk1 regulation and autophagy induction in response to nutrient signalling.

/pdf/ampk-and-mtor-regulate-autophagy-through-direct-14wqsz16ke.pdf

AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.

For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Metformin is a widely used drug for treatment of type 2 diabetes with no defined cellular mechanism of action. Its glucose-lowering effect results from decreased hepatic glucose production and increased glucose utilization. Metformin's beneficial effects on circulating lipids have been linked to reduced fatty liver. AMP-activated protein kinase (AMPK) is a major cellular regulator of lipid and glucose metabolism. Here we report that metformin activates AMPK in hepatocytes; as a result, acetyl-CoA carboxylase (ACC) activity is reduced, fatty acid oxidation is induced, and expression of lipogenic enzymes is suppressed. Activation of AMPK by metformin or an adenosine analogue suppresses expression of SREBP-1, a key lipogenic transcription factor. In metformin-treated rats, hepatic expression of SREBP-1 (and other lipogenic) mRNAs and protein is reduced; activity of the AMPK target, ACC, is also reduced. Using a novel AMPK inhibitor, we find that AMPK activation is required for metformin's inhibitory effect on glucose production by hepatocytes. In isolated rat skeletal muscles, metformin stimulates glucose uptake coincident with AMPK activation. Activation of AMPK provides a unified explanation for the pleiotropic beneficial effects of this drug; these results also suggest that alternative means of modulating AMPK should be useful for the treatment of metabolic disorders.

/pdf/role-of-amp-activated-protein-kinase-in-mechanism-of-3m7xecnts5.pdf

Role of AMP-activated protein kinase in mechanism of metformin action

The epidemic of type 2 diabetes and impaired glucose tolerance is one of the main causes of morbidity and mortality worldwide. In both disorders, tissues such as muscle, fat and liver become less responsive or resistant to insulin. This state is also linked to other common health problems, such as obesity, polycystic ovarian disease, hyperlipidaemia, hypertension and atherosclerosis. The pathophysiology of insulin resistance involves a complex network of signalling pathways, activated by the insulin receptor, which regulates intermediary metabolism and its organization in cells. But recent studies have shown that numerous other hormones and signalling events attenuate insulin action, and are important in type 2 diabetes.

/pdf/insulin-signalling-and-the-regulation-of-glucose-and-lipid-6bdhstccqq.pdf

Insulin signalling and the regulation of glucose and lipid metabolism

Exercise increases skeletal muscle glucose uptake, but the underlying mechanisms are only partially understood. The atypical protein kinase C (PKC) isoforms λ and ζ (PKC‐ λ / ζ ) have been shown to be necessary for insulin‐, AICAR‐, and metformin‐stimulated glucose uptake in skeletal muscle, but not for treadmill exercise‐stimulated muscle glucose uptake. To investigate if PKC‐ λ / ζ activity is required for contraction‐stimulated muscle glucose uptake, we used mice with tibialis anterior muscle‐specific overexpression of an empty vector (WT), wild‐type PKC‐ ζ (PKC‐ ζ WT), or an enzymatically inactive T410A‐PKC‐ ζ mutant (PKC‐ ζ T410A). We also studied skeletal muscle‐specific PKC‐ λ knockout (M λ KO) mice. Basal glucose uptake was similar between WT, PKC‐ ζ WT, and PKC‐ ζ T410A tibialis anterior muscles. In contrast, in situ contraction‐stimulated glucose uptake was increased in PKC‐ ζ T410A tibialis anterior muscles compared to WT or PKC‐ ζ WT tibialis anterior muscles. Furthermore, in vitro contraction‐stimulated glucose uptake was greater in soleus muscles of M λ KO mice than WT controls. Thus, loss of PKC‐ λ / ζ activity increases contraction‐stimulated muscle glucose uptake. These data clearly demonstrate that PKC‐ λ  ζ activity is not necessary for contraction‐stimulated glucose uptake.

/pdf/contraction-stimulates-muscle-glucose-uptake-independent-of-qflzmq29gc.pdf

Contraction stimulates muscle glucose uptake independent of atypical PKC

A Novel Role for Adipose Tissue in Exercise‐Induced Improvements in Glucose Homeostasis

Aging is a major risk factor for type 2 diabetes (T2D) and cellular senescence plays a critical role in β-cell dysfunction leading to T2D. Exercise is a central component in the treatment of T2D and has been shown to attenuate many of the biochemical changes that occur with aging including senescence, but nothing is known about the effects of exercise on β-cells and their aging process. To test the hypothesis that exercise decreases β-cell senescence we used two mouse models of insulin resistance: high-fat diet (HFD, 60% kcal from fat, 6 wks) and insulin receptor antagonist S961 (40nM/wk for 2 wks) . Exercise (treadmill, 1 h/day, 5 times/wk for 2 wks at 17 cm/s and 5% incline) prevented entrance of β-cells into senescence and reversed established senescence in both males and females across the lifespan (started at 1.5-9m or 14m old mice) as seen by a decrease in one or several of the following markers: βGal, p21Cip1 and p16Ink4a. This decrease in senescence was accompanied by improved glucose stimulated insulin secretion characterized by recovery of glucose responsiveness in islets from HFD and S961 exercised animals. Islets cultured with serum from trained animals showed decreased p21Cip1 mRNA, indicating a circulatory factor (s) was responsible for these effects. Exercise increased serum glucagon expression, identified as such factor. Incubation of human and mouse islets with glucagon (1nM) decreased p21Cip1 mRNA, and using AMPK agonist (1uM C99) and antagonist (5uM Compound C) , we found that AMPK mediates the glucagon-stimulated decrease in p21Cip1. Human cadaveric donor islets treated with serum obtained before and after 10-weeks of exercise-training in people with T2D demonstrated that training decreased senescence markers through AMPK activation.
 In conclusion, exercise has anti-aging effects on mouse and human pancreatic islets by decreasing senescence and improving function. This is a novel mechanism of the effects of exercise on β-cell aging with implications for the treatment of T2D.
 
 
 P.Carapeto: None. J.Kahng: None. A.T.Alves wagner: None. R.Middelbeek: Research Support; Novo Nordisk. M.F.Hirshman: Stock/Shareholder; Abbott, AbbVie Inc., Amgen Inc., Colgate-Palmolive, Eli Lilly and Company, Medtronic. L.J.Goodyear: None. C.Aguayo-mazzucato: Consultant; eGenesis.
 
 
 
 American Diabetes Association (1-17-PMF-009) ; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001; Short-Term Research Experience for Underrepresented Persons (1R25DK113652) ; NIDDK grant (K23-DK114550) ; NIH grant (R01DK099511) and the Joslin Diabetes Center DRC (P30 DK36836) .


199-OR: Exercise Decreases Mouse and Human Islet Senescence through Glucagon-Mediated AMPK Activation

Recent studies suggest that exercise-induced circulating factors are critical for the health benefits of exercise. In rodent models, we have identified NEGR1 as a cell adhesion molecule that is essential for remodeling white adipose tissue (WAT) innervation via sympathetic neurite outgrowth, and that NEGR1 is a secreted factor that may be involved inter-tissue communication. In humans, exercise training increases NEGR1 mRNA in subcutaneous WAT, but whether exercise regulates circulating NEGR1 in humans is not known. Here, we investigated the effects of a single bout of maximal exercise and exercise training on plasma NEGR1 in two different cohorts of obese subjects. Acute exercise (single bout until VO2MAX achieved) increased NEGR1 in plasma in all subjects (p<0.05; n=M3/F4). For the training study, obese subjects were randomized to one of three protocols: two different high intensity interval training (HIIT) protocols and one moderate continuous training (MCT) (Table), and blood samples were taken four days after the last training session. 1-HIIT and 4-HIIT similarly increased plasma NEGR1 concentrations, while there was no significant effect of MCT on NEGR1. In conclusion, we identify NEGR1 as a novel exercise-induced circulating factor in human subjects. We hypothesize that NEGR1 plays a role in tissue cross-talk mediating neuronal refinement in several tissues.
 
 
 
 P.Nigro: None. N.P.Carbone: None. C.Bueno junior: None. M.Vamvini: None. L.Simpson: None. G.A.Hansbury: None. M.F.Hirshman: Stock/Shareholder; Abbott, AbbVie Inc., Amgen Inc., Colgate-Palmolive, Eli Lilly and Company, Medtronic. R.J.W.Middelbeek: Research Support; Novo Nordisk. L.Goodyear: None.
 
 
 
 National Institutes of Health (R01DK099511, R01DK101043, K23DK114550, 5T32DK00726042, F32DK12643201); Joslin Diabetes Center (P30DK36836)


Michael F. Hirshman

Papers

Contraction stimulates muscle glucose uptake independent of atypical PKC

A Novel Role for Adipose Tissue in Exercise‐Induced Improvements in Glucose Homeostasis

199-OR: Exercise Decreases Mouse and Human Islet Senescence through Glucagon-Mediated AMPK Activation

596-P: Exercise Increases Plasma Neuronal Growth Regulator 1 (NEGR1) Concentrations in Human Subjects

Moderate-intensity endurance training improves late phase β-cell function in adults with type 2 diabetes