Design of Gadoteridol-Loaded Cationic Liposomal Adjuvant CAF01 for MRI of Lung Deposition of Intrapulmonary Administered Particles.

Designing effective and safe tuberculosis (TB) subunit vaccines for inhalation requires identification of appropriate antigens and adjuvants and definition of the specific areas to target in the lungs. Magnetic resonance imaging (MRI) enables high spatial resolution, but real-time anatomical and functional MRI of lungs is challenging. Here, we describe the design of a novel gadoteridol-loaded cationic adjuvant formulation 01 (CAF01) for MRI-guided vaccine delivery of the clinically tested TB subunit vaccine candidate H56/CAF01. Gadoteridol-loaded CAF01 liposomes were engineered by using a quality-by-design approach to (i) increase the mechanistic understanding of formulation factors governing the loading of gadoteridol and (ii) maximize the loading of gadoteridol in CAF01, which was confirmed by cryotransmission electron microscopy. The encapsulation efficiency and loading of gadoteridol were highly dependent on the buffer pH due to strong attractive electrostatic interactions between gadoteridol and the cationic lipid component. Optimal gadoteridol loading of CAF01 liposomes showed good in vivo stability and safety upon intrapulmonary administration into mice while generating 1.5-fold MRI signal enhancement associated with approximately 30% T1 relaxation change. This formulation principle and imaging approach can potentially be used for other mucosal nanoparticle-based formulations, species, and lung pathologies, which can readily be translated for clinical use.

Restricting glycolysis impairs brown adipocyte glucose and oxygen consumption.

During thermogenic activation, brown adipocytes take up large amounts of glucose. In addition, cold stimulation leads to an upregulation of glycolytic enzymes. Here we have investigated the importance of glycolysis for brown adipocyte glucose consumption and thermogenesis. Using siRNA-mediated knockdown in mature adipocytes, we explored the effect of glucose transporters and glycolytic enzymes on brown adipocyte functions such as consumption of glucose and oxygen. Basal oxygen consumption in brown adipocytes was equally dependent on glucose and fatty acid oxidation, whereas isoproterenol (ISO)-stimulated respiration was fueled mainly by fatty acids, with a significant contribution from glucose oxidation. Knockdown of glucose transporters in brown adipocytes not only impaired ISO-stimulated glycolytic flux but also oxygen consumption. Diminishing glycolytic flux by knockdown of the first and final enzyme of glycolysis, i.e., hexokinase 2 (HK2) and pyruvate kinase M (PKM), respectively, decreased glucose uptake and ISO-stimulated oxygen consumption. HK2 knockdown had a more severe effect, which, in contrast to PKM knockdown, could not be rescued by supplementation with pyruvate. Hence, brown adipocytes rely on glucose consumption and glycolytic flux to achieve maximum thermogenic output, with glycolysis likely supporting thermogenesis not only by pyruvate formation but also by supplying intermediates for efferent metabolic pathways.

Perturbations of NAD+ salvage systems impact mitochondrial function and energy homeostasis in mouse myoblasts and intact skeletal muscle.

Nicotinamide adenine dinucleotide (NAD+) can be synthesized by nicotinamide phosphoribosyltransferase (NAMPT). We aimed to determine the role of NAMPT in maintaining NAD+ levels, mitochondrial function, and metabolic homeostasis in skeletal muscle cells. We generated stable Nampt knockdown (sh Nampt KD) C2C12 cells using a shRNA lentiviral approach. Moreover, we applied gene electrotransfer to express Cre recombinase in tibialis anterior muscle of floxed Nampt mice. In sh Nampt KD C2C12 myoblasts, Nampt and NAD+ levels were reduced by 70% and 50%, respectively, and maximal respiratory capacity was reduced by 25%. Moreover, anaerobic glycolytic flux increased by 55%, and 2-deoxyglucose uptake increased by 25% in sh Nampt KD cells. Treatment with the NAD+ precursor nicotinamide riboside restored NAD+ levels in sh Nampt cells and increased maximal respiratory capacity by 18% and 32% in control and sh Nampt KD cells, respectively. Expression of Cre recombinase in muscle of floxed Nampt mice reduced NAMPT and NAD+ levels by 38% and 43%, respectively. Glucose uptake increased by 40%, and mitochondrial complex IV respiration was compromised by 20%. Hypoxia-inducible factor (HIF)-1α-regulated genes and histone H3 lysine 9 (H3K9) acetylation, a known sirtuin 6 (SIRT6) target, were increased in shNampt KD cells. Thus, we propose that the shift toward glycolytic metabolism observed, at least in part, is mediated by the SIRT6/HIF1α axis. Our findings suggest that NAMPT plays a key role for maintaining NAD+ levels in skeletal muscle and that NAMPT deficiency compromises oxidative phosphorylation capacity and alters energy homeostasis in this tissue.

Quantitative Gd-DOTA uptake from cerebrospinal fluid into rat brain using 3D VFA-SPGR at 9.4T.

PURPOSE: We propose a quantitative technique to assess solute uptake into the brain parenchyma based on dynamic contrast-enhanced MRI (DCE-MRI). With this approach, a small molecular weight paramagnetic contrast agent (Gd-DOTA) is infused in the cerebral spinal fluid (CSF) and whole brain gadolinium concentration maps are derived. METHODS: We implemented a 3D variable flip angle spoiled gradient echo (VFA-SPGR) longitudinal relaxation time (T1) technique, the accuracy of which was cross-validated by way of inversion recovery rapid acquisition with relaxation enhancement (IR-RARE) using phantoms. Normal Wistar rats underwent Gd-DOTA infusion into CSF via the cisterna magna and continuous MRI for approximately 130 min using T1-weighted imaging. Dynamic Gd-DOTA concentration maps were calculated and parenchymal uptake was estimated. RESULTS: In the phantom study, T1 discrepancies between the VFA-SPGR and IR-RARE sequences were approximately 6% with a transmit coil inhomogeneity correction. In the in vivo study, contrast transport profiles indicated maximal parenchymal retention of approximately 19% relative to the total amount delivered into the cisterna magna. CONCLUSION: Imaging strategies for accurate 3D contrast concentration mapping at 9.4T were developed and whole brain dynamic concentration maps were derived to study solute transport via the glymphatic system. The newly developed approach will enable future quantitative studies of the glymphatic system in health and disease states. Magn Reson Med 79:1568-1578, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Elevated arterial lactate delays recovery of intracellular muscle pH after exercise.

PURPOSE: We evaluated muscle proton elimination following similar exercise in the same muscle group following two exercise modalities. METHODS: Seven rowers performed handgrip or rowing exercise for ~ 5 min. The intracellular response of the wrist flexor muscles was evaluated by 31P nuclear magnetic resonance spectroscopy, while arterial and venous forearm blood was collected. RESULTS: Rowing and handgrip reduced intracellular pH to 6.3 ± 0.2 and 6.5 ± 0.1, arterial pH to 7.09 ± 0.03 and 7.40 ± 0.03 and venous pH to 6.95 ± 0.06 and 7.20 ± 0.04 (P < 0.05), respectively. Arterial and venous lactate increased to 17.5 ± 1.6 and 20.0 ± 1.6 mM after rowing while only to 2.6 ± 0.8 and 6.8 ± 0.8 mM after handgrip exercise. Arterio-venous concentration difference of bicarbonate and phosphocreatine recovery kinetics (T50% rowing 1.5 ± 0.7 min; handgrip 1.4 ± 1.0 min) was similar following the two exercise modalities. Yet, intramuscular pH recovery in the forearm flexor muscles was 3.5-fold slower after rowing than after handgrip exercise (T50% rowing of 2 ± 0.1 vs. 7 ± 0.3 min for handgrip). CONCLUSION: Rowing delays intracellular-pH recovery compared with handgrip exercise most likely because rowing, as opposed to handgrip exercise, increases systemic lactate concentration. Thus the intra-to-extra-cellular lactate gradient is small after rowing. Since this lactate gradient is the main driving force for intracellular lactate removal in muscle and, since pHi normalization is closely related to intracellular lactate removal, rowing results in a slower pHi recovery compared to handgrip exercise.

Unchanged mitochondrial phenotype, but accumulation of lipids in the myometrium in obese pregnant women.

KEY POINTS: Obesity during pregnancy and childbirth is associated with labour dystocia leading to instrumental or operative delivery, but the underlying pathophysiological mechanisms remain unclear and insufficient uterine contractility has been suggested. This study examined whether reduced myometrial mitochondrial capacity or quantity could contribute as a pathophysiological mechanism to labour dystocia. Data did not support reduced myometrial mitochondrial capacity or quantity in the myometrium at term in obese women, but a reduced myocyte density with increased triglyceride content was demonstrated, which could lead to poorer uterine contractility. These results add to the understanding of systemic effects of obesity, placing also the myometrium at term as an affected non-adipose tissue. ABSTRACT: Obesity is known to increase the risk of labour dystocia and insufficient energy supply, due to reduced mitochondrial capacity or quantity, could be a possible mechanism leading to reduced efficiency of uterine contractility during labour. In the present study of 36 women having an elective Caesarean section at term, obesity did not change mitochondrial phenotype in the myometrial myocyte obtained from uterine biopsies taken at delivery. Respiration rates in isolated mitochondria were unaffected by obesity. No indication of reduced content, investigated by quantification of the complexes of the respiratory chain, or altered regulation, examined by myometrial mRNA levels of genes related to mitochondrial biogenesis and inflammation, was detected. Yet we found increased myometrial triglyceride content in the obese group (2.39 ± 0.26 vs. 1.56 ± 0.20 mm, P = 0.024), while protein content and citrate synthase activity per gram wet weight myometrium were significantly lower in the obese (109.2 ± 7.2 vs. 139.4 ± 5.6 mg g-1 , P = 0.002, and 24.8 ± 1.0 vs. 29.6 ± 1.4 U g-1 wet wt, P = 0.008, respectively). These differences were substantiated by our histological findings where staining for nuclei, cytoplasm, glycogen and collagen supported the idea of a smaller muscle content in the myometrium in obese women. In conclusion no indication of myometrial mitochondrial dysfunction in the isolated state was found, but the observed increase of lipid content might play a role in the pathophysiological mechanisms behind labour dystocia in obese women.

Gestational Protein Restriction in Wistar Rats; Effect of Taurine Supplementation on Properties of Newborn Skeletal Muscle.

Taurine ameliorates changes occurring in newborn skeletal muscle as a result of gestational protein restriction in C57BL/6 mice, but taurine supplementation effects may be exaggerated in C57BL/6 mice due to their inherent excessive taurinuria.We examined if maternal taurine supplementation could ameliorate changes in gene expression levels, properties of mitochondria, myogenesis, and nutrient transport and sensing, in male newborn skeletal muscle caused by a maternal low protein (LP) diet in Wistar rats.LP diet resulted in an 11% non-significant decrease in birth weight, which was not rescued by taurine supplementation (LP-Tau). LP-Tau offspring had significantly lower birth weight compared to controls. Gene expression profiling revealed 895 significantly changed genes, mainly an LP-induced down-regulation of genes involved in protein translation. Taurine fully or partially rescued 32% of these changes, but with no distinct pattern as to which genes were rescued.Skeletal muscle taurine content in LP-Tau offspring was increased, but no changes in mRNA levels of the taurine synthesis pathway were observed. Taurine transporter mRNA levels, but not protein levels, were increased by LP diet.Nutrient sensing signaling pathways were largely unaffected in LP or LP-Tau groups, although taurine supplementation caused a decrease in total Akt and AMPK protein levels. PAT4 amino acid transporter mRNA was increased by LP, and normalized by taurine supplementation.In conclusion, gestational protein restriction in rats decreased genes involved in protein translation in newborn skeletal muscle and led to changes in nutrient transporters. Taurine partly rescued these changes, hence underscoring the importance of taurine in development.

Acute disruption of glucagon secretion or action does not improve glucose tolerance in an insulin-deficient mouse model of diabetes.

AIMS/HYPOTHESIS: Normal glucose metabolism depends on pancreatic secretion of insulin and glucagon. The bihormonal hypothesis states that while lack of insulin leads to glucose underutilisation, glucagon excess is the principal factor in diabetic glucose overproduction. A recent study reported that streptozotocin-treated glucagon receptor knockout mice have normal glucose tolerance. We investigated the impact of acute disruption of glucagon secretin or action in a mouse model of severe diabetes by three different approaches: (1) alpha cell elimination; (2) glucagon immunoneutralisation; and (3) glucagon receptor antagonism, in order to evaluate the effect of these on glucose tolerance. METHODS: Severe diabetes was induced in transgenic and wild-type mice by streptozotocin. Glucose metabolism was investigated using OGTT in transgenic mice with the human diphtheria toxin receptor expressed in proglucagon producing cells allowing for diphtheria toxin (DT)-induced alpha cell ablation and in mice treated with either a specific high affinity glucagon antibody or a specific glucagon receptor antagonist. RESULTS: Near-total alpha cell elimination was induced in transgenic mice upon DT administration and resulted in a massive decrease in pancreatic glucagon content. Oral glucose tolerance in diabetic mice was neither affected by glucagon immunoneutralisation, glucagon receptor antagonism, nor alpha cell removal, but did not deteriorate further compared with mice with intact alpha cell mass. CONCLUSIONS/INTERPRETATION: Disruption of glucagon action/secretion did not improve glucose tolerance in diabetic mice. Near-total alpha cell elimination may have prevented further deterioration. Our findings support insulin lack as the major factor underlying hyperglycaemia in beta cell-deficient diabetes.

Effect of high-fat diet on rat myometrium during pregnancy-isolated myometrial mitochondria are not affected.

Laboring women with elevated body mass index (BMI) have an increased risk of inefficient uterine labor contractions, and despite the significance of mitochondria in the production of energy to drive uterine contractions, mitochondrial function in the myometrium with reference to the BMI has not been explored. The objective of this study was to determine whether obesity prior to and during gestation affects oxidative capacity and/or morphology of mitochondria in the myometrium at term in an animal model. Rat dams were fed for 47 days prior to impregnation and during gestation with either (1) a regular chow diet, (2) a low-fat high-carbohydrate diet, or (3) a high-fat low-carbohydrate diet (n = 10 in each group). On day 20 of gestation, corresponding to term pregnancy, total hysterectomy was performed with subsequent examination of the function and morphology of myometrial mitochondria. Body composition was regularly assessed by quantitative magnetic resonance imaging, and blood sampling was done prior to diet assignment, impregnation, and hysterectomy. Dams on the high-fat low-carbohydrate diet achieved higher fat percentage compared to rats on the regular chow diet (p < 0.05). Maximal oxygen consumption, phosphate/oxygen ratio, or the amount of mitochondria per gram of myometrium did not differ between the three feeding groups. Electron microscopic examinations did not reveal any morphological differences in mitochondria between groups; however, a previously undescribed subsarcolemmal localization of the mitochondria in the myocyte was identified. We did not find evidence of altered myometrial mitochondrial function or morphology in this animal model of obesity prior to and during pregnancy.

Global gene expression profiling of brown to white adipose tissue transformation in sheep reveals novel transcriptional components linked to adipose remodeling.

BACKGROUND: Large mammals are capable of thermoregulation shortly after birth due to the presence of brown adipose tissue (BAT). The majority of BAT disappears after birth and is replaced by white adipose tissue (WAT). RESULTS: We analyzed the postnatal transformation of adipose in sheep with a time course study of the perirenal adipose depot. We observed changes in tissue morphology, gene expression and metabolism within the first two weeks of postnatal life consistent with the expected transition from BAT to WAT. The transformation was characterized by massively decreased mitochondrial abundance and down-regulation of gene expression related to mitochondrial function and oxidative phosphorylation. Global gene expression profiling demonstrated that the time points grouped into three phases: a brown adipose phase, a transition phase and a white adipose phase. Between the brown adipose and the transition phase 170 genes were differentially expressed, and 717 genes were differentially expressed between the transition and the white adipose phase. Thirty-eight genes were shared among the two sets of differentially expressed genes. We identified a number of regulated transcription factors, including NR1H3, MYC, KLF4, ESR1, RELA and BCL6, which were linked to the overall changes in gene expression during the adipose tissue remodeling. Finally, the perirenal adipose tissue expressed both brown and brite/beige adipocyte marker genes at birth, the expression of which changed substantially over time. CONCLUSIONS: Using global gene expression profiling of the postnatal BAT to WAT transformation in sheep, we provide novel insight into adipose tissue plasticity in a large mammal, including identification of novel transcriptional components linked to adipose tissue remodeling. Moreover, our data set provides a useful resource for further studies in adipose tissue plasticity.

FoxO3A promotes metabolic adaptation to hypoxia by antagonizing Myc function.

Exposure of metazoan organisms to hypoxia engages a metabolic switch orchestrated by the hypoxia-inducible factor 1 (HIF-1). HIF-1 mediates induction of glycolysis and active repression of mitochondrial respiration that reduces oxygen consumption and inhibits the production of potentially harmful reactive oxygen species (ROS). Here, we show that FoxO3A is activated in hypoxia downstream of HIF-1 and mediates the hypoxic repression of a set of nuclear-encoded mitochondrial genes. FoxO3A is required for hypoxic suppression of mitochondrial mass, oxygen consumption, and ROS production and promotes cell survival in hypoxia. FoxO3A is recruited to the promoters of nuclear-encoded mitochondrial genes where it directly antagonizes c-Myc function via a mechanism that does not require binding to the consensus FoxO recognition element. Furthermore, we show that FoxO3A is activated in human hypoxic tumour tissue in vivo and that FoxO3A short-hairpin RNA (shRNA)-expressing xenograft tumours are decreased in size and metabolically changed. Our findings define a novel mechanism by which FoxO3A promotes metabolic adaptation and stress resistance in hypoxia.

Aberrant expression of miR-218 and miR-204 in human mesial temporal lobe epilepsy and hippocampal sclerosis-convergence on axonal guidance.

OBJECTIVE: Mesial temporal lobe epilepsy (MTLE) is one of the most common types of the intractable epilepsies and is most often associated with hippocampal sclerosis (HS), which is characterized by pronounced loss of hippocampal pyramidal neurons. microRNAs (miRNAs) have been shown to be dysregulated in epilepsy and neurodegenerative diseases, and we hypothesized that miRNAs could be involved in the pathogenesis of MTLE and HS. METHODS: miRNA expression was quantified in hippocampal specimens from human patients using miRNA microarray and quantitative real-time polymerase chain reaction RT-PCR, and by RNA-seq on fetal brain specimens from domestic pigs. In situ hybridization was used to show the spatial distribution of miRNAs in the human hippocampus. The potential effect of miRNAs on targets genes was investigated using the dual luciferase reporter gene assay. RESULTS: miRNA expression profiling showed that 25 miRNAs were up-regulated and 5 were down-regulated in hippocampus biopsies of MTLE/HS patients compared to controls. We showed that miR-204 and miR-218 were significantly down-regulated in MTLE and HS, and both were expressed in neurons in all subfields of normal hippocampus. Moreover, miR-204 and miR-218 showed strong changes in expression during fetal development of the hippocampus in pigs, and we identified four target genes, involved in axonal guidance and synaptic plasticity, ROBO1, GRM1, SLC1A2, and GNAI2, as bona fide targets of miR-218. GRM1 was also shown to be a direct target of miR-204. SIGNIFICANCE: miR-204 and miR-218 are developmentally regulated in the hippocampus and may contribute to the molecular mechanisms underlying the pathogenesis of MTLE and HS.

Acute and perinatal programming effects of a fat-rich diet on rat muscle mitochondrial function and hepatic lipid accumulation.

OBJECTIVE: Maternal high-fat intake during pregnancy may have long-term consequences in the offspring. Since this might relate to the capacity of mitochondrial metabolic adaptation and hepatic lipid metabolism, we investigated how maternal high-fat intake affected mitochondrial function and hepatic steatosis in the offspring. DESIGN: Sprague-Dawley rats were fed a high-fat (20% w/w) or a control diet (chow, C) from 10 days before pregnancy and throughout lactation. At weaning the litters were split into two groups; one was continued on the maternal diet and the other was fed low-fat chow. SAMPLE: Skeletal muscle mitochondria and liver lipids. METHODS: Mitochondrial respiration and hepatic lipid content were determined during and after weaning, on days 20 and 70 postpartum. MAIN OUTCOME MEASURES: Mitochondrial function and hepatic lipids. RESULTS: At 20 days, maternal high-fat diet caused increased Vo2max with pyruvate as substrate (p=0.047), at 70 days, pups born by C-dams, but not those born by high-fat-dams, showed increased oxidation of palmitoylcarnitine in the absence of ADP (p=0.018). Rates of ADP-stimulated oxygen consumption, maximal respiratory capacity and mitochondrial respiratory control ratio with pyruvate, increased post weaning (p<0.001), whereas respiratory control ratio with palmitoylcarnitine decreased (p=0.013). The increase in respiratory control ratio was most pronounced in pups from C-dams (p=0.05). The high-fat-diet caused pronounced hepatic steatosis in pups at weaning (p<0.001), without concomitant ceramide accumulation, while high-fat-feeding after weaning induced triacylglycerol and ceramide accumulation (p<0.01), regardless of maternal diet. CONCLUSION: Intake of a fat-rich diet during pregnancy and lactation reduced the age-induced increases in un-coupled fat oxidation.

Retinoic acid has different effects on UCP1 expression in mouse and human adipocytes.

BACKGROUND: Increased adipose thermogenesis is being considered as a strategy aimed at preventing or reversing obesity. Thus, regulation of the uncoupling protein 1 (UCP1) gene in human adipocytes is of significant interest. Retinoic acid (RA), the carboxylic acid form of vitamin A, displays agonist activity toward several nuclear hormone receptors, including RA receptors (RARs) and peroxisome proliferator-activated receptor δ (PPARδ). Moreover, RA is a potent positive regulator of UCP1 expression in mouse adipocytes. RESULTS: The effects of all-trans RA (ATRA) on UCP1 gene expression in models of mouse and human adipocyte differentiation were investigated. ATRA induced UCP1 expression in all mouse white and brown adipocytes, but inhibited or had no effect on UCP1 expression in human adipocyte cell lines and primary human white adipocytes. Experiments with various RAR agonists and a RAR antagonist in mouse cells demonstrated that the stimulatory effect of ATRA on UCP1 gene expression was indeed mediated by RARs. Consistently, a PPARδ agonist was without effect. Moreover, the ATRA-mediated induction of UCP1 expression in mouse adipocytes was independent of PPARγ coactivator-1α. CONCLUSIONS: UCP1 expression is differently affected by ATRA in mouse and human adipocytes. ATRA induces UCP1 expression in mouse adipocytes through activation of RARs, whereas expression of UCP1 in human adipocytes is not increased by exposure to ATRA.

The effect of long-term taurine supplementation and fructose feeding on glucose and lipid homeostasis in Wistar rats.

The nonprotein amino acid taurine has been shown to counteract the negative effects of a high-fructose diet in rats with regard to insulin resistance and dyslipidemia. Here we examined the long-term (26 weeks) effects of oral taurine supplementation (2% in the drinking water) in fructose-fed Wistar rats.The combination of fructose and taurine caused a significant increase in fasting glucose compared to the control diet without changing hepatic phosphoenol pyruvate carboxykinase mRNA levels. The combination of fructose and taurine also improved glucose tolerance compared to control. Neither a high-fructose diet nor taurine supplementation induced significant changes in body weight, body fat or total calorie intake, fasting insulin levels, HOMA-IR, or insulin-induced Akt phosphorylation in skeletal muscle.Fructose alone caused a decrease in liver triglyceride content, with taurine supplementation preventing this. There was no effect of long-term fructose diet and/or taurine supplementation on plasma triglycerides, plasma nonesterified fatty acids, as well as plasma HDL, LDL, and total cholesterol.In conclusion, the study suggests that long-term taurine supplementation improves glucose tolerance and normalize hepatic triglyceride content following long-term fructose feeding. However, as the combination of taurine and fructose also increased fasting glucose levels, the beneficial effect of taurine supplementation towards amelioration of glucose intolerance and insulin resistance may be questionable.

Progression of type 2 diabetes in GK rats affects muscle and liver mitochondria differently: pronounced reduction of complex II flux is observed in liver only.

Impaired mitochondrial function is implicated in the development of type 2 diabetes mellitus (T2DM). This was investigated in mitochondria from skeletal muscle and liver of the Goto-Kakizaki (GK) rat, which spontaneously develops T2DM with age. The early and the manifest stage of T2DM was studied in 6- and 16-wk-old GK rats, respectively. In GK16 compared with GK6 animals, a decrease in state 3 respiration with palmitoyl carnitine (PC) as substrate was observed in muscle. Yet an increase was seen in liver. To test the complex II contribution to the state 3 respiration, succinate was added together with PC. In liver mitochondria, this resulted in an ∼50% smaller respiratory increase in the GK6 group compared with control and no respiratory increase at all in the GK16 animals. Yet no difference between groups was seen in muscle mitochondria. RCR and P/O ratio was increased (P < 0.05) in liver but unchanged in muscle in both GK groups. We observed increased lipid peroxidation and decreased Akt phosphorylation in liver with the progression of T2DM but no change in muscle. We conclude that, during the progression of T2DM in GK rats, liver mitochondria are affected earlier and/or more severely than muscle mitochondria. Succinate dehydrogenase flux in the presence of fatty acids was reduced severely in liver but not in muscle mitochondria during manifest T2DM. The observations support the notion that T2DM pathogenesis is initiated in the liver and that only later are muscle mitochondria affected.

Effects of high-fat overfeeding on mitochondrial function, glucose and fat metabolism, and adipokine levels in low-birth-weight subjects.

Low birth weight (LBW) is associated with an increased risk of insulin resistance and downregulation of oxidative phosphorylation (OXPHOS) genes when exposed to a metabolic challenge of high-fat overfeeding (HFO). To elaborate further on the differential effects of HFO in LBW subjects, we measured in vivo mitochondrial function, insulin secretion, hepatic glucose production, and plasma levels of key regulatory hormones before and after 5 days of HFO in 20 young LBW and 26 normal-birth-weight (NBW) men. The LBW subjects developed peripheral insulin resistance after HFO due to impaired endogenous glucose storage (9.42 ± 4.19 vs. 5.91 ± 4.42 mg·kg FFM(-1)·min(-1), P = 0.01). Resting muscle phosphorcreatine and total ATP in muscle increased significantly after HFO in LBW subjects only, whereas additional measurements of mitochondrial function remained unaffected. Despite similar plasma FFA levels, LBW subjects displayed increased fat oxidation during insulin infusion compared with normal-birth-weight (NBW) subjects after HFO (0.37 ± 0.35 vs. 0.17 ± 0.33 mg·kg FFM(-1)·min(-1), P = 0.02). In contrast to NBW subjects, the plasma leptin levels of LBW subjects did not increase, and the plasma gastric inhibitory polypeptide (GIP) as well as pancreatic polypeptide (PP) levels increased less in LBW compared with NBW subjects during HFO. In conclusion, HFO unmasks dissociation between insulin resistance and mitochondrial dysfunction in LBW subjects, suggesting that insulin resistance may be a cause, rather than an effect, of impaired muscle OXPHOS gene expression and mitochondrial dysfunction. Reduced increments in response to HFO of fasting plasma leptin, PP, and GIP levels may contribute to insulin resistance, lower satiety, and impaired insulin secretion in LBW subjects.

Three-dimensional reconstructions of intrahepatic bile duct tubulogenesis in human liver.

BACKGROUND: During liver development, intrahepatic bile ducts are thought to arise by a unique asymmetric mode of cholangiocyte tubulogenesis characterized by a series of remodeling stages. Moreover, in liver diseases, cells lining the Canals of Hering can proliferate and generate new hepatic tissue. The aim of this study was to develop protocols for three-dimensional visualization of protein expression, hepatic portal structures and human hepatic cholangiocyte tubulogenesis. RESULTS: Protocols were developed to digitally visualize portal vessel branching and protein expression of hepatic cell lineage and extracellular matrix deposition markers in three dimensions. Samples from human prenatal livers ranging from 7 weeks + 2 days to 15½ weeks post conception as well as adult normal and acetaminophen intoxicated liver were used. The markers included cytokeratins (CK) 7 and 19, the epithelial cell adhesion molecule (EpCAM), hepatocyte paraffin 1 (HepPar1), sex determining region Y (SRY)-box 9 (SOX9), laminin, nestin, and aquaporin 1 (AQP1).Digital three-dimensional reconstructions using CK19 as a single marker protein disclosed a fine network of CK19 positive cells in the biliary tree in normal liver and in the extensive ductular reactions originating from intrahepatic bile ducts and branching into the parenchyma of the acetaminophen intoxicated liver. In the developing human liver, three-dimensional reconstructions using multiple marker proteins confirmed that the human intrahepatic biliary tree forms through several developmental stages involving an initial transition of primitive hepatocytes into cholangiocytes shaping the ductal plate followed by a process of maturation and remodeling where the intrahepatic biliary tree develops through an asymmetrical form of cholangiocyte tubulogenesis. CONCLUSIONS: The developed protocols provide a novel and sophisticated three-dimensional visualization of vessels and protein expression in human liver during development and disease.

Effect of revascularization on lower extremity muscle function in combined type 2 diabetes and critical limb threatening ischemia.

BACKGROUND: Critical limb-threatening ischemia (CLTI) and type 2 diabetes (T2D) frequently co-exist and often with less favorable outcome after revascularization. The objective was to evaluate the effects of revascularization on muscle function, perfusion and mitochondrial respiration in patients with combined CLTI and T2D. METHODS: A prospective translational observational study. Two groups of patients facing unilateral peripheral revascularization was included: Patients suffering from combined disease with CLTI+T2D (N.=14) and patients suffering from CLTI (N.=15). During pedal exercise testing, calf muscle perfusion was monitored with near-infrared spectroscopy (NIRS) and leg arterial volume flow in the common femoral artery with duplex ultrasound. Calf muscle biopsy and subsequent assessment of mitochondrial respiratory capacity on isolated muscle fibers was performed. Tests was performed before and six weeks after revascularization. RESULTS: After revascularization, patients CLTI+T2D improved in muscle force from 8.48 kg (CI: 7.49-9.46) to 13.11 kg (CI: 11.58-14.63), (P<0.001). Conversely, muscle force in patients suffering from non-diabetic CLTI decreased from 10.03 kg (CI: 9.1-10.96) to 9.73 kg (CI: 8.77-10.69), (P=0.042). Muscle oxygenation during exercise improved more in the CLTI+T2D group (6.36 µM/kg/s [CI: 5.71-7.01] compared to 2.11 µM/kg/s [CI:1.38-2.83] in the CLTI group; P=0.002). No improvement or difference between groups regarding mitochondrial function was detected. CONCLUSIONS: Patients with combined CLTI+T2D, had an unsuspected better effect of revascularization compared to patients with non-diabetic CLTI, in terms of increased muscle force and improved muscle perfusion. Further studies are needed to elucidate the apparent interaction of the CLTI and T2D syndromes.