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. 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 vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased 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. 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. 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 autophagy assays, we hope to encourage technical innovation in the field.

Guidelines for the use and interpretatoin of assays for monitoring autophagy

After a general overview of lipid biochemistry, this chapter is structured into three main sections. The first one follows the journey of lipids from feed intake, to digestion and absorption, the metabolic fates of lipids, including catabolic and anabolic processes, and interactions with the microbiome. The second section expands on some specific lipid classes characterized by unique nutritional roles, such as sterols, phospholipids, and pigments. The final section covers a series of broader aspects of fish lipid nutrition in aquaculture, analyzing resource availability, challenges and opportunities, consumers' nutritional expectations, and the interactions between lipids in aquaculture feeds and the environment. 

The lipids

Kisspeptin has recently emerged as a key regulator of the mammalian reproductive axis. It is known that kisspeptin, acting centrally via the kisspeptin receptor, stimulates secretion of gonadotrophin releasing hormone (GnRH). Loss of kisspeptin signaling causes hypogonadotrophic hypogonadism in humans and other mammals. Kisspeptin interacts with other neuropeptides such as neurokinin B and dynorphin, to regulate GnRH pulse generation. In addition, a growing body of evidence suggests that kisspeptin signaling be regulated by nutritional status and stress. Kisspeptin may also represent a novel potential therapeutic target in the treatment of fertility disorders. Early human studies suggest that peripheral exogenous kisspeptin administration stimulates gonadotrophin release in healthy adults and in patients with certain forms of infertility. This review aims to concisely summarize what is known about kisspeptin as a regulator of reproductive function, and provide an update on recent advances within this field.

/pdf/comprehensive-review-on-kisspeptin-and-its-role-in-20jnntlo9o.pdf

Comprehensive Review on Kisspeptin and Its Role in Reproductive Disorders

The effect of dietary fats on cardiometabolic diseases, including cardiovascular diseases and type 2 diabetes mellitus, has generated tremendous interest. Many earlier investigations focused on total fat and conventional fat classes (such as saturated and unsaturated fats) and their influence on a limited number of risk factors. However, dietary fats comprise heterogeneous molecules with diverse structures, and growing research in the past two decades supports correspondingly complex health effects of individual dietary fats. Moreover, health effects of dietary fats might be modified by additional factors, such as accompanying nutrients and food-processing methods, emphasizing the importance of the food sources. Accordingly, the rapidly increasing scientific findings on dietary fats and cardiometabolic diseases have generated debate among scientists, caused confusion for the general public and present challenges for translation into dietary advice and policies. This Review summarizes the evidence on the effects of different dietary fats and their food sources on cell function and on risk factors and clinical events of cardiometabolic diseases. The aim is not to provide an exhaustive review but rather to focus on the most important evidence from randomized controlled trials and prospective cohort studies and to highlight current areas of controversy and the most relevant future research directions for understanding how to improve the prevention and management of cardiometabolic diseases through optimization of dietary fat intake.

Dietary fats and cardiometabolic disease: mechanisms and effects on risk factors and outcomes

[Ca2 +]i signalling is a key regulatory mechanism in sperm function. In mammalian sperm the Ca2 +-permeable plasma membrane ion channel CatSper is central to [Ca2 +]i signalling, but there is good evidence that Ca2 + stored in intracellular organelles is also functionally important. Here we briefly review the current understanding of the diversity of Ca2 + stores and the mechanisms for the regulation of their activity. We then consider the evidence for the involvement of these stores in [Ca2 +]i signalling in mammalian (primarily human) sperm, the agonists that may activate these stores and their role in control of sperm function. Finally we consider the evidence that membrane Ca2 + channels and stored Ca2 + may play discrete roles in the regulation of sperm activities and propose a mechanism by which these different components of the sperm Ca2 +-signalling apparatus may interact to generate complex and spatially diverse [Ca2 +]i signals.

/pdf/regulation-and-roles-of-ca2-stores-in-human-sperm-19wptwmnws.pdf

Regulation and roles of Ca2+ stores in human sperm

Kisspeptin acts as an upstream regulator of the hypothalamus-pituitary-gonad axis, which is one of the main regulatory systems for mammalian reproduction. Kiss1 and its receptor Kiss1r (also known as G protein-coupled receptor 54 (Gpr54)) are expressed in various organs, but their functions are not well understood. The purpose of this study was to investigate the expression profiles and functions of kisspeptin and KISS1R in the reproductive tissues of imprinting control region mice. To identify the expression pattern and location of kisspeptin and KISS1R in gonads, testes and ovarian tissues were examined by immunohistochemical or immunofluorescent staining. Kisspeptin and KISS1R were expressed primarily in Leydig cells and seminiferous tubules respectively. KISS1R was specifically localized in the acrosomal region of spermatids and mature spermatozoa. Kisspeptin, but not KISS1R, was expressed in the cumulus-oocyte complex and oviductal epithelium of ovarian and oviductal tissues. The sperm intracellular calcium concentrations significantly increased in response to treatment with kisspeptin 10 in Fluo-4-loaded sperm. The IVF rates decreased after treatment of sperm with the kisspeptin antagonist peptide 234. These results suggest that kisspeptin and KISS1R might be involved in the fertilization process in the female reproductive tract. In summary, this study indicates that kisspeptin and KISS1R are expressed in female and male gametes, respectively, and in mouse reproductive tissues. These data strongly suggest that the kisspeptin system could regulate mammalian fertilization and reproduction.

https://rep.bioscientifica.com/downloadpdf/journals/rep/147/6/835.pdf

Kisspeptin modulates fertilization capacity of mouse spermatozoa.

Hypoxia regulates cell proliferation and steroidogenesis through protein kinase A signaling in bovine corpus luteum

Previous studies have demonstrated that saturated fatty acids (SFAs) are more lipotoxic than unsaturated fatty acids (UFAs) in inhibiting hepatic autophagy and promoting non-alcoholic steatohepatitis (NASH). However, there have been few studies have investigated the effects of carbon chain length on SFA-induced autophagy impairment and lipotoxicity. To investigate whether SFAs with shorter carbon chain lengths have differential effects on hepatic autophagy and NASH development, we partially replaced lard with coconut oil to elevate the ratio of medium-chain fatty acids (MCFAs) to long-chain fatty acids (LCFAs) in a mouse high-fat diet (HFD) and fed mice for 16 weeks. In addition, we treated HepG2 cells with different combinations of fatty acids to study the mechanisms of MCFAs-mediated hepatic protections. Our results showed that increasing dietary MCFA/LCFA ratio mitigated HFD-induced Type 2 diabetes and NASH in mice. Importantly, we demonstrated that increased MCFA ratio exerted its protective effects by restoring Rubicon-suppressed autophagy. Our study suggests that the relative amount of LCFAs and MCFAs in the diet, in addition to the amount of SFAs, can significantly contribute to autophagy impairment and hepatic lipotoxicity. Collectively, we propose that increasing dietary MCFAs could be an alternative therapeutic and prevention strategy for Type 2 diabetes and NASH.

/pdf/increasing-dietary-medium-chain-fatty-acid-ratio-mitigates-vh0ca80atr.pdf

Increasing Dietary Medium-Chain Fatty Acid Ratio Mitigates High-fat Diet-Induced Non-Alcoholic Steatohepatitis by Regulating Autophagy.

Background Kisspeptin, encoded by the Kiss1 gene, has many forms including kisspeptin54, kisspeptin14, kisspeptin13, and kisspeptin10, and all these peptides have the same affinity to their receptor KISS1R encoded by the Kiss1r gene. The KISS1–KISS1R system was discovered in neurons, and many reports stress on their function in the brain. However, recent studies have shown that Kiss1 and Kiss1r are expressed in the testes. The goal of this study was to demonstrate the roles of Kiss1 and Kiss1r in testicular function, especially their steroidogenic activity. Methods Kisspeptin10 and the kisspeptin10 antagonist peptide234 were used to determine their effect on testosterone production. Moreover, expression of steroidogenic genes in mouse testes and their gonadosomatic index (weight of the testes divided by the total body weight) and also serum testosterone level were studied between the ages of 2 weeks and 15 weeks. Results Kisspeptin10 and peptide234 did not affect testosterone production in primary Leydig cells from adult mice. Kiss1 and Esr1 expression also increased during puberty. The peak gonadosomatic index occurred at 4 weeks of age, and serum testosterone levels plateaued after the age of 4 weeks. Conclusion Our results suggest that kisspeptin10 does not affect steroidogenesis in adult Leydig cells, but its pattern of expression follows the stages of testicular development. Future studies should determine if kisspeptin regulates testicular development during puberty.

Kisspeptin expression in mouse Leydig cells correlates with age.

Valproic acid (VPA) is one of the most widely used antiepilepsy drugs. However, several side effects, including weight gain and fatty liver, have been reported in patients following VPA treatment. In this study, we explored the molecular mechanisms of VPA-induced hepatic steatosis using FL83B cell line-based in vitro model. Using fluorescent lipid staining technique, we found that VPA enhanced oleic acid- (OLA-) induced lipid accumulation in a dose-dependent manner in hepatocytes; this may be due to upregulated lipid uptake, triacylglycerol (TAG) synthesis, and lipid droplet formation. Real-time PCR results showed that, following VPA treatment, the expression levels of genes encoding cluster of differentiation 36 (Cd36), low-density lipoprotein receptor-related protein 1 (Lrp1), diacylglycerol acyltransferase 2 (Dgat2), and perilipin 2 (Plin2) were increased, that of carnitine palmitoyltransferase I a (Cpt1a) was not affected, and those of acetyl-Co A carboxylase α (Acca) and fatty acid synthase (Fasn) were decreased. Furthermore, using immunofluorescence staining and flow cytometry analyses, we found that VPA also induced peroxisome proliferator-activated receptor γ (PPARγ) nuclear translocation and increased levels of cell-surface CD36. Based on these results, we propose that VPA may enhance OLA-induced hepatocyte steatosis through the upregulation of PPARγ- and CD36-dependent lipid uptake, TAG synthesis, and lipid droplet formation.

/pdf/study-of-valproic-acid-enhanced-hepatocyte-steatosis-19m5625unv.pdf

Meng-Chieh Hsu

Papers

Kisspeptin modulates fertilization capacity of mouse spermatozoa.

Hypoxia regulates cell proliferation and steroidogenesis through protein kinase A signaling in bovine corpus luteum

Increasing Dietary Medium-Chain Fatty Acid Ratio Mitigates High-fat Diet-Induced Non-Alcoholic Steatohepatitis by Regulating Autophagy.

Kisspeptin expression in mouse Leydig cells correlates with age.

Study of Valproic Acid-Enhanced Hepatocyte Steatosis.