Timing of acute cold exposure determines UCP1 and FGF21 expression - Possible interactions between the thermal environment, thermoregulatory responses, and peripheral clocks.

Thermoregulation is synchronized across the circadian cycle to uphold thermal homeostasis. To test if time-of-day matters for the response to environmental cold exposure, mice were acclimated to thermoneutrality (27 °C) for 2 months were subjected acutely (8 h) to cold ambient conditions (15 °C), whereas controls were maintained at thermoneutral conditions. The thermal exposure was tested in separate groups (N = 8) at three distinct time-of-day periods: in the LIGHT phase (L); the DARK phase (D); and a mix of the two (D + L). The magnitude of UCP1 protein and mRNA induction in brown adipose tissue (BAT) in response to acute cold exposure was time-of-day sensitive, peaking in LIGHT, whereas lower induction levels were observed in D + L, and DARK. Plasma levels of FGF21 were induced 3-fold by acute cold exposure at LIGHT and D + L, compared to the time-matched thermoneutral controls, whereas cold in DARK did not cause a significant increase of FGF21 plasma levels. Cold exposure affected, in BAT, the temporal mRNA expression patterns of core circadian clock components: Bmal1, Clock, Per1, Per3, Cry1, Cry2 Nr1d1, and Nr1d2, but in the liver, none of the transcripts were modified. Behavioral assessment using the Thermal Gradient Test (TGT) showed that acute cold exposure reduced cold sensitivity in D + L, but not in DARK. RNA-seq analyses of somatosensory neurons in DRG highlighted the role of the core circadian components in these cells, as well as transcriptional changes due to acute cold exposure. This elucidates the sensory system as a gauge and potential regulator of thermoregulatory responses based on circadian physiology. In conclusion, acute cold exposure elicits time-of-day specific effects on thermoregulatory pathways, which may involve underlying changes in thermal perception. These results have implications for efforts aimed at reducing risks associated with the organization of shift work in cold environments.

Altered hepatic lipid droplet morphology and lipid metabolism in fasted Plin2-null mice.

Perilipin 2 (Plin2) binds to the surface of hepatic lipid droplets (LDs) with expression levels that correlate with triacylglyceride (TAG) content. We investigated if Plin2 is important for hepatic LD storage in fasted or high-fat diet-induced obese Plin2+/+ and Plin2-/- mice. Plin2-/- mice had comparable body weights, metabolic phenotype, glucose tolerance, and circulating TAG and total cholesterol levels compared with Plin2+/+ mice, regardless of the dietary regime. Both fasted and high-fat fed Plin2-/- mice stored reduced levels of hepatic TAG compared with Plin2+/+ mice. Fasted Plin2-/- mice stored fewer but larger hepatic LDs compared with Plin2+/+ mice. Detailed hepatic lipid analysis showed substantial reductions in accumulated TAG species in fasted Plin2-/- mice compared with Plin2+/+ mice, whereas cholesteryl esters and phosphatidylcholines were increased. RNA-Seq revealed minor differences in hepatic gene expression between fed Plin2+/+ and Plin2-/- mice, in contrast to marked differences in gene expression between fasted Plin2+/+ and Plin2-/- mice. Our findings demonstrate that Plin2 is required to regulate hepatic LD size and storage of neutral lipid species in the fasted state, while its role in obesity-induced steatosis is less clear.

Fatty acids abrogate the growth-suppressive effects induced by inhibition of cholesterol flux in pancreatic cancer cells.

BACKGROUND: Despite therapeutic advances, the prognosis of pancreatic ductal adenocarcinoma (PDAC) remains extremely poor. Metabolic reprogramming is increasingly recognized as a key contributor to tumor progression and therapy resistance in PDAC. One of the main metabolic changes essential for tumor growth is altered cholesterol flux. Targeting cholesterol flux appears an attractive therapeutic approach, however, the complex regulation of cholesterol balance in PDAC cells remains poorly understood. METHODS: The lipid content in human pancreatic duct epithelial (HPDE) cells and human PDAC cell lines (BxPC-3, MIA PaCa-2, and PANC-1) was determined. Cells exposed to eight different inhibitors targeting different regulators of lipid flux, in the presence or absence of oleic acid (OA) stimulation were assessed for changes in viability, proliferation, migration, and invasion. Intracellular content and distribution of cholesterol was assessed. Lastly, proteome profiling of PANC-1 exposed to the sterol O-acyltransferase 1 (SOAT1) inhibitor avasimibe, in presence or absence of OA, was performed. RESULTS: PDAC cells contain more free cholesterol but less cholesteryl esters and lipid droplets than HPDE cells. Exposure to different lipid flux inhibitors increased cell death and suppressed proliferation, with different efficiency in the tested PDAC cell lines. Avasimibe had the strongest ability to suppress proliferation across the three PDAC cell lines. All inhibitors showing cell suppressive effect disturbed intracellular cholesterol flux and increased cholesterol aggregation. OA improved overall cholesterol balance, reduced free cholesterol aggregation, and reversed cell death induced by the inhibitors. Treatment with avasimibe changed the cellular proteome substantially, mainly for proteins related to biosynthesis and metabolism of lipids and fatty acids, apoptosis, and cell adhesion. Most of these changes were restored by OA. CONCLUSIONS: The study reveals that disturbing the cholesterol flux by inhibiting the actions of its key regulators can yield growth suppressive effects on PDAC cells. The presence of fatty acids restores intracellular cholesterol balance and abrogates the alternations induced by cholesterol flux inhibitors. Taken together, targeting cholesterol flux might be an attractive strategy to develop new therapeutics against PDAC. However, the impact of fatty acids in the tumor microenvironment must be taken into consideration.

Mechanisms Behind NAFLD: a System Genetics Perspective.

PURPOSE OF REVIEW: To summarize the key factors contributing to the onset and progress of nonalcoholic fatty liver disease (NAFLD) and put them in a system genetics context. We particularly focus on how genetic regulation of hepatic lipids contributes to NAFLD. RECENT FINDINGS: NAFLD is characterized by excessive accumulation of fat in the liver. This can progress to steatohepatitis (inflammation and hepatocyte injury) and eventually, cirrhosis. The severity of NAFLD is determined by a combination of factors including obesity, insulin resistance, and lipotoxic lipids, along with genetic susceptibility. Numerous studies have been conducted on large human cohorts and mouse panels, to identify key determinants in the genome, transcriptome, proteome, lipidome, microbiome and different environmental conditions contributing to NAFLD. We review common factors contributing to NAFLD and put them in a systems genetics context. In particular, we describe how genetic regulation of liver lipids contributes to NAFLD. The combination of an unhealthy lifestyle and genetic predisposition increases the likelihood of accumulating lipotoxic specie lipids that may be one of the driving forces behind developing severe forms of NAFLD.

Obesity Is Associated with Distorted Proteoglycan Expression in Adipose Tissue.

Proteoglycans are central components of the extracellular matrix (ECM) and binding partners for inflammatory chemokines. Morphological differences in the ECM and increased inflammation are prominent features of the white adipose tissues in patients with obesity. The impact of obesity and weight loss on the expression of specific proteoglycans in adipose tissue is not well known. This study aimed to investigate the relationship between adiposity and proteoglycan expression. We analyzed transcriptomic data from two human bariatric surgery cohorts. In addition, RT-qPCR was performed on adipose tissues from female and male mice fed a high-fat diet. Both visceral and subcutaneous adipose tissue depots were analyzed. Adipose mRNA expression of specific proteoglycans, proteoglycan biosynthetic enzymes, proteoglycan partner molecules, and other ECM-related proteins were altered in both human cohorts. We consistently observed more profound alterations in gene expression of ECM targets in the visceral adipose tissues after surgery (among others VCAN (p = 0.000309), OGN (p = 0.000976), GPC4 (p = 0.00525), COL1A1 (p = 0.00221)). Further, gene analyses in mice revealed sex differences in these two tissue compartments in obese mice. We suggest that adipose tissue repair is still in progress long after surgery, which may reflect challenges in remodeling increased adipose tissues. This study can provide the basis for more mechanistic studies on the role of proteoglycans in adipose tissues in obesity.

Consumption of salmon fishmeal increases hepatic cholesterol content in obese C57BL/6 J mice.

PURPOSE: By-products from farmed fish contain large amounts of proteins and may be used for human consumption. The purpose of this study was to investigate cardiometabolic effects and metabolic tolerance in mice consuming fishmeal from salmon by-products, salmon filet or beef. METHODS: Female C57BL/6J mice were fed chow, as a healthy reference group, or a high-fat diet for 10 weeks to induce obesity and glucose intolerance. Obese mice were subsequently given isocaloric diets containing 50% of the dietary protein from salmon fishmeal, salmon filet or beef for 10 weeks. Mice were subjected to metabolic phenotyping, which included measurements of body composition, energy metabolism in metabolic cages and glucose tolerance. Lipid content and markers of hepatic toxicity were determined in plasma and liver. Hepatic gene and protein expression was determined with RNA sequencing and immunoblotting. RESULTS: Mice fed fishmeal, salmon filet or beef had similar food intake, energy consumption, body weight gain, adiposity, glucose tolerance and circulating levels of lipids and hepatic toxicity markers, such as p-ALT and p-AST. Fishmeal increased hepatic cholesterol levels by 35-36% as compared to salmon filet (p = 0.0001) and beef (p = 0.005). This was accompanied by repressed expression of genes involved in steroid and cholesterol metabolism and reduced levels of circulating Pcsk9. CONCLUSION: Salmon fishmeal was well tolerated, but increased hepatic cholesterol content. The high cholesterol content in fishmeal may be responsible for the effects on hepatic cholesterol metabolism. Before introducing fishmeal from salmon by-products as a dietary component, it may be advantageous to reduce the cholesterol content in fishmeal.

Broad histone H3K4me3 domains in mouse oocytes modulate maternal-to-zygotic transition.

Maternal-to-zygotic transition (MZT) is essential for the formation of a new individual, but is still poorly understood despite recent progress in analysis of gene expression and DNA methylation in early embryogenesis. Dynamic histone modifications may have important roles in MZT, but direct measurements of chromatin states have been hindered by technical difficulties in profiling histone modifications from small quantities of cells. Recent improvements allow for 500 cell-equivalents of chromatin per reaction, but require 10,000 cells for initial steps or require a highly specialized microfluidics device that is not readily available. We developed a micro-scale chromatin immunoprecipitation and sequencing (µChIP-seq) method, which we used to profile genome-wide histone H3 lysine methylation (H3K4me3) and acetylation (H3K27ac) in mouse immature and metaphase II oocytes and in 2-cell and 8-cell embryos. Notably, we show that ~22% of the oocyte genome is associated with broad H3K4me3 domains that are anti-correlated with DNA methylation. The H3K4me3 signal becomes confined to transcriptional-start-site regions in 2-cell embryos, concomitant with the onset of major zygotic genome activation. Active removal of broad H3K4me3 domains by the lysine demethylases KDM5A and KDM5B is required for normal zygotic genome activation and is essential for early embryo development. Our results provide insight into the onset of the developmental program in mouse embryos and demonstrate a role for broad H3K4me3 domains in MZT.

Plin2 deletion increases cholesteryl ester lipid droplet content and disturbs cholesterol balance in adrenal cortex.

Cholesteryl esters (CEs) are the water-insoluble transport and storage form of cholesterol. Steroidogenic cells primarily store CEs in cytoplasmic lipid droplet (LD) organelles, as contrasted to the majority of mammalian cell types that predominantly store triacylglycerol (TAG) in LDs. The LD-binding Plin2 binds to both CE- and TAG-rich LDs, and although Plin2 is known to regulate degradation of TAG-rich LDs, its role for regulation of CE-rich LDs is unclear. To investigate the role of Plin2 in the regulation of CE-rich LDs, we performed histological and molecular characterization of adrenal glands from Plin2+/+ and Plin2-/- mice. Adrenal glands of Plin2-/- mice had significantly enlarged organ size, increased size and numbers of CE-rich LDs in cortical cells, elevated cellular unesterified cholesterol levels, and increased expression of macrophage markers and genes facilitating reverse cholesterol transport. Despite altered LD storage, mobilization of adrenal LDs and secretion of corticosterone induced by adrenocorticotropic hormone stimulation or starvation were similar in Plin2+/+ and Plin2-/- mice. Plin2-/- adrenals accumulated ceroid-like structures rich in multilamellar bodies in the adrenal cortex-medulla boundary, which increased with age, particularly in females. Finally, Plin2-/- mice displayed unexpectedly high levels of phosphatidylglycerols, which directly paralleled the accumulation of these ceroid-like structures. Our findings demonstrate an important role of Plin2 for regulation of CE-rich LDs and cellular cholesterol balance in the adrenal cortex.

The Glutamine Transporter Slc38a1 Regulates GABAergic Neurotransmission and Synaptic Plasticity.

GABA signaling sustains fundamental brain functions, from nervous system development to the synchronization of population activity and synaptic plasticity. Despite these pivotal features, molecular determinants underscoring the rapid and cell-autonomous replenishment of the vesicular neurotransmitter GABA and its impact on synaptic plasticity remain elusive. Here, we show that genetic disruption of the glutamine transporter Slc38a1 in mice hampers GABA synthesis, modifies synaptic vesicle morphology in GABAergic presynapses and impairs critical period plasticity. We demonstrate that Slc38a1-mediated glutamine transport regulates vesicular GABA content, induces high-frequency membrane oscillations and shapes cortical processing and plasticity. Taken together, this work shows that Slc38a1 is not merely a transporter accumulating glutamine for metabolic purposes, but a key component regulating several neuronal functions.

Loss of perilipin 2 in cultured myotubes enhances lipolysis and redirects the metabolic energy balance from glucose oxidation towards fatty acid oxidation.

Lipid droplet (LD) coating proteins are essential for the formation and stability of intracellular LDs. Plin2 is an abundant LD coating protein in skeletal muscle, but its importance for muscle function is unclear. We show that myotubes established from Plin2-/- mice contain reduced content of LDs and accumulate less oleic acid (OA) in triacylglycerol (TAG) due to elevated LD hydrolysis in comparison with Plin2+/+ myotubes. The reduced ability to store TAG in LDs in Plin2-/- myotubes is accompanied by a shift in energy metabolism. Plin2-/- myotubes are characterized by increased oxidation of OA, lower glycogen synthesis, and reduced glucose oxidation in comparison with Plin2+/+ myotubes, perhaps reflecting competition between FAs and glucose as part of the Randle cycle. In accord with these metabolic changes, Plin2-/- myotubes have elevated expression of Ppara and Ppargc1a, transcription factors that stimulate expression of genes important for FA oxidation, whereas genes involved in glucose uptake and oxidation are suppressed. Loss of Plin2 had no impact on insulin-stimulated Akt phosphorylation. Our results suggest that Plin2 is essential for protecting the pool of skeletal muscle LDs to avoid an uncontrolled hydrolysis of stored TAG and to balance skeletal muscle energy metabolism.

Cysteine deprivation prevents induction of peroxisome proliferator-activated receptor gamma-2 and adipose differentiation of 3T3-L1 cells.

Plasma cysteine is strongly associated with body fat mass in human cohorts and diets low in cysteine prevents fat accumulation in mice. It is unclear if plasma cysteine affects fat development or if fat accumulation raises plasma cysteine. To determine if cysteine affects adipogenesis, we differentiated 3T3-L1 preadipocytes in medium with reduced cysteine. Cells incubated in media with 10-20µM cysteine exhibited reduced capacity to differentiate into triacylglycerol-storing mature adipocytes compared with cells incubated with 50µM cysteine. Low cysteine severely reduced expression of peroxisome proliferator-activated receptor gamma2 (Pparγ2) and its target genes perlipin1 (Plin1) and fatty acid binding protein-4 (Fabp4). Expression of stearoyl-CoA desaturase-1 (Scd1), known to be repressed with cysteine depletion, was also reduced with low cysteine. Medium depletion of the essential amino acids leucine, valine, and isoleucine had only a modest effect on adipocyte specific gene expression and differentiation. Stimulation with the PPARγ agonist BRL-49653 or addition of a hydrogen sulfide donor enhanced differentiation of 3T3-L1 cells cultured in low cysteine. This demonstrates that the ability to induce PPARγ expression is preserved when cells are cultured in low cysteine. It therefore appears that cysteine depletion inhibits adipogenesis by specifically affecting molecular pathways required for induction of PPARγ expression, rather than through a general reduction of global protein synthesis. In conclusion, we show that low extracellular cysteine reduces adipocyte differentiation by interfering with PPARγ2 and PPARγ target gene expression. Our results provide further evidence for the hypothesis that plasma cysteine is a casual determinant for body fat mass.

Perilipin family members preferentially sequester to either triacylglycerol-specific or cholesteryl-ester-specific intracellular lipid storage droplets.

Perilipin family proteins (Plins) coat the surface of intracellular neutral lipid storage droplets in various cell types. Studies across diverse species demonstrate that Plins regulate lipid storage metabolism through recruitment of lipases and other regulatory proteins to lipid droplet surfaces. Mammalian genomes have distinct Plin gene members and additional protein forms derived from specific mRNA splice variants. However, it is not known if the different Plins have distinct functional properties. Using biochemical, cellular imaging and flow cytometric analyses, we now show that within individual cells of various types, the different Plin proteins preferentially sequester to separate pools of lipid storage droplets. By examining ectopically expressed GFP fusions and all endogenous Plin protein forms, we demonstrate that different Plins sequester to different types of lipid droplets that are composed of either triacylcerides or cholesterol esters. Furthermore, Plins with strong association preferences to triacylceride (or cholesterol ester) droplets can re-direct the relative intracellular triacylceride-cholesterol ester balance toward the targeted lipid. Our data suggest diversity of Plin function, alter previous assumptions about shared collective actions of the Plins, and indicate that each Plin can have separate and unique functions.

Regulation of lipid droplets and cholesterol metabolism in adrenal cortical cells.

The adrenal gland is composed of two distinctly different endocrine moieties. The interior medulla consists of neuroendocrine chromaffin cells that secrete catecholamines like adrenaline and noradrenaline, while the exterior cortex consists of steroidogenic cortical cells that produce steroid hormones, such as mineralocorticoids (aldosterone), glucocorticoids (cortisone and cortisol) and androgens. Synthesis of steroid hormones in cortical cells requires substantial amounts of cholesterol, which is the common precursor for steroidogenesis. Cortical cells may acquire cholesterol from de novo synthesis and uptake from circulating low- and high-density lipoprotein particles (LDL and HDL). As cholesterol is part of the plasma membrane in all mammalian cells and an important regulator of membrane fluidity, cellular levels of free cholesterol are tightly regulated. To ensure a robust supply of cholesterol for steroidogenesis and to avoid cholesterol toxicity, cortical cells store large amounts of cholesterol as cholesteryl esters in intracellular lipid droplets. Cortical steroidogenesis relies on both mobilization of cholesterol from lipid droplets and constant uptake of circulating cholesterol to replenish lipid droplet stores. This chapter will describe mechanisms involved in cholesterol uptake, cholesteryl ester synthesis, lipid droplet formation, hydrolysis of stored cholesteryl esters, as well as their impact on steroidogenesis. Additionally, animal models and human diseases characterized by altered cortical cholesteryl ester storage, with or without abnormal steroidogenesis, will be discussed.

Distribution of lipid droplets in hippocampal neurons and microglia: impact of diabetes and exercise.

Neuroinflammation, aging, and neurodegenerative disorders are associated with excessive accumulation of neutral lipids in lipid droplets (LDs) in microglia. Type 2 diabetes mellitus (T2DM) may cause neuroinflammation and is a risk factor for neurodegenerative disorders. Here, we show that hippocampal pyramidal neurons contain smaller, more abundant LDs than their neighboring microglia. The density of LDs varied between pyramidal cells in adjacent subregions, with CA3 neurons containing more LDs than CA1 neurons. Within the CA3 region, a gradual increase in the LD content along the pyramidal layer from the hilus toward CA2 was observed. Interestingly, the high neuronal LD content correlated with less ramified microglial morphotypes. Using the db/db model of T2DM, we demonstrated that diabetes increased the number of LDs per microglial cell without affecting the neuronal LD density. High-intensity interval exercise induced smaller changes in the number of LDs in microglia but was not sufficient to counteract the diabetes-induced changes in LD accumulation. The changes observed in response to T2DM may contribute to the cerebral effects of T2DM and provide a mechanistic link between T2DM and neurodegenerative disorders.

Single-cell m6A mapping in vivo using picoMeRIP-seq.

Current N6-methyladenosine (m6A) mapping methods need large amounts of RNA or are limited to cultured cells. Through optimized sample recovery and signal-to-noise ratio, we developed picogram-scale m6A RNA immunoprecipitation and sequencing (picoMeRIP-seq) for studying m6A in vivo in single cells and scarce cell types using standard laboratory equipment. We benchmark m6A mapping on titrations of poly(A) RNA and embryonic stem cells and in single zebrafish zygotes, mouse oocytes and embryos.

Fatty acids regulate perilipin5 in muscle by activating PPARδ.

The surface of lipid droplets (LDs) in various cell types is coated with perilipin proteins encoded by the Plin genes. Perilipins regulate LD metabolism by selectively recruiting lipases and other proteins to LDs. We have studied the expression of perilipins in mouse muscle. The glycolytic fiber-enriched gastrocnemius muscle expresses predominantly Plin2-4. The oxidative fiber-enriched soleus muscle expresses Plin2-5. Expression of Plin2 and Plin4-5 is elevated in gastrocnemius and soleus muscles from mice fed a high-fat diet. This effect is preserved in peroxisome proliferator-activated receptor (PPAR)α-deficient mice. Mouse muscle derived C2C12 cells differentiated into glycolytic fibers increase transcription of these Plins when exposed to various long chain fatty acids (FAs). To understand how FAs regulate Plin genes, we used specific activators and antagonists against PPARs, Plin promoter reporter assays, chromatin immunoprecipitation, siRNA, and animal models. Our analyses demonstrate that FAs require PPARδ to induce transcription of Plin4 and Plin5. We further identify a functional PPAR binding site in the Plin5 gene and establish Plin5 as a novel direct PPARδ target in muscle. Our study reveals that muscle cells respond to elevated FAs by increasing transcription of several perilipin LD-coating proteins. This induction renders the muscle better equipped to sequester incoming FAs into cytosolic LDs.

The RNA m6A landscape of mouse oocytes and preimplantation embryos.

Despite the significance of N6-methyladenosine (m6A) in gene regulation, the requirement for large amounts of RNA has hindered m6A profiling in mammalian early embryos. Here we apply low-input methyl RNA immunoprecipitation and sequencing to map m6A in mouse oocytes and preimplantation embryos. We define the landscape of m6A during the maternal-to-zygotic transition, including stage-specifically expressed transcription factors essential for cell fate determination. Both the maternally inherited transcripts to be degraded post fertilization and the zygotically activated genes during zygotic genome activation are widely marked by m6A. In contrast to m6A-marked zygotic ally-activated genes, m6A-marked maternally inherited transcripts have a higher tendency to be targeted by microRNAs. Moreover, RNAs derived from retrotransposons, such as MTA that is maternally expressed and MERVL that is transcriptionally activated at the two-cell stage, are largely marked by m6A. Our results provide a foundation for future studies exploring the regulatory roles of m6A in mammalian early embryonic development.

Postprandial Effects of Salmon Fishmeal and Whey on Metabolic Markers in Serum and Gene Expression in Liver Cells.

Fish is considered an important part of a healthy diet, in part due to the content of long chain omega-3 fatty acids. However, both lean and fatty fish have beneficial health effects, suggesting that micronutrients and proteins may play a role. In a randomised, controlled, cross-over trial, five healthy male participants consumed 5.2 g of protein from either salmon fishmeal or whey. Blood samples were taken before and 30 and 60 min after intake. The concentration of glucose, lipids, hormones and metabolites, including 28 different amino acids and derivatives, were measured in serum or plasma. Cultured HepG2 cells were incubated with or without serum from the participants, and transcriptomic profiling was performed using RNA sequencing. The ingestion of both salmon fishmeal and whey reduced the glucose and triglyceride levels in serum. Protein intake, independent of the source, increased the concentration of 22 amino acids and derivatives in serum. Fishmeal increased the concentration of arginine, methionine, serine, glycine, cystathionine and 2-aminobutyric acid more than whey did. Incubation with postprandial serum resulted in large transcriptomic alterations in serum-fasted HepG2 cells, with the differential expression of >4500 protein coding genes. However, when comparing cells cultivated in fasting serum to postprandial serum after the ingestion of fishmeal and whey, we did not detect any differentially regulated genes, neither with respect to the protein source nor with respect to the time after the meal. The comparable nutrigenomic effects of fishmeal and whey do not change the relevance of fish by-products as an alternative food source.

Extracellular vesicles and microRNAs are altered in response to exercise, insulin sensitivity and overweight.

Extracellular vesicles induced by exercise have emerged as potential mediators of tissue crosstalk. Extracellular vesicles and their cargo miRNAs have been linked to dysglycemia and obesity in animal models, but their role in humans is unclear. AIM: The aim of the study was to characterize the miRNA content in plasma extracellular vesicle isolates after acute and long-term exercise and to study associations between extracellular vesicle miRNAs, mRNA expression in skeletal muscle and adipose tissue, and cardiometabolic risk factors. METHODS: Sedentary men with or without dysglycemia and overweight underwent an acute bicycle test and a 12-week exercise intervention with extensive metabolic phenotyping. Gene expression in m. vastus lateralis and subcutaneous adipose tissue was measured with RNA sequencing. Extracellular vesicles were purified from plasma with membrane affinity columns or size exclusion chromatography. RESULTS: Extracellular vesicle miRNA profiling revealed a transient increase in the number of miRNAs after acute exercise. We identified miRNAs, such as miR-652-3p, that were associated to insulin sensitivity and adiposity. By performing explorative association analyses, we identified two miRNAs, miR-32-5p and miR-339-3p, that were strongly correlated to an adipose tissue macrophage signature. CONCLUSION: Numerous miRNAs in plasma extracellular vesicle isolates were increased by exercise, and several miRNAs correlated to insulin sensitivity and adiposity. Our findings warrant future studies to characterize exercise-induced extracellular vesicles and cargo miRNA to clarify where exercise-induced extracellular vesicles originate from, and to determine whether they influence metabolic health or exercise adaptation.

Glycated Proteins, Glycine, Acetate, and Monounsaturated Fatty Acids May Act as New Biomarkers to Predict the Progression of Type 2 Diabetes: Secondary Analyses of a Randomized Controlled Trial.

Food protein or food-derived peptides may regulate blood glucose levels; however, studies have shown inconsistent results. The aim of the present study was to characterize subgroups of individuals with increased risk of type 2 diabetes (T2D) and to investigate the cardiometabolic effects of fish protein in the same subgroups. We first divided participants into high insuliniAUC and low insuliniAUC subjects based on their insulin incremental area under the curve (iAUC) levels after a 2 h oral glucose tolerance test (OGTT), and secondly based on whether they had received 5.2 g salmon fish protein or placebo for 8 weeks, in a previously conducted randomized controlled trial (RCT). We then profiled these groups by analyzing plasma metabolomics and peripheral blood mononuclear cell (PBMC) gene expression. Compared to the low insuliniAUC group, the high insuliniAUC group had higher plasma concentrations of monounsaturated fatty acids (MUFAs) and glycated proteins (GlycA) and lower concentrations of glycine and acetate. After intervention with fish protein compared to placebo, however, only acetate was significantly increased in the low insuliniAUC group. In conclusion, we identified metabolic biomarkers known to be associated with T2D; also, intervention with fish protein did not affect cardiometabolic risk markers in subgroups with increased risk of T2D.