Determination of mechanical and rheological properties of a cell-loaded peptide gel during ECM production.

The development of an injectable biomaterial that supports cell survival and maintains or promotes nucleus pulposus (NP) phenotype could aid delivery of cells to degenerated NPs causing low back pain. Mesenchymal cells were loaded and grown in a synthetic peptide gel, PuraMatrix®. Cells were observed within the gels over 0-28 days, and accumulation of glycosaminoglycans were detected by histological staining. The mechanical properties of the cell-loaded constructs, and the change of the mechanical properties were studied using stress relaxation of the gels under compression and confinement. The PuraMatrix® gel was shown to relax fast on compression indicating that the fluid could easily flow out of the gel, and thus indicating the presence of large pores/voids. The presence of these pores/voids was further supported by high mobility of dextran molecules, determined using fluorescence recovery after photo bleaching. The stress required to deform the cell-loaded constructs to a specific strain increases at day 21, at which point the presence of glycosaminoglycans within the cell-loaded constructs was also observed. The results provide evidence of changes in mechanical properties of the PuraMatrix® matrix upon excretion of the extracellular matrix by the cells.

Fat and fat-free mass of healthy Swedish children show tracking during early life, but there are differences.

AIM: Obesity may start early in life. We investigated relationships between size and body composition variables in infancy and at 4 years of age using valid estimates of body composition. The results were compared to those obtained when body mass index (BMI) was used to estimate body fatness at 4 years. METHODS: Using air displacement plethysmography, size, fat mass and fat-free mass were studied, between 2007 and 2015, in 253 full-term healthy Swedish children at 1 week, 12 weeks and 4 years of age. RESULTS: Positive associations between variables in infancy and at 4 years were found at 1 and 12 weeks for weight, height, BMI, fat-free mass and fat-free mass index (p ≤ 0.002) and for fat mass, per cent body fat and fat mass index (p ≤ 0.04) at 12 weeks. Fat mass gained during infancy correlated positively (p ≤ 0.031) with per cent fat mass, fat mass index and BMI, all at 4 years. In girls, gains in fat-free mass during infancy correlated with BMI (p = 0.0005) at 4 years. CONCLUSION: The results provide information regarding body composition trajectories during early life and demonstrate limitations of BMI as a proxy for body fatness when relating early weight gain to variables, relevant for later obesity risk.

Correction to: Compressive loading of the murine tibia reveals site-specific micro-scale differences in adaptation and maturation rates of bone.

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Correlates of ideal cardiovascular health in European adolescents: The HELENA study.

BACKGROUND AND AIMS: The ideal cardiovascular health (iCVH) construct consists of 4 health behaviors (smoking status, body mass index, physical activity and diet) and 3 health factors (total cholesterol, blood pressure and fasting glucose). A greater number of iCVH components in adolescence are related to better cardiovascular health, but little is known about the correlates of iCVH in adolescents. Thus, the aim of the study was to examine correlates of iCVH in European adolescents. METHODS AND RESULTS: The study comprised 637 European adolescents with complete iCVH data. Participants were part of the Healthy Lifestyle in Europe by Nutrition in Adolescence (HELENA) study, a cross-sectional, multicenter study conducted in 9 different European countries. Correlates investigated were sex and age, family affluence scale, maternal education, geographic location, sleep time, television viewing, duration of pregnancy, birth weight and breastfeeding. Younger adolescents, those whose mothers had medium/high education or those who watched television less than 2 h per day had a greater number of iCVH components compared to those who were older, had a mother with low education or watched television 2 h or more daily (P ≤ 0.01). CONCLUSION: Since in our study older adolescents had worse iCVH than younger adolescents, early promotion of cardiovascular health may be important. Future studies may also investigate the usefulness of limiting television viewing to promote iCVH. Finally, since adolescents of mothers with low education had poorer iCVH, it may be of special interest to tailor public health promotion to adolescents from families with low socioeconomic status.

Longitudinal assessment of body composition in healthy Swedish children from 1 week until 4 years of age.

BACKGROUND/OBJECTIVES: Knowledge of longitudinal body composition development is required to identify the mechanisms behind childhood overweight and obesity and to prevent these conditions. However, accurate data on this development in early childhood are lacking. Our aim was to describe the longitudinal body composition development in healthy young Swedish children. SUBJECTS/METHODS: Body size and composition were assessed in 26 children using air-displacement plethysmography (1 and 12 weeks and 4.4 years of age) and isotope dilution (1.5 and 3 years of age) and compared with available reference data. RESULTS: Body fat (%) for boys (n=16) was 12.8±3.9 (1 week), 25.6±4.8 (12 weeks), 28.2±3.8 (1.5 years), 27.3±5.1 (3 years) and 26.1±3.5 (4.4 years). For girls (n=10) these values were 15.3±2.9, 25.7±3.9, 27.9±3.3, 26.3±7.2 and 26.0±5.3, respectively. These values were above the Fomon reference values at 1.5 years of age and later and higher than the Butte reference (P<0.05) for boys at 1.5 years of age. At all ages the coefficients of variation were higher for body fat (%) (12-30%) than for BMI (4-11%). CONCLUSIONS: At 4 years of age our children had more body fat than indicated by reference data. This high level may have already been established at 1.5 years of age but our small sample and the lack of appropriate reference data limit the possibility of drawing firm conclusions. Our results demonstrate the limitations of BMI when investigating overweight and obesity in early life and highlight the need for appropriate reference body composition data in infants and young children.European Journal of Clinical Nutrition advance online publication, 23 August 2017; doi:10.1038/ejcn.2017.125.

Parental body mass index and its association with body composition, physical fitness and lifestyle factors in their 4-year-old children: results from the MINISTOP trial.

BACKGROUND/OBJECTIVES: To examine the association between parental body mass index (BMI) and their offspring's body composition, physical fitness and lifestyle factors (that is, sedentary time, physical activity and diet). SUBJECTS/METHODS: A total of 307 preschoolers (4.5±0.1 years) and their parents (fathers: 38.1±5.1 years and mothers: 35.6±4.2 years) participated in this study. Parental BMI was calculated using self-reported weight and height. Preschoolers body composition was assessed using: BMI, fat mass percentage, fat mass index, fat-free mass index (measured via air-displacement plethysmography) and waist circumference. Physical fitness was assessed by the PREFIT fitness battery. Lifestyle factors were assessed using the ActiGraph wGT3x-BT (sedentary time and physical activity), and the mobile-phone based tool for energy balance in children (diet). RESULTS: Parental BMI were positively associated with their offspring's BMI (paternal BMI: standardised beta, ß=0.233, P<0.001; maternal BMI: ß=0.186, P=0.001), fat mass index (paternal BMI: ß=0.130, P=0.026; maternal BMI: ß=0.163, P=0.005), fat-free mass index (paternal BMI: ß=0.214, P<0.001; maternal BMI: ß=0.119, P=0.036) and waist circumference (paternal BMI: ß=0.178, P=0.001; maternal BMI: ß=0.179, P=0.001). A negative association was found between maternal BMI and their offspring's standing long jump test (ß=-0.132, P=0.022). Paternal BMI was associated with their offspring's sedentary time (ß=0.100, P=0.026), whereas parental BMI was not associated with neither physical activity nor diet (all P⩾0.104). CONCLUSIONS: Parental BMI was positively associated with their offspring's BMI, fat as well as fat-free mass index and waist circumference. Moreover, a higher paternal and maternal BMI were related to higher levels of sedentary time and a lower performance in the standing long jump test of their offspring, respectively.

Seasonal patterns in self-reported peripartum depressive symptoms.

BACKGROUND: In the peripartum period, the literature on seasonality in depression is still scarce and studies present varying findings. The aims of this study were to investigate whether seasonal patterns in postpartum depressive symptoms previously identified in a Swedish study could be replicated in a larger study, as well as to assess seasonal patterns in depressive symptoms during pregnancy. METHODS: This was a nested case-control study comprised of 4129 women who participated in the BASIC project and gave birth at Uppsala University Hospital, Uppsala, Sweden, between February 2010 and December 2015. RESULTS: Women who gave birth in October-December 2011 had an increased odds of depressive symptoms at 6 weeks postpartum, when compared with women giving birth in April-June 2011 (aOR=2.42; 95% CI: 1.12-5.26). The same pattern was found among women with a history of depression. No other seasonal patterns for depressive symptoms during pregnancy or at 6 weeks postpartum were identified. CONCLUSIONS: In general, no consistent seasonal patterns were found in peripartum depressive symptoms. Whether the seasonal patterns found in some studies during certain years may be due to other factors relating to specific years and seasons, such as extreme climatic conditions or other particular events, warrants further investigation.

Compressive loading of the murine tibia reveals site-specific micro-scale differences in adaptation and maturation rates of bone.

Loading increases bone mass and strength in a site-specific manner; however, possible effects of loading on bone matrix composition have not been evaluated. Site-specific structural and material properties of mouse bone were analyzed on the macro- and micro/molecular scale in the presence and absence of axial loading. The response of bone to load is heterogeneous, adapting at molecular, micro-, and macro-levels. INTRODUCTION: Osteoporosis is a degenerative disease resulting in reduced bone mineral density, structure, and strength. The overall aim was to explore the hypothesis that changes in loading environment result in site-specific adaptations at molecular/micro- and macro-scale in mouse bone. METHODS: Right tibiae of adult mice were subjected to well-defined cyclic axial loading for 2 weeks; left tibiae were used as physiologically loaded controls. The bones were analyzed with µCT (structure), reference point indentation (material properties), Raman spectroscopy (chemical), and small-angle X-ray scattering (mineral crystallization and structure). RESULTS: The cranial and caudal sites of tibiae are structurally and biochemically different within control bones. In response to loading, cranial and caudal sites increase in cortical thickness with reduced mineralization (-14 and -3%, p < 0.01, respectively) and crystallinity (-1.4 and -0.3%, p < 0.05, respectively). Along the length of the loaded bones, collagen content becomes more heterogeneous on the caudal site and the mineral/collagen increases distally at both sites. CONCLUSION: Bone structure and composition are heterogeneous, finely tuned, adaptive, and site-specifically responsive at the micro-scale to maintain optimal function. Manipulation of this heterogeneity may affect bone strength, relative to specific applied loads.

The direction of human mesenchymal stem cells into the chondrogenic lineage is influenced by the features of hydrogel carriers.

Low back pain is a major public health issue in the Western world, one main cause is believed to be intervertebral disc (IVD) degeneration. To halt/diminish IVD degeneration, cell therapy using different biomaterials e.g. hydrogels as cell carriers has been suggested. In this study, two different hydrogels were examined (in vitro) as potential cell carriers for human mesenchymal stem cells (hMSCs) intended for IVD transplantation. The aim was to investigate cell-survival and chondrogenic differentiation of hMSCs when cultured in hydrogels Puramatrix® or Hydromatrix® and potential effects of stimulation with growth hormone (GH). hMSCs/hydrogel cultures were investigated for cell-viability, attachment, gene expression of chondrogenic markers SOX9, COL2A1, ACAN and accumulation of extracellular matrix (ECM). In both hydrogel types, hMSCs were viable for 28days, expressed integrin ß1 which indicates adhesion of hMSCs. Differentiation was observed into chondrocyte-like cells, in a higher extent in hMSCs/Hydromatrix® cultures when compared to hMSCs/Puramatrix® hydrogel cultures. Gene expression analyses of chondrogenic markers verified results. hMSCs/hydrogel cultures stimulated with GH displayed no significant effects on chondrogenesis. In conclusion, both hydrogels, especially Hydromatrix® was demonstrated as a promising cell carrier in vitro for hMSCs, when directed into chondrogenesis. This knowledge could be useful in biological approaches for regeneration of degenerated human IVDs.

Indications of that migration of stem cells is influenced by the extra cellular matrix architecture in the mammalian intervertebral disk region.

Disk-degeneration is believed a major cause for lumbar pain. Previously, potential stem cell niches in the intervertebral disk (IVD) region, located adjacent to epiphyseal plate, was reported. The aim of the study was to examine migration of mesenchymal stem cells (MSCs), extracellular matrix (ECM) architecture in a potential cellular migration route (CMR; area located between the niche and IVD) and in the IVD in non-degenerated lapine- and in human degenerated IVD tissues. Human MSCs (n=3), human degenerated IVD tissues (n=10) and lapine IVDs (n=10) were collected. The samples were examined by immunohistochemistry for stem cell markers; CD90, OCT3/4, pre-chondrocytic marker; GDF5, catabolic markers; MMP9, MMP13, inflammatory marker; IL1R, cellular migration markers; SNAI1, SNAI2, adhesion markers; ß1-INTEGRIN and DDR2. In addition, gene-expression analyses (Real time PCR) were performed on additional samples. Further, time lapse studies were performed with hMSCs cultured on aligned COLL-I-fibers-coated glass-slides in DMEM-LG, 10% human serum containing fibroblast growth factor (bFGF). Presence of stem cells (CD90+, OCT3/4+), pre-chondocytic cells (GDF5+) and cells positive for migration markers (SNAI1+, SNAI2+), catabolic markers (MMP9+, MMP13+), inflammatory marker (IL1R+), adhesion markers (DDR2+, B1-INTEGRIN+) were detected (gene- and protein level) in investigated CMR and IVD regions. In the time lapse studies, MSCs alignment and protrusions were observed orientated in the same direction as collagen fibers. Results display influence of ECM collagen architecture and collagen fiber spatial direction on migration of stem cells. The results can be useful when developing tissue-engineering strategies for disk-degeneration.

Moderate physical exercise results in increased cell activity in articular cartilage of the knee joint in rats.

BACKGROUND: Moderate exercise regimens have shown minor positive effects on matrix turnover in articular cartilage (AC), while effects at cellular level, e.g. proliferation, are scarcely described. AIM: The aim of this study was to investigate the effects of moderate exercise on cell proliferation and recruitment of cells possibly active in regeneration in different regions of cartilage in the rat knee joint. METHODS: Eighteen rats were orally given 5-bromo-2-deoxyuridine (BrdU) for 14 days for in vivo DNA labeling. Nine rats underwent treadmill training for 50 min/day, 5 days/week (exercise group), and 9 rats served as controls (no exercise). Animals were sacrificed after 14, 56 and 105 days, and knee joints were harvested. BrdU+ cells were visualized immunohistochemically (IHC) and counted in AC, posterior stem cell niche (PN), potential migration route (PMR; area between PN and the AC border), potential migration area (PMA; region between PN and AC including PN) and epiphyseal cartilage plate (EP) of the tibia and femur. RESULTS: Compared to controls, in the exercise group BrdU+ cells/mm(2) were increased on days 14 (p = 0.022) and 105 (p = 0.045) in AC of the tibia and on day 105 (p = 0.014) in AC of the femur. BrdU+ cell numbers were increased in the PMR region of the tibia on days 14 (p = 0.023) and 105 (p = 0.0018) and in the PMR region of the femur on day 105 (p = 0.0099) as well as in the PMA region of the tibia (p = 0.0008) and femur (p = 0.0080) on day 105. No significant differences in BrdU+ cells/mm(2) were seen in PN or EP between the groups at any time point. Regarding collagen 2A1 expression and proteoglycan accumulation, no significant differences between groups were detected. CONCLUSIONS: The results indicate increased cell activity in AC in response to physical exercise and may help to understand the complexity of AC regeneration in the normal mammal knee joint.

Biocompatibility and resorption of a radiopaque premixed calcium phosphate cement.

Calcium phosphate cements (CPC) are used as bone void filler in various orthopedic indications; however, there are some major drawbacks regarding mixing, transfer, and injection of traditional CPC. By using glycerol as mixing liquid, a premixed calcium phosphate cement (pCPC), some of these difficulties can be overcome. In the treatment of vertebral fractures the handling characteristics need to be excellent including a high radio-opacity for optimal control during injection. The aim of this study is to evaluate a radiopaque pCPC regarding its resorption behavior and biocompatibility in vivo. pCPC and a water-based CPC were injected into a Ø 4-mm drilled femur defect in rabbits. The rabbits were sacrificed after 2 and 12 weeks. Cross sections of the defects were evaluated using histology, electron microscopy, and immunohistochemical analysis. Signs of inflammation were evaluated both locally and systemically. The results showed a higher bone formation in the pCPC compared to the water-based CPC after 2 weeks by expression of RUNX-2. After 12 weeks most of the cement had been resorbed in both groups. Both materials were considered to have a high biocompatibility since no marked immunological response was induced and extensive bone ingrowth was observed. The conclusion from the study was that pCPC with ZrO(2) radiopacifier is a promising alternative regarding bone replacement material and may be suggested for treatment of, for example, vertebral fractures based on its high biocompatibility, fast bone ingrowth, and good handling properties.

Investigation of different cell types and gel carriers for cell-based intervertebral disc therapy, in vitro and in vivo studies.

Biological treatment options for the repair of intervertebral disc damage have been suggested for patients with chronic low back pain. The aim of this study was to investigate possible cell types and gel carriers for use in the regenerative treatment of degenerative intervertebral discs (IVD). In vitro: human mesenchymal cells (hMSCs), IVD cells (hDCs), and chondrocytes (hCs) were cultivated in three gel types: hyaluronan gel (Durolane®), hydrogel (Puramatrix®), and tissue-glue gel (TISSEEL®) in chondrogenic differentiation media for 9 days. Cell proliferation and proteoglycan accumulation were evaluated with microscopy and histology. In vivo: hMSCs or hCs and hyaluronan gel were co-injected into injured IVDs of six minipigs. Animals were sacrificed at 3 or 6 months. Transplanted cells were traced with anti-human antibodies. IVD appearance was visualized by MRI, immunohistochemistry, and histology. Hyaluronan gel induced the highest cell proliferation in vitro for all cell types. Xenotransplanted hMSCs and hCs survived in porcine IVDs for 6 months and produced collagen II in all six animals. Six months after transplantation of cell/gel, pronounced endplate changes indicating severe IVD degeneration were observed at MRI in 1/3 hC/gel, 1/3 hMSCs/gel and 1/3 gel only injected IVDs at MRI and 1/3 hMSC/gel, 3/3 hC/gel, 2/3 gel and 1/3 injured IVDs showed positive staining for bone mineralization. In 1 of 3 discs receiving hC/gel, in 1 of 3 receiving hMSCs/gel, and in 1 of 3 discs receiving gel alone. Injected IVDs on MRI results in 1 of 3 hMSC/gel, in 3 of 3 hC/gel, in 2 of 3 gel, and in 1 of 3 injured IVDs animals showed positive staining for bone mineralization. The investigated hyaluronan gel carrier is not suitable for use in cell therapy of injured/degenerated IVDs. The high cell proliferation observed in vitro in the hyaluronan could have been a negative factor in vivo, since most cell/gel transplanted IVDs showed degenerative changes at MRI and positive bone mineralization staining. However, this xenotransplantation model is valuable for evaluating possible cell therapy strategies for human degenerated IVDs.

Nitric oxide increases cyclic GMP levels, AMP-activated protein kinase (AMPK)alpha1-specific activity and glucose transport in human skeletal muscle.

AIMS/HYPOTHESIS: We investigated the direct effect of a nitric oxide donor (spermine NONOate) on glucose transport in isolated human skeletal muscle and L6 skeletal muscle cells. We hypothesised that pharmacological treatment of human skeletal muscle with N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine (spermine NONOate) would increase intracellular cyclic GMP (cGMP) levels and promote glucose transport. METHODS: Skeletal muscle strips were prepared from vastus lateralis muscle biopsies obtained from seven healthy men. Muscle strips were incubated in the absence or presence of 5 mmol/l spermine NONOate or 120 nmol/l insulin. The L6 muscle cells were treated with spermine NONOate (20 micromol/l) and incubated in the absence or presence of insulin (120 nmol/l). The direct effect of spermine NONOate and insulin on glucose transport, cGMP levels and signal transduction was determined. RESULTS: In human skeletal muscle, spermine NONOate increased glucose transport 2.4-fold (p < 0.05), concomitant with increased cGMP levels (80-fold, p < 0.001). Phosphorylation of components of the canonical insulin signalling cascade was unaltered by spermine NONOate exposure, implicating an insulin-independent signalling mechanism. Consistent with this, spermine NONOate increased AMP-activated protein kinase (AMPK)-alpha1-associated activity (1.7-fold, p < 0.05). In L6 muscle cells, spermine NONOate increased glucose uptake (p < 0.01) and glycogen synthesis (p < 0.001), an effect that was in addition to that of insulin. Spermine NONOate also elicited a concomitant increase in AMPK and acetyl-CoA carboxylase phosphorylation. In the presence of the guanylate cyclase inhibitor LY-83583 (10 micromol/l), spermine NONOate had no effect on glycogen synthesis and AMPK-alpha1 phosphorylation. CONCLUSIONS/INTERPRETATION: Pharmacological treatment of skeletal muscle with spermine NONOate increases glucose transport via insulin-independent signalling pathways involving increased intracellular cGMP levels and AMPK-alpha1-associated activity.

Premixed acidic calcium phosphate cement: characterization of strength and microstructure.

By using a premixed calcium phosphate cement (CPC), the handling properties of the cement are drastically improved, which is a challenge for traditional injectable CPCs. Previously premixed cements have been based on apatitic cements. In this article, acidic cement has been developed and evaluated. Monocalcium phosphate monohydrate and beta-tricalcium phosphate were mixed with glycerol to form a paste. As the paste does not contain water, no setting reaction starts and thus the working time is indefinite. Powder/liquid ratios (P/L) of 2.25, 3.5 and 4.75 were evaluated. Setting time (ST) and compressive strength (CS) were measured after 1 day, 1 week and 4 weeks in phosphate buffered saline (PBS) solution, and the corresponding microstructure was evaluated using electron microscopy and X-ray diffraction. The ST started when the cements were placed in PBS and ranged from 28 to 75 min, higher P/L gave a lower ST. Higher P/L also gave a higher CS, which ranged from 2 to 16 MPa. The microstructure mainly consisted of monetite, 1-5 microm in grain size. After 4 weeks in PBS, the strength increased. As acidic cements are resorbed faster in vivo, this cement should allow faster bone regeneration than apatitic cements. Premixed cements show a great handling benefit when compared with normal CPCs and can be formulated with similar ST and mechanical properties.

Human disk cells from degenerated disks and mesenchymal stem cells in co-culture result in increased matrix production.

Transplantation of mesenchymal stem cells (MSCs) has been suggested for disk degeneration, which is characterized by dysfunctional cells and low proteoglycan production. The aim of this study was to examine the effects of a 3D co-culture system using human disk cells (DCs) and MSCs on collagen and proteoglycan production. DCs and MSCs were expanded in monolayer and grown in pellet cultures for 7, 14 and 28 days and analyzed for hydroxyproline (HP), reflecting total collagen production, and glycosaminoglycan (GAG) accumulation. DCs and MSCs co-cultured at different ratios (25/75, 50/50 and 75%/25%) were examined for GAG accumulation. Collagen type II expression was analyzed immunohistochemically. In a second series, conditioned media were added to pellet cultures of degenerated DCs or MSCs. DCs from degenerated disks and MSCs demonstrated lower total collagen production than non-degenerated DC pellets. GAG production was comparable in DCs and MSCs, except in the youngest donor, with MSC producing about 10 times higher GAG/DNA. Co-cultures resulted in approximately 1.5 times higher GAG/DNA production than DCs. Increased collagen type II expression was seen in co-cultures compared to DC or MSC culture alone, except in the case with highly active MSCs. No positive effect of conditioned media was seen. In conclusion, co-culture of MSCs with degenerated DCs increased proteoglycan and collagen-type ceII production, indicating that in future clinical therapy MSCs can be transplanted without pre-differentiation in vitro. The lack of effect of conditioned media suggests that the positive effect of co-culture on matrix production is not due to soluble factors.

Role of AMP-activated protein kinase in the coordinated expression of genes controlling glucose and lipid metabolism in mouse white skeletal muscle.

AIMS/HYPOTHESIS: AMP-activated protein kinase (AMPK) regulates metabolic adaptations in skeletal muscle. The aim of this study was to investigate whether AMPK modulates the expression of skeletal muscle genes that have been implicated in lipid and glucose metabolism under fed or fasting conditions. METHODS: Two genetically modified animal models were used: AMPK gamma3 subunit knockout mice (Prkag3(-/-)) and skeletal muscle-specific transgenic mice (Tg-Prkag3(225Q)) that express a mutant (R225Q) gamma3 subunit. Levels of mRNA transcripts of genes involved in lipid and glucose metabolism in white gastrocnemius muscles of these mice (under fed or 16-h fasting conditions) were assessed by quantitative real-time PCR. RESULTS: Wild-type mice displayed a coordinated increase in the transcription of skeletal muscle genes encoding proteins involved in lipid/oxidative metabolism (lipoprotein lipase, fatty acid transporter, carnitine palmitoyl transferase-1 and citrate synthase) and glucose metabolism (glycogen synthase and lactate dehydrogenase) in response to fasting. In contrast, these fasting-induced responses were impaired in Prkag3(-/-) mice. The transcription of genes involved in lipid and oxidative metabolism was increased in the skeletal muscle of Tg-Prkag3(225Q) mice compared with that in wild-type mice. Moreover, the expression of the genes encoding hexokinase II and 6-phosphofrucktokinase was decreased in Tg-Prkag3(225Q) mice after fasting. CONCLUSIONS/INTERPRETATION: AMPK is involved in the coordinated transcription of genes critical for lipid and glucose metabolism in white glycolytic skeletal muscle.

5-Aminoimidazole-4-carboxamide ribonucleoside treatment improves glucose homeostasis in insulin-resistant diabetic (ob/ob) mice.

AIMS/HYPOTHESIS: The 5'AMP-activated protein kinase is an important mediator of muscle contraction-induced glucose transport and a target for pharmacological treatment of Type II (non-insulin-dependent) diabetes mellitus. The 5'AMP-activated protein kinase can be activated by 5-aminoimidazole-4-carboxamide ribonucleoside. We hypothesised that 5-aminoimidazole-4-carboxamide ribonucleoside treatment could restore glucose homeostasis in ob/ob mice. METHODS: Lean and ob/ob mice were given 5-aminoimidazole-4-carboxamide ribonucleoside (1 mg.g body wt(-1).day(-1) s.c) or 0.9 % NaCl (vehicle) for 1-7 days. RESULTS: Short-term 5-aminoimidazole-4-carboxamide ribonucleoside treatment normalised glucose concentrations in ob/ob mice within 1 h, with effects persisting over 4 h. After 1 week of daily injections, 5-aminoimidazole-4-carboxamide ribonucleoside treatment corrected hyperglycaemia, improved glucose tolerance, and increased GLUT4 and hexokinase II protein expression in skeletal muscle, but had deleterious effects on plasma non-esterified fatty acids and triglycerides. Treatment with 5-aminoimidazole-4-carboxamide ribonucleoside increased liver glycogen in fasted and fed ob/ob mice and muscle glycogen in fasted, but not fed ob/ob and lean mice. Defects in insulin-stimulated phosphatidylinositol 3-kinase and glucose transport in skeletal muscle from ob/ob mice were not corrected by 5-aminoimidazole-4-carboxamide ribonucleoside treatment. While ex vivo insulin-stimulated glucose transport was reduced in isolated muscle from ob/ob mice, the 5-aminoimidazole-4-carboxamide ribonucleoside stimulated response was normal. CONCLUSION/INTERPRETATION: The 5-aminoimidazole-4-carboxamide ribonucleoside mediated improvements in glucose homeostasis in ob/ob mice can be explained by effects in skeletal muscle and liver. Due to the apparently deleterious effects of 5-aminoimidazole-4-carboxamide ribonucleoside on the blood lipid profile, strategies to develop tissue-specific and pathway-specific activators of 5'AMP-activated protein kinase should be considered in order to improve glucose homeostasis.

Family 7 cellobiohydrolases from Phanerochaete chrysosporium: crystal structure of the catalytic module of Cel7D (CBH58) at 1.32 A resolution and homology models of the isozymes.

Cellobiohydrolase 58 (Cel7D) is the major cellulase produced by the white-rot fungus Phanerochaete chrysosporium, constituting approximately 10 % of the total secreted protein in liquid culture on cellulose. The enzyme is classified into family 7 of the glycosyl hydrolases, together with cellobiohydrolase I (Cel7A) and endoglucanase I (Cel7B) from Trichoderma reesei. Like those enzymes, it catalyses cellulose hydrolysis with net retention of the anomeric carbon configuration. The structure of the catalytic module (431 residues) of Cel7D was determined at 3.0 A resolution using the structure of Cel7A from T. reesei as a search model in molecular replacement, and ultimately refined at 1.32 A resolution. The core structure is a beta-sandwich composed of two large and mainly antiparallel beta-sheets packed onto each other. A long cellulose-binding groove is formed by loops on one face of the sandwich. The catalytic residues are conserved and the mechanism is expected to be the same as for other family members. The Phanerochaete Cel7D binding site is more open than that of the T. reesei cellobiohydrolase, as a result of deletions and other changes in the loop regions, which may explain observed differences in catalytic properties. The binding site is not, however, as open as the groove of the corresponding endoglucanase. A tyrosine residue at the entrance of the tunnel may be part of an additional subsite not present in the T. reesei cellobiohydrolase. The Cel7D structure was used to model the products of the five other family 7 genes found in P. chrysosporium. The results suggest that at least two of these will have differences in specificity and possibly catalytic mechanism, thus offering some explanation for the presence of Cel7 isozymes in this species, which are differentially expressed in response to various growth conditions.