Metabolic characteristics of transmembrane prolyl 4-hydroxylase (P4H-TM) deficient mice.

Transmembrane prolyl 4-hydroxylase (P4H-TM) is an enigmatic enzyme whose cellular function and primary substrate remain to be identified. Its loss-of-function mutations cause a severe neurological HIDEA syndrome with hypotonia, intellectual disability, dysautonomia and hypoventilation. Previously, P4H-TM deficiency in mice was associated with reduced atherogenesis and lower serum triglyceride levels. Here, we characterized the glucose and lipid metabolism of P4h-tm-/- mice in physiological and tissue analyses. P4h-tm-/- mice showed variations in 24-h oscillations of energy expenditure, VO2 and VCO2 and locomotor activity compared to wild-type (WT) mice. Their rearing activity was reduced, and they showed significant muscle weakness and compromised coordination. Sedated P4h-tm-/- mice had better glucose tolerance, lower fasting insulin levels, higher fasting lactate levels and lower fasting free fatty acid levels compared to WT. These alterations were not present in conscious P4h-tm-/- mice. Fasted P4h-tm-/- mice presented with faster hepatic glycogenolysis. The respiratory rate of conscious P4h-tm-/- mice was significantly lower compared to the WT, the decrease being further exacerbated by sedation and associated with acidosis and a reduced ventilatory response to both hypoxia and hypercapnia. P4H-TM deficiency in mice is associated with alterations in whole-body energy metabolism, day-night rhythm of activity, glucose homeostasis and neuromuscular and respiratory functions. Although the underlying mechanism(s) are not yet fully understood, the phenotype appears to have neurological origins, controlled by brain and central nervous system circuits. The phenotype of P4h-tm-/- mice recapitulates some of the symptoms of HIDEA patients, making this mouse model a valuable tool to study and develop tailored therapies.

Interplay between Learning and Voluntary Wheel Running in Male C57BL/6NCrl Mice.

Exercise is shown to improve cognitive function in various human and animal studies. Laboratory mice are often used as a model to study the effects of physical activity and running wheels provide a voluntary and non-stressful form of exercise. The aim of the study was to analyze whether the cognitive state of a mouse is related to its wheel-running behavior. Twenty-two male C57BL/6NCrl mice (9.5 weeks old) were used in the study. The cognitive function of group-housed mice (n = 5-6/group) was first analyzed in the IntelliCage system followed by individual phenotyping with the PhenoMaster with access to a voluntary running wheel. The mice were divided into three groups according to their running wheel activity: low, average, and high runners. The learning trials in the IntelliCage showed that the high-runner mice exhibited a higher error rate at the beginning of learning trials but improved their outcome and learning performance more compared to the other groups. The high-runner mice ate more compared to the other groups in the PhenoMaster analyses. There were no differences in the corticosterone levels between the groups, indicating similar stress responses. Our results demonstrate that high-runner mice exhibit enhanced learning capabilities prior to access to voluntary running wheels. In addition, our results also show that individual mice react differently when introduced to running wheels, which should be taken into consideration when choosing animals for voluntary endurance exercise studies.

Seasonal Regulation of Metabolism: The Effect of Wintertime Fasting and Autumnal Fattening on Key Central Regulators of Metabolism and the Metabolic Profile of the Raccoon Dog (Nyctereutes Procyonoides).

Investigations into the mechanisms regulating obesity are frantic and novel translational approaches are needed. The raccoon dog (Nyctereutes procyonoides) is a canid species representing a promising model to study metabolic regulation in a species undergoing cycles of seasonal obesity and fasting. To understand the molecular mechanisms of metabolic regulation in seasonal adaptation, we analyzed key central nervous system and peripheral signals regulating food intake and metabolism from raccoon dogs after autumnal fattening and winter fasting. Expressions of neuropeptide Y (NPY), orexin-2 receptor (OX2R), pro-opiomelanocortin (POMC) and leptin receptor (ObRb) were analyzed as examples of orexigenic and anorexigenic signals using qRT-PCR from raccoon dog hypothalamus samples. Plasma metabolic profiles were measured with 1H NMR-spectroscopy and LC-MS. Circulating hormones and cytokines were determined with canine specific antibody assays. Surprisingly, NPY and POMC were not affected by the winter fasting nor autumn fattening and the metabolic profiles showed a remarkable equilibrium, indicating conserved homeostasis. However, OX2R and ObRb expression changes suggested seasonal regulation. Circulating cytokine levels were not increased, demonstrating that the autumn fattening did not induce subacute inflammation. Thus, the raccoon dog developed seasonal regulatory mechanisms to accommodate the autumnal fattening and prolonged fasting making the species unique in coping with the extreme environmental challenges.

Role of Brown and Beige Adipose Tissues in Seasonal Adaptation in the Raccoon Dog (Nyctereutes procyonoides).

Brown adipose tissue (BAT) expresses uncoupling protein-1 (UCP1), which enables energy to be exerted towards needed thermogenesis. Beige adipocytes are precursor cells interspersed among white adipose tissue (WAT) that possess similar UCP1 activity and capacity for thermogenesis. The raccoon dog (Nyctereutes procyonoides) is a canid species that utilizes seasonal obesity to survive periods of food shortage in climate zones with cold winters. The potential to recruit a part of the abundant WAT storages as beige adipocytes for UCP1-dependent thermogenesis was investigated in vitro by treating raccoon dog adipocytes with different browning inducing factors. In vivo positron emission tomography/computed tomography (PET/CT) imaging with the glucose analog 18F-FDG showed that BAT was not detected in the adult raccoon dog during the winter season. In addition, UCP1 expression was not changed in response to chronic treatments with browning inducing factors in adipocyte cultures. Our results demonstrated that most likely the raccoon dog endures cold weather without the induction of BAT or recruitment of beige adipocytes for heat production. Its thick fur coat, insulating fat, and muscle shivering seem to provide the adequate heat needed for surviving the winter.

Lack of collagen XVIII leads to lipodystrophy and perturbs hepatic glucose and lipid homeostasis.

KEY POINTS: Extracellular matrix is highly remodelled in obesity and associates with the development of metabolic disorders, such as insulin resistance. Previously, we have shown that the lack of specific collagen XVIII isoforms impairs adipocyte differentiation in mice. Here, we show that mice lacking the medium and long isoforms of collagen XVIII develop insulin resistance and glucose intolerance and show elevated serum triglycerides and fat accumulation in the liver. We report that collagen XVIII-deficient mice have increased heat production at low temperatures. These results reveal a new role for collagen XVIII in the regulation of glucose and lipid metabolism, and they expand the understanding of the development of metabolic disorders. ABSTRACT: Liver and adipose tissues play important roles in the regulation of systemic glucose and lipid metabolism. Extracellular matrix synthesis and remodelling are significantly altered in these tissues in obesity and type 2 diabetes. Collagen XVIII is a ubiquitous extracellular matrix component, and it occurs in three isoforms which differ in terms of molecular size, domain structure and tissue distribution. We recently showed that, in mice, the lack of collagen XVIII, and especially its medium and long isoforms, leads to reduced adiposity and dyslipidaemia. To address the metabolic consequences of these intriguing observations, we assessed whole-body glucose homeostasis in mice challenged with a high-fat diet and in normal physiological conditions. We observed that, in the high caloric diet, the overall adiposity was decreased by 30%, serum triglyceride values were threefold higher and the steatotic area in liver was twofold larger in collagen XVIII knockout mice compared with controls. We demonstrated that mice lacking either all three collagen XVIII isoforms, or specifically, the medium and long isoforms develop insulin resistance and glucose intolerance. Furthermore, we found that ablation of collagen XVIII leads to increased heat production in low temperatures and to reduction of the high blood triglyceride levels of the knockout mice to the level of wild-type mice. Our data indicate that collagen XVIII plays a role in the regulation of glucose tolerance, insulin sensitivity and lipid homeostasis, principally through its ability to regulate the expansion of the adipose tissue. These findings advance the understanding of metabolic disorders.

Cross-linking of sodium caseinate-structured emulsion with transglutaminase alters postprandial metabolic and appetite responses in healthy young individuals.

The physico-chemical and interfacial properties of fat emulsions influence lipid digestion and may affect postprandial responses. The aim of the present study was to determine the effects of the modification of the interfacial layer of a fat emulsion by cross-linking on postprandial metabolic and appetite responses. A total of fifteen healthy individuals (26.5 (sem 6.9) years and BMI 21.9 (sem 2.0) kg/m2) participated in a cross-over design experiment in which they consumed two isoenergetic (1924 kJ (460 kcal)) and isovolumic (250 g) emulsions stabilised with either sodium caseinate (Cas) or transglutaminase-cross-linked sodium caseinate (Cas-TG) in a randomised order. Blood samples were collected from the individuals at baseline and for 6 h postprandially for the determination of serum TAG and plasma NEFA, cholecystokinin (CCK), glucagon-like peptide 1 (GLP-1), glucose and insulin responses. Appetite was assessed using visual analogue scales. Postprandial TAG and NEFA responses and gastric emptying (GE) rates were comparable between the emulsions. CCK increased more after the ingestion of Cas-TG than after the ingestion of Cas (P< 0.05), while GLP-1 responses did not differ between the two test emulsions. Glucose and insulin profiles were lower after consuming Cas-TG than after consuming Cas (P< 0.05). The overall insulin, glucose and CCK responses, expressed as areas above/under the curve, did not differ significantly between the Cas and Cas-TG meal conditions. Satiety ratings were reduced and hunger, desire to eat and thirst ratings increased more after the ingestion of Cas-TG than after the ingestion of Cas (P< 0.05). The present results suggest that even a subtle structural modification of the interfacial layer of a fat emulsion can alter the early postprandial profiles of glucose, insulin, CCK, appetite and satiety through decreased protein digestion without affecting significantly on GE or overall lipid digestion.

Effect of Aerobic Exercise and Time-Restricted Feeding on Metabolic Markers and Circadian Rhythm in Mice Fed with the High-Fat Diet.

SCOPE: Diet and exercise are significant players in obesity and metabolic diseases. Time-restricted feeding (tRF) has been shown to improve metabolic responses by regulating circadian clocks but whether it acts synergically with exercise remains unknown. It is hypothesized that forced exercise alone or combined with tRF alleviates obesity and its metabolic complications. METHODS AND RESULTS: Male C57bl6 mice are fed with high-fat or a control diet for 12 weeks either ad libitum or tRF for 10 h during their active period. High-fat diet (HFD)-fed mice are divided into exercise (treadmill for 1 h at 12 m min-1 alternate days for 9 weeks and 16 m min-1 daily for the following 3 weeks) and non-exercise groups. tRF and tRF-Ex significantly decreased body weight, food intake, and plasma lipids, and improved glucose tolerance. However, exercise reduced only body weight and plasma lipids. tRF and tRF-Ex significantly downregulated Fasn, Hmgcr, and Srebp1c, while exercise only Hmgcr. HFD feeding disrupted clock genes, but exercise, tRF, and tRF-Ex coordinated the circadian clock genes Bmal1, Per2, and Rev-Erbα in the liver, adipose tissue, and skeletal muscles. CONCLUSION: HFD feeding disrupted clock genes in the peripheral organs while exercise, tRF, and their combination restored clock genes and improved metabolic consequences induced by high-fat diet feeding.

Are skeletal muscle FNDC5 gene expression and irisin release regulated by exercise and related to health?

Recently, contradictory findings have been reported concerning the function of irisin and its precursor gene, skeletal muscle FNDC5, in energy homeostasis, and the associated regulatory role of exercise and PGC-1α. We therefore evaluated whether muscle FNDC5 mRNA and serum irisin are exercise responsive and whether PGC-1α expression is associated with FNDC5 expression. The male subjects in the study performed single exercises: (1) 1 h low-intensity aerobic exercise (AE) (middle-aged, n = 17), (2) a heavy-intensity resistance exercise (RE) bout (young n = 10, older n = 11) (27 vs. 62 years), (3) long-term 21 weeks endurance exercise (EE) training alone (twice a week, middle-aged, n = 9), or (4) combined EE and RE training (both twice a week, middle-aged, n = 9). Skeletal muscle mRNA expression was analysed by quantitative PCR and serum irisin by ELISA. No significant changes were observed in skeletal muscle PGC-1α, FNDC5 and serum irisin after AE, EE training or combined EE + RE training. However, a single RE bout increased PGC-1α by 4-fold in young and by 2-fold in older men, while FNDC5 mRNA only increased in young men post-RE, by 1.4-fold. Changes in PGC-1α or serum irisin were not consistently accompanied by changes in FNDC5. In conclusion, for the most part, neither longer-term nor single exercise markedly increases skeletal muscle FNDC5 expression or serum irisin. Therefore their changes in response to exercise are probably random and not consistent excluding the confirmation of any definitive link between exercise and FNDC5 expression and irisin release in humans. Moreover, irisin and FNDC5 were not associated with glucose tolerance and being overweight, or with metabolic disturbances, respectively. Finally, factor(s) other than PGC-1α and transcription may regulate FNDC5 expression.

PCSK9 Levels and Metabolic Profiles in Elderly Subjects with Different Glucose Tolerance under Statin Therapy.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) degrades low-density lipoprotein cholesterol (LDL-C) receptors, and thus regulates the LDL-C levels in the circulation. Type 2 diabetics often have elevated LDL-C levels. However, the functions of PCSK9 in patients with alterations of glu-cose metabolism and statin therapy are still unclear. METHOD: we investigated a large cohort of 608 subjects, born in 1945 in Oulu, Finland (Oulu Cohort 1945). We studied the effects of PSCK9 lev-els with different glucose tolerances (normal glucose tolerance (NGT), prediabetes (PreDM) or type 2 diabetes (T2D)) with and without statin medication, and analyzed clinical data, NMR metabolomics and PCSK9 plasma levels. RESULTS: PCSK9 plasma levels did not significantly differ between the three groups. Statin therapy significantly increased the PCSK9 levels in NGT, PreDM and T2D groups compared with subjects with no statins. In the NGT group, negative associations between PCSK9 and LDL-C, intermediate-density lipoprotein cholesterol (IDL-C), very low-density lipoprotein cholesterol (VLDL-C), total cholesterol and LDL and IDL triglycerides were observed under statin medication. In contrast, in the PreDM and T2D groups, these associa-tions were lost. CONCLUSIONS: our data suggest that in subjects with abnormal glucose metabolism and statin therapy, the significant PCSK9-mediated effects on the lipid metabolites are lost com-pared to NGT subjects, but statins reduced the LDL-C and VLDL-C levels.

Adipose gene expression profiles reveal insights into the adaptation of northern Eurasian semi-domestic reindeer (Rangifer tarandus).

Reindeer (Rangifer tarandus) are semi-domesticated animals adapted to the challenging conditions of northern Eurasia. Adipose tissues play a crucial role in northern animals by altering gene expression in their tissues to regulate energy homoeostasis and thermogenic activity. Here, we perform transcriptome profiling by RNA sequencing of adipose tissues from three different anatomical depots: metacarpal (bone marrow), perirenal, and prescapular fat in Finnish and Even reindeer (in Sakha) during spring and winter. A total of 16,212 genes are expressed in our data. Gene expression profiles in metacarpal tissue are distinct from perirenal and prescapular adipose tissues. Notably, metacarpal adipose tissue appears to have a significant role in the regulation of the energy metabolism of reindeer in spring when their nutritional condition is poor after winter. During spring, genes associated with the immune system are upregulated in the perirenal and prescapular adipose tissue. Blood and tissue parameters reflecting general physiological and metabolic status show less seasonal variation in Even reindeer than in Finnish reindeer. This study identifies candidate genes potentially involved in immune response, fat deposition, and energy metabolism and provides new information on the mechanisms by which reindeer adapt to harsh arctic conditions.

HIF-P4H-2 inhibition enhances intestinal fructose metabolism and induces thermogenesis protecting against NAFLD.

Non-alcoholic fatty liver disease (NAFLD) parallels the global obesity epidemic with unmet therapeutic needs. We investigated whether inhibition of hypoxia-inducible factor prolyl 4-hydroxylase-2 (HIF-P4H-2), a key cellular oxygen sensor whose inhibition stabilizes HIF, would protect from NAFLD by subjecting HIF-P4H-2-deficient (Hif-p4h-2gt/gt) mice to a high-fat, high-fructose (HFHF) or high-fat, methionine-choline-deficient (HF-MCD) diet. On both diets, the Hif-p4h-2gt/gt mice gained less weight and had less white adipose tissue (WAT) and its inflammation, lower serum cholesterol levels, and lighter livers with less steatosis and lower serum ALT levels than the wild type (WT). The intake of fructose in majority of the Hif-p4h-2gt/gt tissues, including the liver, was 15-35% less than in the WT. We found upregulation of the key fructose transporter and metabolizing enzyme mRNAs, Slc2a2, Khka, and Khkc, and higher ketohexokinase activity in the Hif-p4h-2gt/gt small intestine relative to the WT, suggesting enhanced metabolism of fructose in the former. On the HF-MCD diet, the Hif-p4h-2gt/gt mice showed more browning of the WAT and increased thermogenesis. A pharmacological pan-HIF-P4H inhibitor protected WT mice on both diets against obesity, metabolic dysfunction, and liver damage. These data suggest that HIF-P4H-2 inhibition could be studied as a novel, comprehensive treatment strategy for NAFLD. KEY MESSAGES: • HIF-P4H-2 inhibition enhances intestinal fructose metabolism protecting the liver. • HIF-P4H-2 inhibition downregulates hepatic lipogenesis. • Induced browning of WAT and increased thermogenesis can also mediate protection. • HIF-P4H-2 inhibition offers a novel, comprehensive treatment strategy for NAFLD.

Association of Physical Activity With Telomere Length Among Elderly Adults - The Oulu Cohort 1945.

Introduction: Physical activity (PA) has been associated with telomere shortening. The association of PA intensity or volume with telomere length (TL) is nonetheless unclear. The aim of our study was to investigate the associations of exercise intensity and volume with TL in elderly adults from Northern Finland (65° latitude North). Methods: Seven hundred elderly subjects born in 1945 in the Oulu region were investigated. PA was measured during a 2-week period with a wrist-worn accelerometer. In addition, a questionnaire was used to assess sedentary time and to achieve a longitudinal PA history and intensity. Relative telomere lengths (RTL) were determined from frozen whole blood samples using a qPCR-based method. Results: Relative telomere lengths were significantly longer in women than men and negatively correlated with age in both genders (men r = -0.210, p = 0.000, women r = -0.174, and p = 0.000). During the 2-week study period, women took more steps than men (p = 0.001), but the association between steps and RTL was only seen in men (p = 0.05). Total steps taken (r = 0.202 and p = 0.04) and sedentary time (r = -0.247 and p = 0.007) significantly correlated with RTLs in 70-year old subjects. Moderate PA was associated with RTL in subjects with the highest quartile of moderate PA compared to the three lower quartiles (p-values: 0.023 between 4th and 1st, 0.04 between 4th and 2nd, and 0.027 between 4th and 3rd) in the 70-year old subjects. Conclusion: Women had longer RTL and a higher step count compared to men. However, exercise volume and RTL correlated positively only in men. Surprisingly, age correlated negatively with RTL already within an age difference of 2 years. This suggests that telomere attrition rate may accelerate in older age. Moderate physical activity at the time of study was associated with RTL.

The Circadian Clock Protein CRY1 Is a Negative Regulator of HIF-1α.

The circadian clock and the hypoxia-signaling pathway are regulated by an integrated interplay of positive and negative feedback limbs that incorporate energy homeostasis and carcinogenesis. We show that the negative circadian regulator CRY1 is also a negative regulator of hypoxia-inducible factor (HIF). Mechanistically, CRY1 interacts with the basic-helix-loop-helix domain of HIF-1α via its tail region. Subsequently, CRY1 reduces HIF-1α half-life and binding of HIFs to target gene promoters. This appeared to be CRY1 specific because genetic disruption of CRY1, but not CRY2, affected the hypoxia response. Furthermore, CRY1 deficiency could induce cellular HIF levels, proliferation, and migration, which could be reversed by CRISPR/Cas9- or short hairpin RNA-mediated HIF knockout. Altogether, our study provides a mechanistic explanation for genetic association studies linking a disruption of the circadian clock with hypoxia-associated processes such as carcinogenesis.

Plasma Orexin-A Levels Do Not Undergo Circadian Rhythm in Young Healthy Male Subjects.

Orexin-A (OXA) has been originally isolated from a precursor peptide prepro-orexin from the lateral hypothalamus. The orexin system has been attributed to important functions in sleep, arousal and regulation of energy homeostasis. In addition to its high levels in cerebrospinal fluid, OXA is present in blood. However, reported peptide concentrations in plasma vary significantly depending on the method used. Therefore, a specific and sensitive OXA radioimmunoassay (RIA) with solid phase extraction method was developed to determine whether plasma OXA concentrations is affected by acute feeding and/or wake and sleep in young healthy males. Blood samples were collected for 24 h from nine healthy males (aged 20-24 years; BMI 20.7-26.5) every 2 h starting at 11 a.m. Food was served at 12 p.m, 5:30 p.m, 8 p.m and 8 a.m and the sleep time was between 10 p.m and 7 a.m. Plasma samples were analyzed in addition for cortisol and melatonin levels. Blood pressure was monitored through the experimental period. OXA antibody was raised in rabbits. OXA antiserum had only minor cross-reactivity with prepro-orexin precursor (<0.001%), amino-terminal peptide (<0.001%), carboxy-terminal peptide (0.001%), and orexin-B (0.3%) with high sensitivity (0.15 pg/tube). Plasma OXA levels varied between 0.5 and 16 pg/ml in seven subjects and were undetectable (below 0.5 pg/ml) in two subjects. The OXA concentrations did not correlate to feeding nor wake/sleep, whereas cortisol, melatonin and mean arterial blood pressure presented a clear circadian rhythm in each subject. In conclusion, OXA is present in blood in low amounts and its levels do not follow autonomic nor neuroendocrine circadian rhythms. Thereby, studies examining regulatory mechanisms and influences of OXA from blood samples should interpret results very cautiously.

A multipurpose vector system for the screening of libraries in bacteria, insect and mammalian cells and expression in vivo.

We have constructed a novel tetra-promoter vector (pBVboostFG) system that enables screening of gene/cDNA libraries for functional genomic studies. The vector enables an all-in-one strategy for gene expression in mammalian, bacterial and insect cells and is also suitable for direct use in vivo. Virus preparation is based on an improved mini Tn7 transpositional system allowing easy and fast production of recombinant baculoviruses with high diversity and negligible background. Cloning of the desired DNA fragments or libraries is based on the recombination system of bacteriophage lambda. As an example of the utility of the vector, genes or cDNAs of 18 different proteins were cloned into pBVboostFG and expressed in different hosts. As a proof-of-principle of using the vector for library screening, a chromophoric Thr65-Tyr-Gly67-stretch of enhanced green fluorescent protein was destroyed and subsequently restored by novel PCR strategy and library screening. The pBVboostFG enables screening of genome-wide libraries, thus making it an efficient new platform technology for functional genomics.

Low level activity thresholds for changes in NMR biomarkers and genes in high risk subjects for Type 2 Diabetes.

Our objectives were to determine if there are quantitative associations between amounts and intensities of physical activities (PA) on NMR biomarkers and changes in skeletal muscle gene expressions in subjects with high risk for type 2 diabetes (T2D) performing a 3-month PA intervention. We found that PA was associated with beneficial biomarker changes in a factor containing several VLDL and HDL subclasses and lipids in principal component analysis (P = <0.01). Division of PA into quartiles demonstrated significant changes in NMR biomarkers in the 2nd - 4th quartiles compared to the 1st quartile representing PA of less than 2850 daily steps (P = 0.0036). Mediation analysis of PA-related reductions in lipoproteins showed that the effects of PA was 4-15 times greater than those of body weight or fat mass reductions. In a subset study in highly active subjects' gene expressions of oxidative fiber markers, Apo D, and G0/G1 Switch Gene 2, controlling insulin signaling and glucose metabolism were significantly increased. Slow walking at speeds of 2-3 km/h exceeding 2895 steps/day attenuated several circulating lipoprotein lipids. The effects were mediated rather by PA than body weight or fat loss. Thus, lower thresholds for PA may exist for long term prevention of cardio-metabolic diseases in sedentary overweight subjects.

The effects of group and single housing and automated animal monitoring on urinary corticosterone levels in male C57BL/6 mice.

Mice are used extensively in physiological research. Automated home-cage systems have been developed to study single-housed animals. Increased stress by different housing conditions might affect greatly the results when investigating metabolic responses. Urinary corticosteroid concentration is considered as a stress marker. The aim of the study was to compare the effects of different housing conditions and an automated home-cage system with indirect calorimetry located in an environmental chamber on corticosterone levels in mice. Male mice were housed in different conditions and in automated home-cage system to evaluate the effects of housing and measuring conditions on urine corticosterone levels. Corticosterone levels in single-housed mice in the laboratory animal center were consistently lower compared with the group-housed mice. Single-housed mice in a separate, small animal unit showed a rise in their corticosterone levels a day after they were separated to their individual cages, which decreased during the following 2 days. The corticosterone levels of group-housed mice in the same unit were increased during the first 7 days and then decreased. On day 7, the corticosterone concentrations of group-housed mice were significantly higher compared with that of single-housed mice, including the metabolic measurement protocol. In conclusion, single housing caused less stress when compared with group-housed mice. In addition, the urine corticosterone levels were decreased in single-housed mice before the metabolic measurement started. Thus, stress does not affect the results when utilizing the automated system for measuring metabolic parameters like food and water intake and calorimetry.

Fat mass- and obesity-associated gene Fto affects the dietary response in mouse white adipose tissue.

Common variants of human fat mass- and obesity-associated gene Fto have been linked with higher body mass index, but the biological explanation for the link has remained obscure. Recent findings suggest that these variants affect the homeobox protein IRX3. Here we report that FTO has a role in white adipose tissue which modifies its response to high-fat feeding. Wild type and Fto-deficient mice were exposed to standard or high-fat diet for 16 weeks after which metabolism, behavior and white adipose tissue morphology were analyzed together with adipokine levels and relative expression of genes regulating white adipose tissue adipogenesis and Irx3. Our results indicate that Fto deficiency increases the expression of genes related to adipogenesis preventing adipocytes from becoming hypertrophic after high-fat diet. In addition, we report a novel finding of increased Irx3 expression in Fto-deficient mice after high-fat feeding indicating a complex link between FTO, IRX3 and fat metabolism.

HIF prolyl 4-hydroxylase-2 inhibition improves glucose and lipid metabolism and protects against obesity and metabolic dysfunction.

Obesity is a major public health problem, predisposing subjects to metabolic syndrome, type 2 diabetes, and cardiovascular diseases. Specific prolyl 4-hydroxylases (P4Hs) regulate the stability of the hypoxia-inducible factor (HIF), a potent governor of metabolism, with isoenzyme 2 being the main regulator. We investigated whether HIF-P4H-2 inhibition could be used to treat obesity and its consequences. Hif-p4h-2-deficient mice, whether fed normal chow or a high-fat diet, had less adipose tissue, smaller adipocytes, and less adipose tissue inflammation than their littermates. They also had improved glucose tolerance and insulin sensitivity. Furthermore, the mRNA levels of the HIF-1 targets glucose transporters, glycolytic enzymes, and pyruvate dehydrogenase kinase-1 were increased in their tissues, whereas acetyl-CoA concentration was decreased. The hepatic mRNA level of the HIF-2 target insulin receptor substrate-2 was higher, whereas that of two key enzymes of fatty acid synthesis was lower. Serum cholesterol levels and de novo lipid synthesis were decreased, and the mice were protected against hepatic steatosis. Oral administration of an HIF-P4H inhibitor, FG-4497, to wild-type mice with metabolic dysfunction phenocopied these beneficial effects. HIF-P4H-2 inhibition may be a novel therapy that not only protects against the development of obesity and its consequences but also reverses these conditions.