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CD163, a scavenger receptor with anti-inflammatory function expressed exclusively on monocytes/macrophages, is dysregulated in cases of diabetes complications. This study aimed to characterize circulating CD163+ monocytes in the presence (D+Comps) or absence (D−Comps) of diabetes-related complications. RNA-sequencing and mass cytometry were conducted on CD163+ monocytes in adults with long-duration diabetes and D+Comps or D−Comps. Out of 10,868 differentially expressed genes identified between D+Comps and D−Comps, 885 were up-regulated and 190 were down-regulated with a ≥ 1.5-fold change. In D+Comps, 'regulation of centrosome cycle' genes were enriched 6.7-fold compared to the reference genome. MIR27A, MIR3648-1, and MIR23A, the most up-regulated and CD200R1, the most down-regulated gene, were detected in D+Comps from the list of 75 'genes of interest'. CD163+ monocytes in D+Comps had a low proportion of recruitment markers CCR5, CD11b, CD11c, CD31, and immune regulation markers CD39 and CD86. A gene–protein network identified down-regulated TLR4 and CD11b as 'hub-nodes'. In conclusion, this study reports novel insights into CD163+ monocyte dysregulation in diabetes-related complications. Enriched centrosome cycle genes and up-regulated miRNAs linked to apoptosis, coupled with down-regulated monocyte activation, recruitment, and immune regulation, suggest functionally distinct CD163+ monocytes in cases of diabetes complications. Further investigation is needed to confirm their role in diabetes-related tissue damage. [ABSTRACT FROM AUTHOR]

Research has described that adiponectin plays a key role in cardiomyocytes metabolism, however, the effects of exercise during obesity on cardiac adiponectin levels is unclear. To investigate the effects of constant-moderate endurance (END) and high-intensity interval training (HIIT), on heart adiponectin levels in mice. Two experiments were conducted: (1) preventive (EX1): 10 week-old male mice were fed standard (CHOW) or high-fat diet (HFD;45% fat) and simultaneously trained with END and HIIT for 10 weeks; (2) Treatment (EX2): after 10 weeks of dietary intervention, another cohort of 10 week-old mice were trained by both programmes for 10 weeks. In EX1, END and HIIT decreased low-molecular weight adiponectin (∼0.5-fold; p < 0.05) and increased GLUT4 levels (∼2-fold; p <.05). In EX2, HFD significantly decreased high-molecular weight adiponectin (∼0.7-fold; p <.05), and END reversed this change. Discussion and conclusion: HFD and exercise influence heart adiponectin isoforms and therefore might impact cardiomyocyte metabolism. [ABSTRACT FROM AUTHOR]

Transforming growth factor beta (TGFβ) is a versatile cytokine. Although a profibrotic role of TGFβ is well established, its effect on tissue inhibitor of metalloproteinase (TIMPs) and inflammatory mediators are incompletely described. This study investigates the profibrotic and pro-inflammatory role of TGFβ1 during adipocyte differentiation. NIH3T3L1 cells were used for the in vitro study and were differentiated by adding a standard differentiation mix either with rosiglitazone (R-Diff) or without (S-Diff). Recombinant TGFβ1 (2 ng/mL) was added to the undifferentiated preadipocyte during the commitment stage and at the terminal differentiation stage. TGFβ1 treatment significantly decreased adiponectin mRNA at both early commitment (>300 fold) and terminal differentiated cells [S-Diff (~33%) or R-Diff (~20%)]. TGFβ1 upregulated collagen VI mRNA and its regulators connective tissue growth factor (CCN2/CTGF), TIMP1 and TIMP3 mRNA levels in undifferentiated preadipocytes and adipocytes at commitment stage. But in the terminal differentiated adipocytes, changes in mRNA and protein of collagen VI and TIMP3 mRNA were not observed despite an increase in CCN2/CTGF, TIMP1 mRNA. Although TGFβ1 upregulated interleukin-6 (IL6) and monocyte chemoattractant protein-1 (MCP1) mRNA at all stages of differentiation, decreased tumor necrosis factor-α (TNFα) mRNA was observed early in adipocyte differentiation. This study highlights the complex role of TGFβ1 on extracellular matrix (ECM) remodeling and inflammatory markers in stimulating both synthetic and inhibitory markers of fibrosis at different stages of adipocyte differentiation. [ABSTRACT FROM AUTHOR]

Wang X, McLennan SV, Allen T J, Tsoutsman T, Semsarian C, Twigg SM. Adverse effects of high glucose and free fatty acid on cardiomyocytes are mediated by connective tissue growth factor. Am J Physiol Cell Physiol 297: C1490-C1500, 2009. First published July 22, 2009; doi: 10.1152/ajpcell.00049.2009.--Diabetic cardiomyopathy is characterized by interstitial fibrosis and cardiomyocyte hypertrophy and apoptosis. Also known as CCN2, connective tissue growth factor (CTGF) is implicated in the fibrosis; however, whether it contributes to cardiomyocytes changes and adverse effects of high glucose and lipids on these cells remains unknown. Hearts from streptozotocin-induced diabetic rats had elevated CTGF and changes of pathological myocardial hypertrophy, fibrosis, and cardiomyocyte apoptosis. Rat H9c2 cardiomyocytes were then treated with recombinant human (rh)CTGF, high glucose, or the saturated free fatty acid palmitate. Each reagent induced cell hypertrophy, as indicated by the ratio of total protein to cell number, cell size, and gene expression of cardiac hypertrophy marker genes atrial natriuretic peptide (ANP), and [alpha]-skeletal actin. Each treatment also caused apoptosis measured by increased caspase3/7 activity, apoptotic cells by transferase-mediated dUTP nick end labeling (TUNEL) assay, and lower viable cell number. Further studies showed CTGF mRNA was rapidly induced by high glucose and palmitate in H9c2 cells and in mouse neonatal cardiomyocyte primary cultures, small interfering RNA against CTGF blocked the high glucose and palmitate induction of hypertrophy and apoptosis. In addition, these CTGF effects were through the tyrosine kinase A (TrkA) receptor with tyrosine kinase activity, which has previously been implicated in CTGF signaling: TrkA was phosphorylated by CTGF, and a specific TrkA blocker abrogated CTGF-induced effects on hypertrophy and apoptosis. For the first time in any system, fatty acid is newly identified as a regulator of CTGF, and this work implicates autocrine CTGF as a mediator of adverse effects of high glucose and fatty acids in cardiomyocytes. diabetic cardiomyopathy; apoptosis; hypertrophy doi: 10.1152/ajpcell.00049.2009

Infiltration of macrophages to the kidney is a feature of early diabetic nephropathy. For this to happen monocytes must become activated, migrate from the circulation, and infiltrate the mesangium. This process involves degradation of extracellular matrix, a process mediated by matrix metalloproteinases (MMPs). In the present study we investigate the expression of proinflammatory cytokines TNF-[alpha], IL-6, and MMP-9 in glomeruli of control and diabetic rodents and use an in vitro coculture system to examine whether factors secreted by mesangial cells in response to a diabetic milieu can induce monocyte MMP-9 expression and infiltration. After 8 wk of diabetes, the glomerular level of TNF-[alpha], IL-6, and macrophage number and colocalization of MMP-9 with macrophage were increased (P < 0.01). Coculture of THP1 monocytes and glomerular mesangial cells in 5 or 25 mM glucose increased MMP-9 (5 mM: 65% and 25 mM: 112%; P < 0.05) and conditioned media degradative activity (5 mM: 30.0% and 25 mM: 33.5%: P < 0.05). These effects were reproduced by addition of mesangial cell conditioned medium to THP1 cells. High glucose (25 mM) increased TNF-[alpha], IL-6, and monocyte chemoattractant protein-1 in mesangial cell conditioned medium. These cytokines all increased adhesion and differentiation of THP1 cells (P < 0.05), but only TNF-[alpha] and IL-6 increased MMP-9 expression (50- and 60-fold, respectively; P < 0.05). Our results show that mesangial cell-secreted factors increase monocyte adhesion, differentiation, MMP expression, and degradative capacity. High glucose could augment these effects by increasing mesangial cell proinflammatory cytokine secretion. This mesangial cell-monocyte interaction may be important in activating monocytes to migrate from the circulation to the kidney in the early stages of diabetic nephropathy. matrix metalloproteinases; matrix degradation; cell adhesion and infiltration; inflammation response doi: 10.1152/ajprenal.00074.2009.

Adipocyte differentiation is a key process implicated in the pathogenesis of obesity and insulin resistance. Its regulation is triggered by a cascade of transcription factors, including the CCAAT/enhancer binding proteins (C/EBPs) and peroxisome proliferator-activated receptor-[gamma] (PPAR[gamma]). Growth factors such as transforming growth factor-[beta]1 (TGF-[beta]1) are known to inhibit adipocyte differentiation in vitro, via the C/EBP pathway, and in vivo, but whether a downstream mediator of TGF-[beta]1, connective tissue growth factor (CTGF), also known as CCN2, has a similar role is unknown. Mouse 3T3-L1 cells were differentiated into adipocytes by using standard methods, and effects and regulation of CTGF were studied. Intervention with recombinant human CTGF during differing stages of differentiation caused an inhibition in the development of the adipocyte phenotype, according to the gene expression of the differentiation markers adiponectin and PPAR[gamma], as well as suppression of lipid accumulation and expression of the lipogenic enzyme glycerol-3-phosphate dehydrogenase. Whereas CTGF gene expression promptly fell by 90% as 3T3-L1 preadipocytes differentiated into mature adipocytes, CTGF mRNA expression was induced by added TGF-[beta]1. CTGF applied to cells early in the course of differentiation inhibited total cell protein levels and nuclear localization of the [beta]-isoform of C/EBP (C/EBP-[beta]) and, subsequently, total cell C/EBP-[alpha] levels. CTGF also inhibited the adipocyte differentiation program in primary cultures of mouse preadipocytes. Expression of CTGF mRNA was twofold higher in the central fat depots of mice compared with subcutaneous fat, suggesting a potential role for CTGF in vivo. In summary, these data show that CTGF inhibits the adipocyte differentiation program. NIH/3T3:CCN2

Diet and/or exercise are cost effective interventions to treat obesity. However, it is unclear if the type of exercise undertaken can prevent the onset of obesity and if it can act through different effects on fat depots. In this study we did not allow obesity to develop so we commenced the high‐fat diet (HFD) and exercise programs concurrently and investigated the effect of endurance exercise (END) and high‐intensity interval training (HIIT) on changes in cellular adipogenesis, thermogenesis, fibrosis, and inflammatory markers in three different fat depots, on a HFD and a chow diet. This was to assess the effectiveness of exercise to prevent the onset of obesity‐induced changes. Mice fed with chow or HFD (45% kcal fat) were trained and performed either END or HIIT for 10 weeks (3 x 40 min sessions/week). In HFD mice, both exercise programs significantly prevented the increase in body weight (END: 17%, HIIT: 20%), total body fat mass (END: 46%, HIIT: 50%), increased lean mass as a proportion of body weight (Lean mass/BW) by 14%, and improved insulin sensitivity by 22%. Further evidence of the preventative effect of exercise was seen significantly decreased markers for adipogenesis, inflammation, and extracellular matrix accumulation in both subcutaneous adipose tissue (SAT) and epididymal adipose tissue (EPI). In chow, no such marked effects were seen with both the exercise programs on all the three fat depots. This study establishes the beneficial effect of both HIIT and END exercise in preventing metabolic deterioration, collagen deposition, and inflammatory responses in fat depots, resulting in an improved whole body insulin resistance in HFD mice. [ABSTRACT FROM AUTHOR]

Transforming growth factor β (TGFβ) a multifunctional cytokine is known to regulate cell proliferation, differentiation, migration and survival. Although there is variable expression of modulators of TGFβ action during differentiation, a differential effect on fat cell metabolism at the different stages of adipocyte differentiation was unclear. In the present study, 3T3L1 cells were used as an in vitro model to study the effect of TGFβ on adipogenic and thermogenic markers at various stages of preadipocyte to mature adipocyte differentiation. As in our earlier studies on the effect of TGFβ on CEBP's, we used a standard differentiation mix, and one with the addition of rosiglitazone. RhTGFβ1 was added to undifferentiated adipocytes (preadipocytes) and to adipocytes at day 0 (commitment stage) as well as day 10 (terminal differentiation). Cellular responses in terms of Pref1, PPARγ, TLE3, PGC1α, PRDM16, UCP1 and UCP2 mRNA levels and selected protein products, were determined. Increases in PPARγ, PRDM16, UCP1 and UCP2 mRNA and decreases in Pref1 are good indicators of successful differentiation. The early addition of rhTGFβ1 during commitment stage decreased PPARγ, PRDM16, TLE3, UCP1 and UCP2 mRNA and decreased PRDM16 protein consistent with our earlier report on the inhibition of CEBP's by TGFβ and CCN2. The addition of rhTGFβ1 to mature adipocyte at day 10 increased UCP1 mRNA and increased PRDM16 and UCP1 proteins. In the present study, our results suggest that TGFβ1 added late enhances the thermogenic potential of mature cells and causes 3T3L1 cells to differentiate to resemble brown or beige rather than white adipose tissue. [ABSTRACT FROM AUTHOR]

The various effects of diabetes and glycation inhibitor, aminoguanidine on the endothelial function in a primate model of type 1 diabetes are studied. The aminoguanidine treatment is shown to lead to endothelial dysfunction in both diabetic animals and controls.

Background: Chronic inflammation is the driver of liver injury and results in progressive fibrosis and eventual cirrhosis with consequences including both liver failure and liver cancer. We have previously described increased expression of the highly multifunctional glycoprotein CD147 in liver injury. This work describes a novel role of CD147 in liver inflammation and the importance of leukocyte aggregates in determining the extent of liver injury. Methods: Non-diseased, progressive injury, and cirrhotic liver from humans and mice were examined using a mAb targeting CD147. Inflammatory cell subsets were assessed by multiparameter flow cytometry. Results: In liver injury, we observe abundant, intrahepatic leukocyte clusters defined as ≥5 adjacent CD45+ cells which we have termed “leukocyte aggregates”. We have shown that these leukocyte aggregates have a significant effect in determining the extent of liver injury. If CD147 is blocked in vivo, these leukocyte aggregates diminish in size and number, together with a marked significant reduction in liver injury including fibrosis. This is accompanied by no change in overall intrahepatic leukocyte numbers. Further, blocking of aggregation formation occurs prior to an appreciable increase in inflammatory markers or fibrosis. Additionally, there were no observed, “off-target” or unpredicted effects in targeting CD147. Conclusion: CD147 mediates leukocyte aggregation which is associated with the development of liver injury. This is not a secondary effect, but a cause of injury as aggregate formation proceeds other markers of injury. Leukocyte aggregation has been previously described in inflammation dating back over many decades. Here we demonstrate that leukocyte aggregates determine the extent of liver injury. [ABSTRACT FROM AUTHOR]

In a mouse model of diet-induced obesity, this study determined if two exercise prescriptions with equivalent time and distance covered, [constant-moderate endurance (END) and high intensity interval training (HIIT)], exert differential metabolic benefits on insulin sensitive tissues. Male 10 week old C57BL/6 mice were fed a high fat diet (HFD; 45% kcal fat) ad libitum for 10 weeks and for a further 10 weeks they underwent END or HIIT training (3 × 40 min sessions/wk). Untrained HFD and chow-fed mice acted as controls. At 30 weeks of age, mice were sacrificed and quadriceps muscle, subcutaneous adipose tissue (SAT) and liver were excised. Neither END nor HIIT altered body weight or composition in HFD mice. In quadriceps , HFD decreased high-molecular weight adiponectin protein, which was normalized by END and HIIT. In contrast, HIIT but not END reversed the HFD-driven decrease in the adiponectin receptor 1 (AdipoR1). In SAT, both programs tended to decrease collagen VI protein (p = 0.07–0.08) in HFD, whereas only HIIT induced an increase in the mRNA (3-fold vs. HFD untrained) and protein (2-fold vs. HFD untrained) of UCP1. In liver, only END reversed collagen I accumulation seen in HFD untrained mice. Our results suggest that HIIT may promote better systemic metabolic changes, compared to END, which may be the result of the normalization of muscle AdipoR1 and increased UCP1 seen in SAT. However, END was more effective in normalizing liver changes, suggesting differential metabolic effects of END and HIIT in different tissues during obesity. [ABSTRACT FROM AUTHOR]

Objective: To determine whether rapid polymerase chain reaction (PCR) testing for influenza and respiratory syncytial viruses (RSV) in emergency departments (EDs) is associated with better patient and laboratory outcomes than standard multiplex PCR testing.Design, Setting: A before-and-after study in four metropolitan EDs in New South Wales.Participants: 1491 consecutive patients tested by standard multiplex PCR during July-December 2016, and 2250 tested by rapid PCR during July-December 2017.Main Outcome Measures: Hospital admissions; ED length of stay (LOS); test turnaround time; patient receiving test result before leaving the ED; ordering of other laboratory tests.Results: Compared with those tested by standard PCR, fewer patients tested by rapid PCR were admitted to hospital (73.3% v 77.7%; P < 0.001) and more received their test results before leaving the ED (67.4% v 1.3%; P < 0.001); the median test turnaround time was also shorter (2.4 h [IQR, 1.6-3.9 h] v 26.7 h [IQR, 21.2-37.8 h]). The proportion of patients admitted to hospital was also lower in the rapid PCR group for both children under 18 (50.6% v 66.6%; P < 0.001) and patients over 60 years of age (84.3% v 91.8%; P < 0.001). Significantly fewer blood culture, blood gas, sputum culture, and respiratory bacterial and viral serology tests were ordered for patients tested by rapid PCR. ED LOS was similar for the rapid (7.4 h; IQR, 5.0-12.9 h) and standard PCR groups (6.5 h; IQR, 4.2-11.9 h; P = 0.27).Conclusion: Rapid PCR testing of ED patients for influenza virus and RSV was associated with better outcomes on a range of indicators, suggesting benefits for patients and the health care system. A formal cost-benefit analysis should be undertaken. [ABSTRACT FROM AUTHOR]

Summary: Context: Telomeres protect chromosomes from damage, and shorter leucocyte telomere length (LTL) is a marker of advancing biological age. The association between testosterone (T) and its bioactive metabolites, dihydrotestosterone (DHT) and oestradiol (E2) with telomere length, particularly in older men, is uncertain. The study aimed to clarify associations of sex hormones with LTL in older men. Participants and methods: We used cross‐sectional data from 2913 men aged 76.7 ± 3.2 years with morning blood samples assayed for T, DHT, E2 (mass spectrometry), and sex hormone‐binding globulin (SHBG, immunoassay), to correlate sex hormones with LTL measured using PCR and expressed as T/S ratio in multivariable linear regression models adjusted for age, cardiometabolic risk factors and cardiovascular disease history. Results: Average difference per decade of age was T −0.46 nmol/L, DHT −0.11 nmol/L, E2 −7.5 pmol/L, SHBG +10.2 nmol/L and LTL (T/S ratio) −0.065. E2 correlated with T/S ratio (r = 0.038, P = 0.039) and SHBG was inversely correlated (r = −0.053, P = 0.004). After multivariable adjustment, E2 was associated with T/S ratio (per 1 SD increase E2: coefficient 0.011, P = 0.043), T and DHT were not associated. When E2 and SHBG were simultaneously included, E2 remained positively (coefficient 0.014, P = 0.014) and SHBG inversely (coefficient −0.013, P = 0.037) associated with T/S ratio. Conclusions: In older men, neither T nor DHT is associated with LTL while E2 is independently associated with LTL and SHBG is inversely associated, thus relating sex hormone exposure to lower biological age. Further research is needed to determine causality and clarify the role of sex hormones in male ageing. [ABSTRACT FROM AUTHOR]

Exercise regimens may have differing effects in the presence of obesity. In addition to being fat derived, adiponectin has recently been described as a myokine that regulates insulin sensitivity, which may link to exercise related metabolic benefits in obesity. Whether skeletal muscle adiponectin varies in different exercise modalities is unclear. This study investigated the comparative effects of 10 weeks of endurance constant-moderate intensity exercise (END) with high intensity interval training (HIIT), on metabolic outcomes, including muscle adiponectin in a mouse model of diet-induced obesity. Tenweek- old male C57BL/6 mice were fed a high-fat diet (HFD) (45% FAT) or standard CHOW diet ab libitum and underwent one of three training regimes: (1) no exercise, (2) END, or (3) HIIT (8 bouts of 2.5 min with eight periods of rest of 2.5 min) for 10 weeks (3 9 40 min sessions/week). Chow-fed mice acted as controls. Compared with HFD alone, both training programs similarly protected against body weight gain (HFD = 45 ± 2; END = 37 ± 2; HIIT = 36 ± 2 g), preserved lean/fat tissue mass ratio (HFD = 0.64 ± 0.09; END = 0.34 ± 0.13; HIIT = 0.33 ± 0.13), and improved blood glucose excursion during an insulin tolerance test (HFD = 411 ± 54; END = 350 ± 57; HIIT = 320 ± 66 arbitrary units [AU]). Alterations in fasting glycemia, insulinemia, and AST/ALT ratios were prevented only by END. END, but not HIIT increased skeletal muscle adiponectin mRNA (14-fold; P < 0.05) and increased protein content of high molecular weight (HMW) adiponectin (3.3-fold), whereas HIIT induced a milder increase (2.4-fold). Compared with HFD, neither END nor HIIT altered circulating low (LMW) or high (HMW) molecular weight adiponectin forms. Furthermore, only END prevented the HFD downregulation of PGC1a (P < 0.05) mRNA levels downstream of muscle adiponectin. These data show that different training programs affect muscle adiponectin to differing degrees. Together these results suggest that END is a more effective regimen to prevent HFD-induced metabolic disturbances in mice. [ABSTRACT FROM AUTHOR]

Background: Cell-free fetal miRNAs have been identified as potential biomarkers for fetal abnormalities and/or placental function. Factors affecting the stability of cell-free fetal miRNA samples (type of collection tube and time interval between sampling and analysis) have not previously been reported.Methods: Blood from pregnant women (n = 12, 18 ± 4 weeks' gestation) was collected into two types of tube (EDTA and RNA BCT) and was stored at different temperatures for up to 72 h. Expression of seven apparently placental specific miRNAs was then measured to compare the effects of sampling and storage. These miRNAs were also assessed in non-pregnant women (n = 9).Results: The quantity of miRNA extracted was not affected by time or tube. Three miRNAs (miR-518b, miR-525 and miR-526a*) were measureable only in pregnant women, but miR-518b was not always present. Detailed study of the two pregnancy specific miRNAs showed no effect of tube type at 4 h. However, variability in miRNA level was observed with increased time and was significant for one miRNA in the BCT tube at >48 h (p < 0.005).Conclusion: Some cffmiRNAs are placental specific, and these samples are stable when analyzed within 48 h of collection in either tube type. © 2017 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

New Findings What is the central question of this study? Non-invasive muscle function tests have not been validated for use in the study of muscle performance in high-fat-fed mice., What is the main finding and its importance? This study shows that grip strength, hang wire and four-limb hanging tests are able to discriminate the muscle performance between chow-fed and high-fat-fed mice at different time points, with grip strength being reliable after 5, 10 and 20 weeks of dietary intervention., Non-invasive tests are commonly used for assessing muscle function in animal models. The value of these tests in obesity, a condition where muscle strength is reduced, is unclear. We investigated the utility of three non-invasive muscle function tests, namely grip strength (GS), hang wire (HW) and four-limb hanging (FLH), in C57BL/6 mice fed chow (chow group, n = 48) or a high-fat diet (HFD group, n = 48) for 20 weeks. Muscle function tests were performed at 5, 10 and 20 weeks. After 10 and 20 weeks, HFD mice had significantly reduced GS (in newtons; mean ± SD: 10 weeks chow, 1.89 ± 0.1 and HFD, 1.79 ± 0.1; 20 weeks chow, 1.99 ± 0.1 and HFD, 1.75 ± 0.1), FLH [in seconds per gram body weight; median (interquartile range): 10 weeks chow, 2552 (1337-4964) and HFD, 1230 (749-1994); 20 weeks chow, 2048 (765-3864) and HFD, 1036 (717-1855)] and HW reaches [ n; median (interquartile range): 10 weeks chow, 4 (2-5) and HFD, 2 (1-3); 20 weeks chow, 3 (1-5) and HFD, 1 (0-2)] and higher falls [ n; median (interquartile range): 10 weeks chow, 0 (0-2) and HFD, 3 (1-7); 20 weeks chow, 1 (0-4) and HFD, 8 (5-10)]. Grip strength was reliable in both dietary groups [intraclass correlation coefficient (ICC) = 0.5-0.8; P < 0.05], whereas FLH showed good reliability in chow (ICC = 0.7; P < 0.05) but not in HFD mice after 10 weeks (ICC < 0.5). Our data demonstrate that non-invasive muscle function tests are valuable and reliable tools for assessment of muscle strength and function in high-fat-fed mice. [ABSTRACT FROM AUTHOR]

Neutrophils are important for wound repair, but their persistence can impair the healing process. Neutrophils express matrix metalloproteinases including MMP-9 and its regulator neutrophil gelatinase associated lipocalin (NGAL). Whether wounding affects neutrophil MMP-9 and NGAL in diabetic animals is not known. Skin wound tissue MMP-9 and NGAL was examined by qRT-PCR and immunohistochemistry in control, diabetic and insulin treated diabetic rats. The temporal expression of MMP-9 and NGAL mRNA, MMP-9 activity and the NGAL/MMP-9 complex was also investigated in an implant model and their circulating neutrophils. The cellular localisation of MMP-9 and NGAL was confirmed by immunofluorescence and the ability of glucose to regulate these factors was examined in isolated neutrophils. In skin wound tissue compared with control, diabetes increased neutrophil infiltration, NGAL mRNA and MMP-9 protein (P<0.05). Diabetes significantly increased implant neutrophil NGAL and MMP-9 protein as well as NGAL mRNA, wound fluid NGAL/MMP-9 complex and MMP-9 activity (all <0.05). Circulating neutrophil MMP-9 and NGAL was also increased in these diabetic animals (P<0.05). These changes were prevented by insulin treatment. Ex vivo, high glucose (25mM) increased neutrophil NGAL and MMP-9 (both by 2 fold, P<0.05). NGAL and MMP-9 are increased in wound and circulating neutrophils in diabetic rodents. These changes and the association between higher NGAL and increased wound fluid MMP-9 activity suggest that increased neutrophil NGAL may contribute to increased MMP-9 in poorly healing diabetic wounds. Whether targeting neutrophil NGAL or MMP-9 can improve diabetic wound healing remains to be investigated. [ABSTRACT FROM AUTHOR]

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Aryl hydrocarbon receptor nuclear translocator (ARNT) is a transcription factor that binds to partners to mediate responses to environmental signals. To investigate its role in the innate immune system, floxed ARNT mice were bred with lysozyme M-Cre recombinase animals to generate lysozyme MARNT (LAR) mice with reduced ARNT expression. Myeloid cells of LAR mice had altered mRNA expression and delayed wound healing. Interestingly, when the animals were rendered diabetic, the difference in wound healing between the LAR mice and their littermate controls was no longer present, suggesting that decreased myeloid cell ARNT function may be an important factor in impaired wound healing in diabetes. Deferoxamine (DFO) improves wound healing by increasing hypoxia-inducible factors, which require ARNT for function. DFO was not effective in wounds of LAR mice, again suggesting that myeloid cells are important for normal wound healing and for the full benefit of DFO. These findings suggest that myeloid ARNT is important for immune function and wound healing. Increasing ARNT and, more specifically, myeloid ARNT may be a therapeutic strategy to improve wound healing. [ABSTRACT FROM AUTHOR]

Background: The classical paradigm of liver injury asserts that hepatic stellate cells (HSC) produce, remodel and turnover the abnormal extracellular matrix (ECM) of fibrosis via matrix metalloproteinases (MMPs). In extrahepatic tissues MMP production is regulated by a number of mechanisms including expression of the glycoprotein CD147. Previously, we have shown that CD147 is expressed on hepatocytes but not within the fibrotic septa in cirrhosis [1]. Therefore, we investigated if hepatocytes produce MMPs, regulated by CD147, which are capable of remodelling fibrotic ECM independent of the HSC. Methods: Non-diseased, fibrotic and cirrhotic livers were examined for MMP activity and markers of fibrosis in humans and mice. CD147 expression and MMP activity were co-localised by in-situ zymography. The role of CD147 was studied in-vitro with siRNA to CD147 in hepatocytes and in-vivo in mice with CCl4 induced liver injury using ãCD147 antibody intervention. Results: In liver fibrosis in both human and mouse tissue MMP expression and activity (MMP-2, -9, -13 and -14) increased with progressive injury and localised to hepatocytes. Additionally, as expected, MMPs were abundantly expressed by activated HSC. Further, with progressive fibrosis there was expression of CD147, which localised to hepatocytes but not to HSC. Functionally significant in-vitro regulation of hepatocyte MMP production by CD147 was demonstrated using siRNA to CD147 that decreased hepatocyte MMP-2 and -9 expression/activity. Further, in-vivo α-CD147 antibody intervention decreased liver MMP-2, -9, -13, -14, TGF-β and α-SMA expression in CCl4 treated mice compared to controls. Conclusion: We have shown that hepatocytes produce active MMPs and that the glycoprotein CD147 regulates hepatocyte MMP expression. Targeting CD147 regulates hepatocyte MMP production both in-vitro and in-vivo, with the net result being reduced fibrotic matrix turnover in-vivo. Therefore, CD147 regulation of hepatocyte MMP is a novel pathway that could be targeted by future anti-fibrogenic agents. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is a prevalent primary liver cancer that is derived from hepatocytes and is characterised by high mortality rate and poor prognosis. While HCC is driven by cumulative changes in the hepatocyte genome, it is increasingly recognised that the liver microenvironment plays a pivotal role in HCC propensity, progression and treatment response. The microenvironmental stimuli that have been recognised as being involved in HCC pathogenesis are diverse and include intrahepatic cell subpopulations, such as immune and stellate cells, pathogens, such as hepatitis viruses, and non-cellular factors, such as abnormal extracellular matrix (ECM) and tissue hypoxia. Recently, a number of novel environmental influences have been shown to have an equally dramatic, but previously unrecognized, role in HCC progression. Novel aspects, including diet, gastrointestinal tract (GIT) microflora and circulating microvesicles, are now being recognized as increasingly important in HCC pathogenesis. This review will outline aspects of the HCC microenvironment, including the potential role of GIT microflora and microvesicles, in providing new insights into tumourigenesis and identifying potential novel targets in the treatment of HCC. [ABSTRACT FROM AUTHOR]

Monocytes express many cell surface markers indicative of their inflammatory and activation status. Whether these markers are affected by diabetes and its complications is not known and was investigated in this study. Blood was obtained from 22 nondiabetic and 43 diabetic subjects with a duration of diabetes >10 years, including 25 without and 18 with clinically significant complications. The number of CD45+CD14+ monocytes and the percentage expressing the proinflammatory marker CD16 were determined by flow cytometry. Other markers of monocyte activation and expression of chemokine receptors were also examined. The relationship betweenmonocyte CD16 and clinical data, selected cytokines, and chemokines was also investigated. Diabetes had no effect on total white cell number but increased monocyte number. Diabetes also significantly decreased the number of CD16+ monocytes but only in those with diabetic complications. Other markers of monocyte activation status and chemokine receptors were not affected by diabetes or complications status. Diabetes induced plasma proinflammatory cytokines and they were lower in diabetic subjects with complications compared to those without complications. These results suggest that the circulating monocyte phenotype is altered by diabetic complications status. These changes may be causally related to and could potentially be used to predict susceptibility to diabetic complications. [ABSTRACT FROM AUTHOR]

Diabetic cardiomyopathy is characterized by interstitial fibrosis and cardiomyocyte hypertrophy and apoptosis. Also known as CCN2, connective tissue growth factor (CTGF) is implicated in the fibrosis; however, whether it contributes to cardiomyocytes changes and adverse effects of high glucose and lipids on these cells remains unknown. Hearts from streptozotocin-induced diabetic rats had elevated CTGF and changes of pathological myocardial hypertrophy, fibrosis, and cardiomyocyte apoptosis. Rat H9c2 cardiomyocytes were then treated with recombinant human (rh)CTGF, high glucose, or the saturated free fatty acid palmitate. Each reagent induced cell hypertrophy, as indicated by the ratio of total protein to cell number, cell size, and gene expression of cardiac hypertrophy marker genes atrial natriuretic peptide (ANP), and a-skeletal actin. Each treatment also caused apoptosis measured by increased caspase3/7 activity, apoptotic cells by transferase-mediated dUTP nick end labeling (TUNEL) assay, and lower viable cell number. Further studies showed CTGF mRNA was rapidly induced by high glucose and palmitate in H9c2 cells and in mouse neonatal cardiomyocyte primary cultures. small interfering RNA against CTGF blocked the high glucose and palmitate induction of hypertrophy and apoptosis. k addition, these CTGF effects were through the tyrosine kinase A (TrkA) receptor with tyrosine kinase activity, which has previously been implicated in CTGF signaling: TrkA was phosphorylated by CTGF, and a specific TrkA blocker abrogated CTGF-induced effects on hypertrophy and apoptosis. For the first time in any system, fatty acid is newly identified as a regulator of CTGF, and this work implicates autocrine CTGF as a mediator of adverse effects of high glucose and fatty acids in cardiomyocytes. [ABSTRACT FROM AUTHOR]

Infiltration of macrophages to the kidney is a feature of early diabetic nephropathy. For this to happen monocytes must become activated, migrate from the circulation, and infiltrate the mesangium. This process involves degradation of extracellular matrix, a process mediated by matrix metalloproteinases (MMPs). In the present study we investigate the expression of proinflammatory cytokines TNF-α, IL-6, and MMP-9 in glomeruli of control and diabetic rodents and use an in vitro coculture system to examine whether factors secreted by mesangial cells in response to a diabetic milieu can induce monocyte MMP-9 expression and infiltration. After 8 wk of diabetes, the glomerular level of TNF-α, IL-6, and macrophage number and colocalization of MMP-9 with macrophage were increased (P < 0.01). Coculture of THP1 monocytes and glomerular mesangial cells in 5 or 25 mM glucose increased MMP-9 (5 mM: 65% and 25 mM: 112%; P < 0.05) and conditioned media degradative activity (5 mM: 30.0% and 25 mM: 33.5%: P < 0.05). These effects were reproduced by addition of mesangial cell conditioned medium to THP 1 cells. High glucose (25 mM) increased TNF-α, IL-6, and monocyte chemoattractant protein-1 in mesangial cell conditioned medium. These cytokines all increased adhesion and differentiation of THP1 cells (P < 0.05), but only TNF-α and IL-6 increased MMP-9 expression (50- and 60-fold, respectively; P < 0.05). Our results show that mesangial cell-secreted factors increase monocyte adhesion, differentiation, MMP expression, and degradative capacity. High glucose could augment these effects by increasing mesangial cell proinflammatory cytokine secretion. This mesangial cell-monocyte interaction may be important in activating monocytes to migrate from the circulation to the kidney in the early stages of diabetic nephropathy. [ABSTRACT FROM AUTHOR]

A diet-induced non-alcoholic fatty liver disease (NAFLD) model causing obesity in rodents was used to examine whether sitagliptin and gliclazide therapies have similar protective effects on pathological liver change. Methods: Male mice were fed a high-fat diet (HFD) or standard chow (Chow) ad libitum for 25 weeks and randomly allocated to oral sitagliptin or gliclazide treatment for the final 10 weeks. Fasting blood glucose and circulating insulin were measured. Inflammatory and fibrotic liver markers were assessed by qPCR. The second messenger ERK and autophagy markers were examined by Western immunoblot. F4/80, collagens and CCN2 were assessed by immunohistochemistry (IHC). Results: At termination, HFD mice were obese, hyperinsulinemic and insulin-resistant but non-diabetic. The DPP4 inhibitor sitagliptin prevented intrahepatic induction of pro-fibrotic markers collagen-IV, collagen-VI, CCN2 and TGF-β1 and pro-inflammatory markers TNF-α and IL-1β more effectively than sulfonylurea gliclazide. By IHC, liver collagen-VI and CCN2 induction by HFD were inhibited only by sitagliptin. Sitagliptin had a greater ability than gliclazide to normalise ERK-protein liver dysregulation. Conclusion: These data indicate that sitagliptin, compared with gliclazide, exhibits greater inhibition of pro-fibrotic and pro-inflammatory changes in an HFD-induced NAFLD model. Sitagliptin therapy, even in the absence of diabetes, may have specific benefits in diet-induced NAFLD. [ABSTRACT FROM AUTHOR]

Foot ulcers and poor wound healing are problematic for patients with diabetes. The beehive protectant Propolis can improve wound healing but whether it can improve healing in diabetic wounds has not been investigated. In this study, the effect of a single application of Propolis on epithelial closure, wound morphology, cellular infiltrate, and blood vessel density were investigated. Diabetes was induced in rats using streptozocin. After 6 weeks, diabetic and control animals were wounded and the wounds were treated with Propolis or saline as control. At days 6 and 12 animals were sacrificed and wounds were excised. Compared with controls, diabetes decreased epithelial closure and reepithelialization but had no effect on wound contraction. These delays were prevented by Propolis. At day 12, the impaired macrophage infiltration (C:1.49±0.09 vs. D:0.25±0.14), persistent neutrophil infiltration (C:0.22±0.19 vs. D:1.33±0.81), and increased myeloperoxidase activity (fourfold) in diabetic wounds were prevented by Propolis. Diabetes had no effect on wound volume, vessel number, or branch points. These novel data indicate that Propolis can accelerate wound healing in diabetes. As neutrophil infiltration is normalized, its mechanism of action may be through anti-inflammatory pathways. This result and the established safety profile of Propolis provide a rationale for studying topical application of this agent in a clinical setting. [ABSTRACT FROM AUTHOR]

The aim of this study was to characterise microvascular blood flow in the skin and to compare it with biomarkers of endothelial dysfunction and tissue inflammation in patients with type 2 diabetes with (n=20) or without (n=20) microvascular complications and 20 control subjects. Microvascular function was measured by laser Doppler velocimetry in combination with iontophoresis of acetylcholine (ACh) and sodium nitroprusside (SNP). Blood was collected for measurement of biomarkers including plasminogen activator inhibitor-1 (PAI-1), soluble intercellular adhesion molecule (sICAM), soluble vascular cell adhesion molecule (sVCAM) and high-sensitivity C-reactive protein (hsCRP).Both ACh and SNP responses fall progressively with the development of diabetes and microvascular complications. For the total cohort, there was a significant overall correlation between ACh and SNP response (r=0.7, p<0.0001), and this relationship was particularly strong in those with microvascular complications. There was a trend towards higher hsCRP levels across the three groups, but no difference in other biomarkers.Abnormalities of microvascular blood flow are evident in diabetes and become more marked with the development of microvascular complications. This relationship was similar to that shown by the marker of inflammation (hsCRP), but stronger than that pertaining to biomarkers of endothelial function. As both ACh and SNP responses are attenuated, the disturbance is not characteristic of endothelial dysfunction alone. [ABSTRACT FROM PUBLISHER]

The prevalence of diabetes is increasing worldwide and has been forecasted to double in the next 20 years. The major increase in morbidity and mortality of diabetes is due to the development of both macro- and microvascular complications, including failure of the wound-healing process. Foot ulcers occur in 15% of all patients with diabetes and precede 84% of all lower-leg amputations. The essential components of diabetic foot ulcer treatment are to reduce foot bearing pressure (in neuropathic ulcers) and to increase blood supply (in the case of vascular ulcers). Antibacterial therapy is also important. Despite optimized treatment, for reasons not completely understood, some ulcers fail to heal. Previous research studies have shown clearly that failure of healing eventually leads to deep-seated infection and amputation. Therefore, impaired wound healing is the pivotal event responsible for most of the morbidity (and mortality) of diabetic foot disease. Improving wound healing in diabetes requires a multidisciplinary approach in terms of clinical management as well as an increased effort aimed at better understanding the pathogenesis of poor wound healing in diabetes. Consequently, a detailed understanding of the wound-healing process in diabetes and how it can be improved is of great importance. However, efforts to develop new therapies are hampered by a lack of knowledge of the molecular mechanisms responsible for the pathologies, as well as a lack of suitable models for the study of chronic wounds. Therefore, this review will address both clinical and biochemical aspects of wound healing in diabetes. [ABSTRACT FROM AUTHOR]

(1) Background: studies on the long-term dynamic changes in fat depot metabolism in response to a high-fat diet (HFD) on hepatic lipid deposition and insulin resistance are sparse. This study investigated the dynamic changes produced by HFD and the production of dysfunctional fat depots on insulin resistance and liver lipid metabolism. (2) Methods: mice fed a chow or HFD (45% kcal fat) diet had three fat depots, liver, and blood collected at 6, 10, 20, and 30 weeks. Anthropometric changes and gene markers for adipogenesis, thermogenesis, ECM remodeling, inflammation, and tissue insulin resistance were measured. (3) Results: early responses to the HFD were increased body weight, minor deposition of lipid in liver, increased adipocyte size, and adipogenesis. Later changes were dysfunctional adipose depots, increased liver fat, insulin resistance (shown by changes in ITT) accompanied by increased inflammatory markers, increased fibrosis (fibrosis > 2-fold, p < 0.05 from week 6), and the presence of crown cells in white fat depots. Later, changes did not increase thermogenic markers in response to the increased calories and decreased UCP1 and PRDM16 proteins in WAT. (4) Conclusions: HFD feeding initially increased adipocyte diameter and number, but later changes caused adipose depots to become dysfunctional, restricting adipose tissue expansion, changing the brown/beige ratios in adipose depots, and causing ectopic lipid deposition and insulin resistance. [ABSTRACT FROM AUTHOR]