Exploring the physiological role of transthyretin in glucose metabolism in the liver

Transthyretin (TTR), a 55 kDa evolutionarily conserved protein, presents altered levels in several conditions, including malnutrition, inflammation, diabetes, and Alzheimer’s Disease. It has been shown that TTR is involved in several functions, such as insulin release from pancreatic ß-cells, recovery of blood glucose and glucagon levels of the islets of Langerhans, food intake, and body weight. Here, the role of TTR in hepatic glucose metabolism was explored by studying the levels of glucose in mice with different TTR genetic backgrounds, namely with two copies of the TTR gene, TTR+/+; with only one copy, TTR+/-; and without TTR, TTR-/-. Results showed that TTR haploinsufficiency (TTR+/-) leads to higher glucose in both plasma and in primary hepatocyte culture media and lower expression of the influx glucose transporters, GLUT1, GLUT3, and GLUT4. Further, we showed that TTR haploinsufficiency decreases pyruvate kinase M type (PKM) levels in mice livers, by qRT-PCR, but it does not affect the hepatic production of the studied metabolites, as determined by 1H NMR. Finally, we demonstrated that TTR increases mitochondrial density in HepG2 cells and that TTR insufficiency triggers a higher degree of oxidative phosphorylation in the liver. Altogether, these results indicate that TTR contributes to the homeostasis of glucose by regulating the levels of glucose transporters and PKM enzyme and by protecting against mitochondrial oxidative stress.This work was supported by Norte-01-0145-FEDER-000008-Porto Neurosciences and Neurologic Disease Research Initiative at I3S, and Pest-OE/SAU/UI0215/2014 at UMIB, supported by Norte Portugal Regional Operational Programme (NORTE2020), under the PORTUGAL 2020 Partnership Agreement, by COMPETE 2020—Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, through the European Regional Development Fund (FEDER), by Portuguese funds through FCT—Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274), and by a grant from Fundação Millennium bcp. Alemi M was a recipient of fellowship by Norte-01-0145-FEDER-000008. Oliveira  was a recipient of fellowship by Norte-01-0145-FEDER-000008. Cardoso I, Oliveira PF, and Alves MG work under the Investigator FCT Program, which is financed by national funds through the Foundation for Science and Technology and co-financed by the European Social Fund (ESF) through the Human Potential Operational Programme (HPOP), type 4.2—Promotion of Scientific Employment

Sperm, metabolic memory and echoes from Lamarck

There is a widespread misconception that only maternal variables affect in utero development.Epigenetic markers carried by the spermatozoon are transmitted to the zygote.Sperm-born epigenetic factors influence in utero development, for various generations.Acquired traits of metabolic disease can be inherited by the offspring via the male gamete.Health assessment of future fathers is essential to predict the offspring's health.</ul

A switch from high-fat to normal diet does not restore sperm quality but prevents metabolic syndrome

In recent decades, the prevalence of metabolic diseases has concomitantly increased with a decline on fertility rates and sperm quality. High-fat diets (HFD) are seldom considered part of the problem, but the molecular mechanisms underlying its effects on male fertility remain poorly understood. Herein we postulated that HFD alter sperm quality. We evaluated the effects of switching from a HFD to a normal diet in early adulthood on metabolic disease onset, testicular metabolism and sperm quality. Thirty-six male C57BL6/J mice were divided in: a control group fed with standard chow; a group fed with HFD for 200 days; and a group fed with HFD for 60 days and then with standard chow (HFDt). Biometric data and whole-body metabolism were assessed. Epididymal sperm was studied for concentration, motility, viability and morphology. 1H-NMR metabolomics approach was performed on testicular extracts to trace the metabolic changes. Diet switch reduced body weight and fat mass, preventing metabolic syndrome onset. However, sperm viability, motility and morphology were deteriorated by HFD consumption and not restored by diet switch. HFD induced irreversible changes in pyruvate and glutamate metabolism, ethanol degradation and ammonia recycling in testis. Furthermore, HFDt changed purine and cysteine metabolism, urea cycle, and glutathione content. Overall, HFD caused irreversible changes in testicular metabolism even after switching to normal diet. HFD feeding until early adulthood decreases sperm quality, which cannot be restored by diet switch or weight loss, even when development of metabolic syndrome is avoided

Metabolic diseases affect male reproduction and induce signatures in gametes that may compromise the offspring health

The most prevalent diseases worldwide are non-communicable such as obesity and type 2 diabetes. Noteworthy, the prevalence of obesity and type 2 diabetes is expected to steadily increase in the next decades, mostly fueled by bad feeding habits, stress, and sedentarism. The reproductive function of individuals is severely affected by abnormal metabolic environments, both at mechanical and biochemical levels. Along with mechanical dysfunctions, and decreased sperm quality (promoted both directly and indirectly by metabolic abnormalities), several studies have already reported the potentially harmful effects of metabolic disorders in the genetic and epigenetic cargo of spermatozoa, and the epigenetic inheritance of molecular signatures induced by metabolic profile (paternal diet, obesity, and diabetes). The inheritance of epigenetic factors towards the development of metabolic abnormalities means that more people in reproductive age can potentially suffer from these disorders and for longer periods. In its turn, these individuals can also transmit this (epi)genetic information to future generations, creating a vicious cycle. In this review, we collect the reported harmful effects related to acquired metabolic disorders and diet in sperm parameters and male reproductive potential. Besides, we will discuss the novel findings regarding paternal epigenetic inheritance, particularly the ones induced by paternal diet rich in fats, obesity, and type 2 diabetes. We analyze the data attained with in vitro and animal models as well as in long-term trans generational population studies. Although the findings on this topic are very recent, epigenetic inheritance of metabolic disease has a huge societal impact, which may be crucial to tackle the `fat epidemic' efficiently

Glycerol and testicular activity: the good, the bad and the ugly

Over the past decades, there have been several studies suggesting that semen quality is declining. Interestingly, these observations are paired with a significant increase in the number of individuals diagnosed with metabolic diseases, including obesity and diabetes mellitus. Hence, it is tempting to hypothesize that obesity and its associated comorbidities and risk factors (such as a hypercaloric diets) impair the homeostasis of the male reproductive health, with a possible direct effect on the testes. The blood and interstitial fluids of obese individuals usually have increased levels of glycerol, notably due to triglyceride and phospholipid catabolism and high fructose intake. Glycerol is metabolized via intermediary metabolism by a group of reactions centred at the glycerol-3-phosphate shuttle, which links the metabolic pathway of glucose, lipids and oxidative phosphorylation, illustrating its high relevance for biological systems. Glycerol enters and exits the cells by the action of specialized carriers, known as aquaglyceroporins, whose functional importance for male reproductive health has emerged in the last few years. Notably, glycerol has antispermatogenic properties. When present in high concentration in the testis, it causes blood-testis barrier disruption, impairing tubular fluid homeostasis. Nevertheless, glycerol metabolism in testicular cells remains a matter of debate. Herein we discuss previous and current research concerning the role of glycerol and its metabolism in testicular cells, and how it can influence testicular activity

H-Ferritin Is Essential for Macrophages' Capacity to Store or Detoxify Exogenously Added Iron

Macrophages are central cells both in the immune response and in iron homeostasis. Iron is both essential and potentially toxic. Therefore, iron acquisition, transport, storage, and release are tightly regulated, by several important proteins. Cytosolic ferritin is an iron storage protein composed of 24 subunits of either the L- or the H-type chains. H-ferritin differs from L-ferritin in the capacity to oxidize Fe2+ to Fe3+. In this work, we investigated the role played by H-ferritin in the macrophages' ability to respond to immune stimuli and to deal with exogenously added iron. We used mice with a conditional deletion of the H-ferritin gene in the myeloid lineage to obtain bone marrow-derived macrophages. These macrophages had normal viability and gene expression under basal culture conditions. However, when treated with interferon-gamma and lipopolysaccharide they had a lower activation of Nitric Oxide Synthase 2. Furthermore, H-ferritin-deficient macrophages had a higher sensitivity to iron-induced toxicity. This sensitivity was associated with a lower intracellular iron accumulation but a higher production of reactive oxygen species. These data indicate that H-ferritin modulates macrophage response to immune stimuli and that it plays an essential role in protection against iron-induced oxidative stress and cell death.Tis work was fnanced by FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE2020 - Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project PTDC/IMI-MIC/1683/2014 (POCI-01-0145-FEDER-016590). PFO and MGA acknowledge FCT for the Investigador FCT 2015. We thank the valuable collaboration of the following i3S Scientifc Platforms: Cell Culture and Genotyping Core Facility (CCGen), [Histology and Electron Microscopy Service (HEMS), and BioSciences Screening], member of the PPBI (PPBI-POCI-01-0145-FEDER-022122)], Animal Facility, and Flow Cytometry Unit (TraCy). We acknowledge Lukas Kuhn (Swiss Institute for Experimental Cancer Research, Lausanne, Switzerland) for kindly providing the frst breeding pairs of Fth1−/− mice. Te authors also acknowledge Marisa Castro, from Departamento de Biologia Molecular from ICBAS, Clara Bento, from i3S, and Edgar Pinto from LAQV – REQUIMTE for technical assistance at diferent stages of the project

Hyperoside Supplementation in Preservation Media Surpasses Vitamin C Protection Against Oxidative Stress-Induced Damages in Human Spermatozoa

Background/Aims: Oxidative Stress (OS) is reported as one of the main causes of male infertility. Infertile couples often resort to assisted reproductive technology (ART) to achieve parenthood. However, preparation for ART protocols increases the exposer of gametes to OS. Thus, it is crucial to find suitable preservation media that can counteract the OS-induced damages in spermatozoa. In this work, we tested and compared the efficiency of vitamin C (VC) and hyperoside (HYP) as potential antioxidant supplements for sperm preservation media. Methods: We evaluated the cytotoxicity of HYP (0, 5, 50, 100, and 500 µM) in spermatozoa. After incubation of sperm cells with VC (600 µM) and HYP (100 and 500 µM), in the presence and absence of H2O2 (300 µM), the following parameters were assessed: total sperm motility and vitality, OS biomarkers expression, total antioxidant capacity (TAC) of the media, percentage of DNA fragmentation, mitochondrial membrane potential (MMP), and metabolite quantification of the media by proton nuclear magnetic resonance (1H-NMR). Results: The supplementation with VC (600 µM) and HYP (100 and 500 µM) did not induce any deleterious effects to the physiology and metabolism of the spermatozoa, after 1-hour of treatment. In the presence of H2O2 (300 µM), both VC and HYP were able to prevent some of the deleterious effects of H2O2 in sperm, which were represented by an increase in sperm motility, a decrease in DNA fragmentation, and a decreasing trend in lipid peroxidation levels. However, these antioxidants were not able to prevent the decrease of MMP associated with H2O2 treatment, nor were able to prevent the conversion of pyruvate into acetate (a reaction promoted by H2O2). Conclusion: The supplementation of sperm preservation media with VC and HYP could be beneficial for the preservation of sperm physiology. From the antioxidant conditions tested, the supplementation of media with HYP (100 µM) demonstrated the best results regarding sperm preservation, evidencing the higher antioxidant capacity of HYP compared to VC. Nevertheless, none of the antioxidants used was able to prevent the metabolic alterations promoted by H2O2 in spermatozoa.This work was supported by Fundação para a Ciência e a Tecnologia - FCT to Sara C. Pereira (2021.05487.BD); David F. Carrageta (SFRH/BD/136779/2018); Marco G. Alves (IFCT2015 and PTDC/MEC-AND/28691/2017); LAQV-REQUIMTE (UIDB/50006/2020); UMIB (UIDB/00215/2020, and UIDP/00215/2020); ITR - Laboratory for Integrative and Translational Research in Population Health (LA/P/0064/2020). Pedro F. Oliveira was funded by national funds through FCT – Fundação para a Ciência e a Tecnologia, I.P., under the Scientific Employment Stimulus - Institutional Call - reference CEECINST/00026/2018

Diet during early life defines testicular lipid content and sperm quality in adulthood

Childhood obesity is a serious concern associated with ill health later in life. Emerging data suggest that obesity has long-term adverse effects upon male sexual and reproductive health but few studies addressed this issue. We hypothesized that exposure to high-fat diet during early life alters testicular lipid content and metabolism leading to permanent damage to sperm parameters. After weaning (day 21 after birth), 36 male mice were randomly divided into 3 groups and fed with different diet regimen for 200 days: CTRL-standard chow; HFD-high-fat diet (Carbohydrate: 35.7%, Protein: 20.5%, Fat: 36.0%); HFDt-high-fat diet for 60 days then replaced by standard chow. Biometric and metabolic data were monitored. Animals were then sacrificed, and tissues collected. Epididymal sperm parameters and endocrine parameters were evaluated. Testicular metabolites were extracted and characterized by 1H-NMR and GC-MS. Testicular mitochondrial and antioxidant activity were evaluated. Our results show that mice fed with high-fat diet, even if only until early adulthood, had lower sperm viability and motility, and higher incidence of head and tail defects. Although diet reversion with weight loss during adulthood prevents the progression of metabolic syndrome, testicular content in fatty acids is irreversibly affected. Excessive fat intake promoted an over-accumulation of pro-inflammatory n-6 polyunsaturated fatty acids in testis, which are strongly correlated with negative effects upon sperm quality. Therefore, the adoption of high-fat diets during early life correlates to irreversible changes in testicular lipid content and metabolism, which are related to permanent damage to sperm quality later in life

Lifestyle, metabolic disorders and male hypogonadism - A one-way ticket?

Hypogonadism is more frequent among men with common metabolic diseases, notably obesity and type 2 diabetes. Indeed, endocrine disruption caused by metabolic diseases can trigger the onset of hypogonadism, although the underlying molecular mechanisms are not entirely understood. Metabolic diseases are closely related to unhealthy lifestyle choices, such as dietary habits and sedentarism. Therefore, hypogonadism is part of a pathological triad gathering unhealthy lifestyle, metabolic disease and genetic background. Additionally, hypogonadism harbors the potential to aggravate underlying metabolic disorders, further sustaining the mechanisms leading to disease. To what extent does lifestyle intervention in men suffering from these metabolic disorders can prevent, improve or reverse hypogonadism, is still controversial. Moreover, recent evidence suggests that the metabolic status of the father is related to the risk of inter and transgenerational inheritance of hypogonadism. In this review, we will address the proposed mechanisms of disease, as well as currently available interventions for hypogonadism

Inheritable testicular metabolic memory of high-fat diet causes transgenerational sperm defects in mice

The consumption of energy-dense diets has contributed to an increase in the prevalence of obesity and its comorbidities worldwide. The adoption of unhealthy feeding habits often occurs at early age, prompting the early onset of metabolic disease with unknown consequences for reproductive function later in life. Recently, evidence has emerged regarding the intergenerational and transgenerational effects of high-fat diets (HFD) on sperm parameters and testicular metabolism. Hereby, we study the impact of high-fat feeding male mice (F-0) on the testicular metabolome and function of their sons (F-1) and grandsons (F-2). Testicular content of metabolites related to insulin resistance, cell membrane remodeling, nutritional support and antioxidative stress (leucine, acetate, glycine, glutamine, inosine) were altered in sons and grandsons of mice fed with HFD, comparing to descendants of chow-fed mice. Sperm counts were lower in the grandsons of mice fed with HFD, even if transient. Sperm quality was correlated to testicular metabolite content in all generations. Principal Component Analysis of sperm parameters and testicular metabolites revealed an HFD-related phenotype, especially in the diet-challenged generation and their grandsons. Ancestral HFD, even if transient, causes transgenerational "inherited metabolic memory" in the testicular tissue, characterized by changes in testicular metabolome and function