NAFLD and therapeutic interventions in gut microbiota
In view of a potentially beneficial role in NAFLD, therapeutic interventions in gut microbiota have attracted great research interest. As mentioned above, gut microbiota may influence energy harvest and affect satiety. Given these roles, randomized studies performed both in adults and children have investigated the role of probiotics and have shown promising results. Probiotics are live microorganisms that provide health benefit to the host when administered in adequate amounts by influencing the intestinal microbial ecology (56). A prebiotic is a nonviable food component that confers a health benefit on the host associated with modulation of the microbiota, (i.e. a fiber). The synergistic combination of prebiotics and probiotics is described as synbiotic (56). Modulations of the gut microbiota with the use of probiotics and/or symbiotics can result in adaptations in regulating guthormones and thereby reduce energy harvest, enhance the feeling of satiety, improve glucosemetabolism and also improve gut barrier function and thereby ameliorate endotoxaemia and inflammation that are often found in obesity and type 2 diabetes (11).
GLUT4 is the primary hormonally-responsive transporter. GLUT4 is found primarily in muscle and adipose tissue, where it is normally sequestered in intracellular vesicles; it is translocated to plasma membrane in response to insulin, resulting in enhanced glucose uptake. In contrast, cortisol decreases the amount of GLUT4 in the plasma membrane. Prolonged high levels of the hormones that affect GLUT4 localization result in effects on GLUT4 gene transcription in the opposite direction of the effects on activity; thus GLUT4 gene transcription is increased by high levels of glucocorticoids and inhibited by high levels of insulin. However, GLUT4 expression is also reduced by low insulin states, such as in muscle during fasting, and in insulin-resistant adipose tissue. The remaining GLUT gene products are much less important for glucose transport. GLUT5 is found in gut, liver and spermatozoa, and is thought to function primarily as a fructose transporter. GLUT6 is thought to be a non-functional pseudogene. GLUT7 is an intracellular liver protein responsible for glucose-6-phosphate transport into the endoplasmic reticulum.
anterior end of the gut. The gut was then weighed, slit longitudinally, spread on an ice-cooled plate and rinsed twice with Hanks’ medium. The mucosa was gently scraped using the edge of a glass slide, and the mucosa thus obtained was transferred immediately to a Petri dish containing modified Hanks’ medium plus 1 mmol l −1 EGTA and 0.05 mg ml −1 collagenase (from Clostridium histolyticum, type IV, Sigma). This suspension was gently aspirated with a glass Pasteur pipette for 15 min to disperse the cells into the medium. The suspension was then filtered through 250 and 72 µm filters. The filtered cell suspension was centrifuged at 80 g for 3 min at 4 °C (Sorvall RC 5B Plus, SS-34 rotor). The cells were washed four times with 10 vols of modified Hanks’ medium plus 1 % defatted bovine serum albumin, pH 7.6 (Hanks’ complete). The final pellet was resuspended in Hanks’ complete to a concentration of 17.5 mg ml −1 . Cell viability was assessed by Trypan Blue exclusion (Mommsen et al. 1994), and only preparations that yielded cell viabilities greater than 90 % were used. The pH of all media was adjusted to 7.6, and no significant changes in pH were observed during any experiment. The cells began to deteriorate 8 h after isolation, as judged by their reduced viability (Trypan Blue exclusion) and loss of shape. All experiments reported here were completed within 5 h of isolation. There was no indication of decreased viability during any of the studies. Cells maintained their distinctive microvillar structure on a portion of the membrane throughout this period.
The gastrointestinal tract has a crucial role in the control of energy homeostasis through its role in the digestion, absorption, and assimilation of ingested nutrients. Furthermore, signals from the gastrointestinal tract are important regulators of gut motility and satiety, both of which have implications for the long-term control of body weight. Among the specialized cell types in the gastrointestinal mucosa, enteroendocrine cells have important roles in
Glucose was administered to the controls at a rate (2 mg/kg/min; 144 +/- 4 mg/min) known to inhibit splanchnic glucose output without influencing peripheral glucose utilization. The obese subjects received glucose at two dose levels (75 and 150 mg/min) which simulated either the rise in insulin or the inhibition in splanchnic glucose production observed in the controls. In the basal state splanchnic glucose production did not differ significantly between obese and control subjects. However splanchnic uptake of lactate, glycerol, alanine, free fatty acids, and oxygen was 50-160% greater in obese subjects. Splanchnic uptake of glucose precursors could account for 33% of hepatic glucose output in the obese group as compared to 19% in controls. The increase in alanine and lactate uptake was due in part, to a 50% increase in splanchnic fractional extraction. Administration of glucose to the control subjects 144 +/- 4 mg/min) resulted in a 50-60% increment in arterial insulin and a 75% reduction in splanchnic glucose output. In the obese group, infusion of glucose at a rate of 75 mg/min resulted in an equivalent rise in arterial […]
subsequently synthesised into a variety of agents including kynurenine via specific metabolic processes. As such, ATD is based on the premise that by reducing the plasma tryptophan to LNAA ratio, the rate of tryptophan subsequently crossing the BBB for further metabolism is also reduced (Hood et al., 2005). As tryptophan is an essential amino acid, ATD is normally achieved by administering an amino acid mix to study participants that contains a large amount of all other LNAAs, but lacks tryptophan. Despite a predominant focus on the effects of ATD on serotonin, this specificity has often come into question over the years and alternative mechanisms mediating the central and peripheral effects of ATD have been speculated upon (van Donkelaar et al., 2011). In support of an alternative/additional mechanism of action, it has been demonstrated in healthy control participants that ATD increases plasma kynurenic acid (Keszthelyi et al., 2012) and decreases plasma kynurenine levels in both healthy controls and female patients with IBS (Kennedy et al., 2015). Of note, ATD concurrently improved visuospatial memory performance in patients with IBS (Kennedy et al., 2015), which has previously been shown to be impaired in this clinical population (Kennedy et al., 2014a). Moreover, an intriguing study further demonstrated that the brain response to visceral pain stimulation in healthy females following ATD reflected the brain response in patients with IBS who underwent the same visceral pain stimulation, but not ATD (Labus et al., 2011). Together these studies lend further support for altered tryptophan metabolism in brain-gut axis dysregulation in IBS.
Rising arterial glucose levels and small decreases in plasma insulin concentrations were found during heavy exercise. Significant arterial-femoral venous differences for glucose were demonstrated both at rest and during exercise, their magnitude increasing with work intensity as well as duration of the exercise performed. Estimated glucose uptake by the leg increased 7-fold after 40 min of light exercise and 10- to 20-fold at moderate to heavy
the environment, also shape the architecture of gut microbiota [5, 7, 9]. The diet and particularly dietary carbohydrates are key determinants for the composition and activity of the intestinal microbiome [1, 5, 7, 10, 11]. At weaning, mammals gradually transition from lac- tose to plant carbohydrates as main source of dietary carbohydrates; this dietary shift also induces a major shift of the intestinal microbiome. In contrast, current swine production systems impose an abrupt transition from sow’s milk to solid food in piglets; this also induces an accelerated succession of microbial communities in weanling piglets [7, 12]. Gut microbial communities play a pivotal role in facilitating adaption of weanling piglets to fibrous feed and in minimizing the risk of colonization by pathogens after weaning . Carbohydrates are the main energy source for pigs; in commercial pig production, car- bohydrates account for more than 60% of the dry matter and 60–70% of the dietary energy intake [14, 15]. How- ever, digestive enzymes secreted by the host do not
That both glycolysis and mitochondrial oxidative phos- phorylation (OXPHOS) play key roles in cancer cells has led to a new research focus into drugs that inhibit both pathways [7,9,10]. Dichloroacetate (DCA), a pyruvate dehydrogenase kinase (PDK) inhibitor that reverses the Warburg effect [11,12], has been demonstrated to inhibit tumor growth in vivo [11,13,14], and induce apoptosis in tumors of GBM patients by normalizing the mitochon- drial activity . In cancer treatment, the mechanism by which DCA induces apoptosis of cancer cells is via an enhancement of a flux of electrons through the electron transport chain (ETC.) resulting in greater depolarization of the mitochondrial membrane potential (which is generally hyperpolarized in tumor cells) and release of cytochrome c followed by subsequent activa- tion of apoptosis . However, there are some conflict- ing reports for DCA’s anti-tumor efficacy in vitro and in vivo . In particular, not all studies found induction of apoptosis with DCA alone at clinical relevant concen- trations when tested in vitro . Improved sensitization of tumor cells to glycolysis inhibition has been achieved by the combination of glycolytic inhibitors and mito- chondrial toxins [18,19]. As a glycolytic inhibitor, DCA has also been reported to be more effective when com- bined with mitochondria-targeted agents [20,21]. Specif- ically, DCA has been demonstrated to sensitize cancer cells towards apoptosis and enhance the effects of sev- eral anti-cancer agents, including arsenic trioxide , cisplatin [22,23] and metformin . In this way, dual targeting of glucosemetabolism, using DCA to restore
An anticipated fate of intestinal glycerol is its reduction to 3-HPA, based on the common presence of genes encoding GDH in metagenomes and its conversion to acrolein, supported by the observation of acrolein transformation products. The het- erocyclic amines PhIP and MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline) are converted to PhIP-M1 and MelQx-M1 by complex colonic microbiota in the presence of glycerol (17, 18). Vanhaecke and coworkers reported that PhIP was transformed to PhIP-M1 by 18 fecal microbiota from individual donors, with PhIP transformation efﬁciencies ranging from 1.8% to 96% (16). Using an in vitro continuous fermentation model, PolyFermS, it could be shown that inactive microbiota can be made to signif- icantly promote HCA transformation by addition of a reuterin-producing, GDH-bearing strain of E. hallii (17, 18). Finally, PhIP-M1 could be recovered from feces of consumers that obtained a single portion of cooked chicken meat containing PhIP (26). Variations in the occurrence and abundances of gut microbes with GDH among individuals and further metabolism of reuterin to 1,3-PD by some strains (20) might be a reason for interindividual variations in acrolein formation-respective HCA degradation proﬁciency and for susceptibility to the development of colorectal cancer.
The prevalence of dyslipidemia is increasing in modern Japan because of westernization of the diet and ezetimibe is a reasonable etiologic therapy for dyslipidemia. Since this drug also improves postprandial metabolism, ezetimibe could be a beneﬁcial treat- ment option for patients who have dyslipidemia accompanied by obesity or metabolic syndrome. The present study had no control group and a limited sample size, as well as investigating rela- tively few laboratory parameters. It is difﬁcult to establish that ezetimibe treatment itself improved postprandial lipid and glu- cose metabolism. A randomized controlled trial should therefore be conducted in the future.
MAIDA SEFEROVIĆ ŠARIĆ
Department of Biology, Faculty of Science, University of Zagreb ABSTRACT
Atherosclerosis is a chronic progressive disease, which develops in childhood, and which encompasses a whole range of processes that result in carotid wall thickening, reduction of its elasticity and development of plaque. When this process affects the carotid arteries, there is a development of subclinical changes on the arterial wall, which can be quantified by measuring the thickness of IMT with an ultrasound. Thyroid hormones affect the cardiovascular system, but the precise mechanisms of their effects on the development of atherosclerosis are not entirely clear. This research included 100 women whose serum concentrations of fT4, TSH, CRP, total cholesterol count, triglycerides, HDL, LDL and its subtype sdLDL (previously suggested to have highly atherogenic properties), as well as carotid IMT thickness using colour Doppler, have been determined. The results of this research confirmed the hypothesis that subjects with subclinical hypothyroidism have an imbalanced lipid metabolism with increased triglycerides, cholesterol/HDL ratio, sdLDL and carotid IMT, when compared with the euthyroid group. Based on these results we can conclude that there is a need to screen, track and eventually treat people showing early, subclinical signs of thyroid gland dysfunction, and that it is necessary, along with conventional risk factors, to measure sdLDL.
In the present study, we aim to gain specific information about the regulatory ele- ments of the human brain in the systemic energy homeostasis. Therefore, we combine mathematical modeling and experimental data. In our novel approach, the integrative behavior of the human whole body energy metabolism is mathematically modeled in a compact dynamical system [11,12]. This model takes into account the central roles of the brain with respect to the systemic energy homeostasis. That is, the brain is consid- ered as regulatory instance and as energy consumer. Energy fluxes and their control signals, such as glucose fluxes and hormonal signals, are integrated in the dynamical system. The peripheral hormone insulin is regarded not only as local signal but also as key feedback signal to the brain [13-15]. Hence, in the mathematical model we integrate the competition for energy between brain and body periphery. There exist numerous mathematical models of human glucosemetabolism, e.g., [16-22]. However, in our novel approach we formulate the cerebral energy content in terms of high-energy phosphate levels comprising energy metabolites that emerge from several energy supplying substrates such as glucose and lactate. The novelty of our approach lies in combining a brain cen- tered mathematical model with experimental data of an euglycemic-hyperinsulinemic clamp to reveal systemic information of the brain energy metabolism. Our approach includes a new parameter estimation method to unfold major features of the energy regulation.
16S rRNA sequencing analysis of fecal samples was performed to determine the bacterial composition of each mouse strain after 7 weeks on an HFD, with or without antibiotic treatment. Despite their different genetic backgrounds and breeding sites, the gut microbiota of both B6J and 129T mice on an HFD were dominat- ed by Firmicutes (74% in B6J and 72% in 129T mice), whereas in 129J mice, Verrucomicrobia accounted for 66% of the bacterial sequences. Vancomycin treatment reduced the relative abun- dance of Firmicutes in B6J mice to 37% (P = 0.009) and in 129T mice to 50% of the untreated HFD levels (P = 0.003), and in both cases, this was associated with an increase in the relative abun- dance of Proteobacteria (Figure 2E). Interestingly, despite the difference in initial bacterial composition in 129J mice, metroni- dazole and vancomycin markedly reduced Verrucomicrobia from 66% to 0% (P = 0.002) and 23% (P = 0.007), respectively, leaving only Proteobacteria and Firmicutes (Figure 2E and Supplemental Figure 1A; supplemental material available online with this arti- cle; doi:10.1172/JCI86674DS1). PCA showed that, despite initial differences in microbiota among these 3 strains, the major deter- minant of the microbial landscape was antibiotic treatment (Sup- plemental Figure 1B). Nonetheless, even on the same HFD and the same antibiotic, each strain had unique microbiotial communities. Modification of gut microbiota by antibiotics improves glucosemetabolism and insulin signaling in B6J mice. In the obesity-prone B6J mice on an HFD, the vancomycin-treated mice gained sig- nificantly more weight than did the metronidazole-treated ani- mals, while placebo-treated mice had weight gains intermediate to those of the 2 groups (Supplemental Figures 2A). Vancomy- cin-treated B6J mice exhibited a significant increase in lean mass (22.8 ± 0.33 g to 24.1 ± 0.27 g; P = 0.002), with no change in fat mass as assessed by dual-energy x-ray absorptiometry (DEXA) (Figure 3A). By comparison, there were no significant differenc- es in weight gain in either the 129T or 129J mice during antibiotic treatment, although there was a small increase in lean mass in the 129T mice on vancomycin (Supplemental Figure 2, B and C, and Figure 3A). Food intake was not altered by antibiotic treatment in mice of any of the strains (Supplemental Figures 2, D and F).
ATP is central to a cell’s energy currency, but too much ATP is not necessarily beneﬁcial (27, 28). In fact, we observed a negative correlation between cellular ATP levels and cellobiose consumption efﬁciency (Fig. 4B). A similar correlation has been reported for glucose as a carbon source, suggesting metabolic uncoupling of energy homeostasis in yeast cells (29). We propose that intracellular glucose concentrations— generated by cellobiose hydrolysis in our experiments—and glucosemetabolism (23) are insufﬁcient to trigger glucose activation of key metabolic pathways and enzyme activity. For example, we found that the ATP-dependent proton pump Pma1 existed in a carbon-starvation-like state during cellobiose fermentation and was partially respon- sible for the aberrant accumulation of ATP. These results suggest that neither intracel- lular glucose nor glucosemetabolism is sufﬁcient to fully activate Pma1. A previous study showed the lack of phosphorylation of S899 and S911/T912 in Pma1, in a hexokinase/glucokinase deletion strain (hxk1 ⌬ hxk2 ⌬ glk1 ⌬ ) provided with glucose, suggesting that phosphorylation of these residues requires glucosemetabolism (30). Together with our results, we propose that the activation of Pma1 through S911 phosphorylation requires both extracellular glucose and glucosemetabolism. Our results reveal that the cellobiose utilization system allows uncoupling of glucosemetabolism and intracellular glucose from extracellular glucose signaling. Future ex- periments will be required to reveal why ATP was not consumed by other cellular processes triggered under starvation (31).
with the body of literature showing acute hypothalamic changes in response to hypoglycemia. Additionally, microdialysis can provide temporal resolution to our understanding of brain glycogen metabolism, but we may require sensitive techniques measuring intracellular glycogen content and enrichment to complement these data with spatial resolution. The measurement of other metabolites in microdialysate will also be necessary to understand the metabolic changes that occur in the VMH. We were capable of measuring trace quantities of unlabeled lactate but not labeled lactate in our plasma samples and both forms of lactate were below detection limits in microdialysate samples. Similarly, glutamate could only be identified in samples spiked with a standard, but not at physiologically relevant concentrations. Since microdialysate volumes were small, it may be necessary to either pool samples for analysis or to optimize sample derivatization for lactate and glutamate GC/MS analysis.
Within a given location, NAD + has two main pools, the free pool and protein-associated pool. Moreover, the ratio of these pools varies across different organelles, cell types, and tissues [ 29 ]. NAD + is predicted to bind >500 proteins involved in the regulation of almost all major biological processes [ 39 , 40 ]. Two principal types of NAD + -dependent signaling reactions have recently been described: the generation of second messenger molecules and the modification of proteins [ 41 , 42 ]. NAD + is a direct precursor of various small molecules such as ADP-ribose (ADPr), cyclic ADP-ribose (cADPr), NAADP and O-acetyl-ADP-ribose (OAADPR); all of which are important second messengers that regulate multiple aspects of biology, including cell survival, apoptosis, and inflammation [ 39 , 43 – 48 ]. In addition, NAD + serves as a substrate for post-translational protein modifications (PTM). In mammals, the two main NAD + -dependent enzyme families are the ADP-ribosyltransferases (ARTs) and the sirtuins (SIRTs) [ 39 , 49 – 52 ]. Both protein families use NAD + as a co-substrate to modify or de-modify target proteins. Interestingly, ADP-ribosylation was the first NAD + -dependent PTM identified [ 53 ]. ADP-ribosylation involves the attachment of one (mono-ADP-ribosylation; MARylation) or several (poly-ADP-ribosylation; PARylation) moieties of ADPr onto specific amino acid acceptor sites of target proteins or onto ribonucleotides [ 54 ]. Depending on their structural similarities to either cholera- or diphtheria toxins, ARTs can be further subdivided into the intracellular ARTDs (Diphteria toxin-like) and the extracellular or membrane-associated ARTCs (Cholera toxin-like). Despite the very low NAD + concentrations in these compartments [ 55 ], ARTCs as well as cADPr hydrolases, such as CD38, can metabolize NAD + in the extracellular space and in serum. Since this review focuses on the influence of ADP-ribosylation on carbohydrate metabolism the reader is referred to other reviews describing extracellular NAD + consumption [ 45 , 56 ]. SIRTs remove acyl marks (most commonly acetylation) from proteins using NAD + as an acceptor, thereby generating OAADPR and NAM [ 39 , 57 ]. SIRTs and ARTs localize to various intracellular compartments and have been experimentally linked to distinct cellular functions. Both enzyme families are associated with the regulation of various physiological processes, including metabolic regulation, DNA damage repair, cell cycle progression, and epithelial-to-mesenchymal transition (EMT) [ 58 – 62 ].
reported in both early pregnancy (2.4%) and term placental fragment cultures (<2%) (197), as well as in primary trophoblast cultures from term placenta (1.8-3%) (148). Interestingly, total glucose uptake was reduced in placentas from GDM mothers (176) and the transplacental glucose flux into the fetal circulation was similar (198) or reduced (176) in comparison with placentas from healthy mothers. Also, maternal GDM independent of insulin therapy led to a lower placental glucose utilisation than in normal controls (198). In addition, early studies found that pyruvate kinase activity, which is insulin-responsive, was increased in GDM placentas in comparison with controls (199). As neonates born to those GDM mothers were significantly heavier (199), increases in pyruvate kinase function could lead to higher fetal supply of pyruvate derived products including lactate and fatty acids. Nevertheless, in placental perfusion experiments in which the newborns’ weights were matched, the lactate release into maternal and fetal circulations was reduced in placentas from GDM pregnancies on diet in comparison with normal controls (176). Whether these differences in placental glucosemetabolism participate in a placental adaptation to prevent fetal excessive growth is undetermined. There is less known about glucose utilisation in isolated trophoblasts in response to increasing culture glucose concentrations. A previous study from our group showed that glycolysis measured by a radioactive tracer method, as described in Chapter 3, did not increase with increasing media glucose concentrations above physiological levels (200). Indeed, glycolysis rates were near maximal at 4 mM glucose and maximum at media glucose concentrations of 8 mM (200). Thus, it is probable that isolated trophoblasts will not increase glucose oxidation or lactate synthesis at high glucose media concentrations, but this still needs confirmation.