Constant-moderate versus high-intensity interval training on heart adiponectin levels in high-fat fed mice: a preventive and treatment approach.

CONTEXT: 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. OBJECTIVE: To investigate the effects of constant-moderate endurance (END) and high-intensity interval training (HIIT), on heart adiponectin levels in mice. MATERIAL AND METHODS: 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. RESULTS: 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.

The Effect of TGFß1 in Adipocyte on Inflammatory and Fibrotic Markers at Different Stages of Adipocyte Differentiation.

Transforming growth factor beta (TGFß) is a versatile cytokine. Although a profibrotic role of TGFß 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 TGFß1 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 TGFß1 (2 ng/mL) was added to the undifferentiated preadipocyte during the commitment stage and at the terminal differentiation stage. TGFß1 treatment significantly decreased adiponectin mRNA at both early commitment (>300 fold) and terminal differentiated cells [S-Diff (~33%) or R-Diff (~20%)]. TGFß1 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 TGFß1 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 TGFß1 on extracellular matrix (ECM) remodeling and inflammatory markers in stimulating both synthetic and inhibitory markers of fibrosis at different stages of adipocyte differentiation.

Sitagliptin Is More Effective Than Gliclazide in Preventing Pro-Fibrotic and Pro-Inflammatory Changes in a Rodent Model of Diet-Induced Non-Alcoholic Fatty Liver Disease.

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-ß1 and pro-inflammatory markers TNF-α and IL-1ß 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.

Targeting CCN2 protects against progressive non-alcoholic steatohepatitis in a preclinical model induced by high-fat feeding and type 2 diabetes.

Type 2 diabetes is an independent risk factor for non-alcoholic steatohepatitis (NASH) progression and its mediators have not been resolved. In this study, a pathogenic role of cellular communication network factor 2 (CCN2) protein in NASH pathology, was investigated in an established preclinical NASH model. Male wild type C57BL/6 mice received either Chow or high fat diet (HFD) for 26 weeks, with some mice in each group randomly selected to receive low dose streptozotocin (STZ: 3 i.p. injections, 65 mg/kg) at 15 weeks to induce type 2 diabetes. In the final 10 of the 26 weeks mice from each group were administered i.p. either rabbit anti-CCN2 neutralizing antibody (CCN2Ab) or as control normal rabbit IgG, at a dose of 150 µg per mouse twice/week. NASH developed in the HFD plus diabetes (HFD+DM) group. Administration of CCN2Ab significantly downregulated collagen I and collagen III mRNA induction and prevented pro-inflammatory MCP-1 mRNA induction in HFD+DM mice. At the protein level, CCN2Ab significantly attenuated collagen accumulation by PSR stain and collagen I protein induction in HFD+DM. Phosphorylation of the pro-fibrotic ERK signalling pathway in liver in HFD+DM was attenuated by CCN2Ab treatment. Intrahepatic CCN1 mRNA was induced, whereas CCN3 was downregulated at both the mRNA and protein levels in HFD+DM. CCN3 down-regulation was prevented by CCN2Ab treatment. This in vivo study indicates that CCN2 is a molecular target in NASH with high fat diet and diabetes, and that regulation of ERK signalling is implicated in this process.

The Effect of a Sustained High-Fat Diet on the Metabolism of White and Brown Adipose Tissue and Its Impact on Insulin Resistance: A Selected Time Point Cross-Sectional Study.

(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.

Exercise induces favorable metabolic changes in white adipose tissue preventing high-fat diet obesity.

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.

Monocyte phenotype as a predictive marker for wound healing in diabetes-related foot ulcers.

AIMS: Delayed healing of diabetes-related foot ulcers (DRFUs) is associated with increased macrophage and matrix metalloproteinases (MMPs) at the wound site. Whether circulating monocyte phenotype and/or MMPs are altered in association with wound healing outcome is unknown, and was investigated in this study. METHODS: Blood was obtained from 21 participants with DRFU, at initial visit (V1), week-4 (V2), and week-8 (V3) for measurement of monocyte number (CD14+), phenotype (CD16, CD163) and chemokine receptors (CCRs) by flow cytometry, and circulating MMPs and TIMP-1 by ELISA. RESULTS: Six wounds healed during the study. At V1, non-classical CD16++ monocytes and MMP-3 were higher in healed vs unhealed (both p < 0.05). At V3, the increased %CD16++ persisted and %CCR2+ was decreased in healed, but no other monocyte markers nor MMP/TIMP differed between groups. Increased wound closure rate (WCR) at V3 correlated with increased %CD16++ monocytes and decreased MMP-2 at V1 or V1 + V2. Receiver operating characteristic (ROC) curves yielded an area-under-the-curve of %CD16++ at V1 of 0.78 to predict ulcer healing at V3. CONCLUSIONS: These results indicate that circulating monocyte phenotype and MMPs alter as DRFUs heal. The relationship of %CD16++ monocytes with WCR and ROC curve suggest a predictive role of %CD16++ monocytes for ulcer healing.

The effect of TGFß1 on thermogenic markers is dependent on the degree of adipocyte differentiation.

Transforming growth factor ß (TGFß) a multifunctional cytokine is known to regulate cell proliferation, differentiation, migration and survival. Although there is variable expression of modulators of TGFß 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 TGFß on adipogenic and thermogenic markers at various stages of preadipocyte to mature adipocyte differentiation. As in our earlier studies on the effect of TGFß on CEBP's, we used a standard differentiation mix, and one with the addition of rosiglitazone. RhTGFß1 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 rhTGFß1 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 TGFß and CCN2. The addition of rhTGFß1 to mature adipocyte at day 10 increased UCP1 mRNA and increased PRDM16 and UCP1 proteins. In the present study, our results suggest that TGFß1 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.

Skeletal muscle adiponectin induction in obesity and exercise.

Recent scientific efforts have focused on the detrimental effects that obesity has on the metabolic function of skeletal muscles and whether exercise can improve this dysfunction. In this regard, adiponectin, with important metabolic functions (e.g. insulin-sensitizer and anti-inflammatory), has been recently described as a myokine that acts in an autocrine/paracrine manner. Earlier studies reported that muscle adiponectin could be induced by pro-inflammatory mediators (e.g. lipopolysaccharide), cytokines, and high-fat diets, providing a protective mechanism of this tissue against metabolic insults. However, when metabolic insults such as high-fat diets are sustained this protective response becomes dysregulated, making the skeletal muscle susceptible to metabolic impairments. Recent studies have suggested that exercise could prevent or even reverse this process. Considering that most scientific knowledge on adiponectin dysregulation in obesity is from the study of adipose tissue, the present review summarizes and discusses the literature available to date regarding the effects of obesity on skeletal muscle adiponectin induction, along with the potential effects of different exercise prescriptions on this response in an obesity context.

Liver enzyme profile and progression in association with thyroid autoimmunity in Graves' disease.

OBJECTIVE: To investigate the associations of Graves' disease (GD) severity, autoimmunity and longitudinal liver enzyme changes with time in a cohort with well-characterized GD. DESIGN: Retrospective cohort study. PATIENTS: Patients diagnosed with Graves' disease, treated at Royal Prince Alfred Hospital Sydney, Adult Thyroid Clinic from 2000 to 2012 inclusive. MEASUREMENTS: Inclusion criteria were patients with a complete set of TSH, FT4, FT3, liver enzymes and TSH receptor antibody (TRAb) results prior to commencement of thionamide therapy. RESULTS: Of the 146 patients who had complete results, 69 (47%) had at least one abnormal liver enzyme. Gamma glutamyltransferase (GGT) was most frequently abnormal (74%), followed by alanine aminotransferase (ALT) (57%), alkaline phosphatase (ALP) (39%) and then aspartate aminotransferase (AST) (29%). Subsequent to thyroid function normalization, 78% of the liver enzymes were normalized, 10% were persistently abnormal and 12% were lost to follow-up. Circulating TRAb, FT3 and FT4 results were categorized into mild, moderate and severe elevations. At univariate regression analyses, TRAb, FT3 and FT4 levels were each significantly associated with abnormal liver enzyme profile. Multivariate regression including TRAB, FT3 and FT4 as independent variables demonstrated FT3 and FT4 were more strongly associated with abnormal liver profile than TRAb. However, the initial FT3 and FT4 levels were not associated with abnormal liver profile in the subgroup with persistently abnormal liver profile. CONCLUSION: Graves' disease is commonly associated with abnormal liver enzymes, and most commonly with abnormal levels of GGT, and that an abnormal liver enzyme profile is more directly linked to the degree of thyrotoxicosis than levels of TRAB.

CD147 mediates intrahepatic leukocyte aggregation and determines the extent of liver injury.

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.

Shortening of telomere length by metabolic factors in diabetes: protective effects of fenofibrate.

People with diabetes mellitus have shorter telomeres compared with non-diabetic subjects. The aim of this study was to investigate an in-vitro model of telomere shortening under diabetes metabolic conditions. The mechanisms of the accelerated telomere length attrition and the potential telomere protective action of fenofibrate with related cellular mechanisms were also examined. Human dermal fibroblasts were passaged and cultured in normal (5.5 mM) or high (25 mM) D-glucose, across 7 days with hydrogen peroxide (H2O2), glucosamine (GA), or glycated albumin (AGEs-BSA). Relative telomere length (RTL) was determined by qPCR. The expression of shelterin complex members which regulate telomere stability were measured by qRT-PCR and Western immunoblot. Culture in high glucose decreased RTL compared with normal glucose: H2O2 and GA lowered the RTL after 7 days (each P < 0.05 vs untreated control), whereas AGEs-BSA had no effect compared with control-BSA. At day 7 the mRNA levels of most shelterin complex members, were induced by H2O2 and to a lesser extent by GA. Trf1 and Trf2 protein were induced by H2O2. Co-treatment with fenofibrate (100 µM) significantly attenuated the reduction in RTL caused by H2O2 and GA and prevented Trf induction by H2O2. However knockdown of Trf1 and Trf2 expression using specific siRNA did not prevent H2O2 effects to lower RTL, thus implicating factors other than these Trfs alone in the fenofibrate protection against the H2O2 induction of RTL lowering. These in vitro findings demonstrate that diabetic conditions can induce telomere shortening and that fenofibrate has protective effects on telomere attrition, through as yet undefined mechanisms.

Constant-Moderate and High-Intensity Interval Training Have Differential Benefits on Insulin Sensitive Tissues in High-Fat Fed Mice.

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.

The impact of rapid molecular diagnostic testing for respiratory viruses on outcomes for emergency department patients.

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.

Impact of Rapid Molecular Diagnostic Testing of Respiratory Viruses on Outcomes of Adults Hospitalized with Respiratory Illness: a Multicenter Quasi-experimental Study.

A standard multiplex PCR offers comprehensive testing for respiratory viruses. However, it has traditionally been performed in a referral laboratory with a lengthy turnaround time, which can reduce patient flow through the hospital. We aimed to determine whether the introduction of a rapid PCR, but with limited targets (Cepheid Xpert Flu/RSV XC), was associated with improved outcomes for adults hospitalized with respiratory illness. A controlled quasi-experimental study was conducted across three hospitals in New South Wales, Australia. Intervention groups received standard multiplex PCR during the preimplementation, July to December 2016 (n = 953), and rapid PCR during the postimplementation, July to December 2017 (n = 1,209). Control groups (preimplementation, n = 937, and postimplementation, n = 1,102) were randomly selected from adults hospitalized with respiratory illness during the same periods. The outcomes were hospital length of stay (LOS) and microbiology test utilization (blood culture, urine culture, sputum culture, and respiratory bacterial and virus serologies). The introduction of rapid PCR was associated with a nonsignificant 8.9-h reduction in median LOS (95% confidence interval [CI], -21.5 h to 3.7 h; P = 0.17) for all patients and a significant 21.5-h reduction in median LOS (95% CI, -36.8 h to -6.2 h; P < 0.01) among patients with positive test results in an adjusted difference-in-differences analysis. For patients receiving test results before disposition, rapid PCR use was associated with a significant reduction in LOS, irrespective of test results. Compared with standard PCR testing, rapid PCR use was significantly associated with fewer blood culture (adjusted odds ratio [aOR], 0.67; 95% CI, 0.5 to 0.82; P < 0.001), sputum culture (aOR, 0.56; 95% CI, 0.47 to 0.68, P < 0.001), bacterial serology (aOR, 0.44; 95% CI, 0.35 to 0.55, P < 0.001) and viral serology (aOR, 0.42; 95% CI, 0.33 to 0.53, P < 0.001) tests, but not with fewer urine culture tests (aOR, 0.94; 95% CI, 0.78 to 1.12, P = 0.48). Rapid PCR testing of adults hospitalized with respiratory illnesses can deliver benefits to patients and reduce resource utilization. Future research should consider a formal economic analysis and assess its potential impacts on clinical decision making.

Cross-sectional associations of sex hormones with leucocyte telomere length, a marker of biological age, in a community-based cohort of older men.

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.

Skeletal muscle adiponectin induction depends on diet, muscle type/activity, and exercise modality in C57BL/6 mice.

Changes in skeletal muscle adiponectin induction have been described in obesity and exercise. However, whether changes are consistent across muscle types and with different exercise modalities, remain unclear. This study compared the effects of diet and two isocaloric training programs on adiponectin induction and its regulators in three muscles: quadriceps (exercising/glycolytic-oxidative), gastrocnemius (exercising/glycolytic), and masseter (nonexercising/glycolytic). Ten-week-old male C57BL/6 mice were fed a high-fat diet (HFD) (45% fat) or standard CHOW diet (12% fat) ad libitum and underwent one of two training regimes: (1) constant-moderate training (END), or (2) high intensity interval training (HIIT) for 10 weeks (3 × 40 min sessions/week). Chow and HFD-fed untrained mice were used as control. Compared with Chow, HFD induced an increase in protein levels of low-molecular weight (LMW) adiponectin in gastrocnemius and masseter (~2-fold; P < 0.05), and a decrease of high-molecular weight adiponectin (HMW-most bioactive form) in quadriceps (~0.5-fold; P < 0.05). Only END prevented these changes (P < 0.05). HFD induced a decrease of adiponectin receptor 1 (AdipoR1) protein in exercising muscles of untrained mice (~0.5-0.8-fold; P < 0.05); notably, END also decreased AdipoR1 protein levels in lean and HFD mice. This type of training also normalized HFD-driven mRNA changes found in some adiponectin downstream factors (sirtuin 1, Pgc-1a, and Ucp2) in the three muscles tested. Our results indicate that diet, muscle type/activity, and exercise modality influences muscle adiponectin profile, and some of its mediators. These parameters should be taken into consideration when investigating this endocrine response of the skeletal muscle, particularly in the context of obesity and metabolic disorders.

Differential metabolic effects of constant moderate versus high intensity interval training in high-fat fed mice: possible role of muscle adiponectin.

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. Ten-week-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 × 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 PGC1α (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.

Utility and reliability of non-invasive muscle function tests in high-fat-fed mice.

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.

Effects of sample processing and storage on the integrity of cell-free miRNAs in maternal plasma.

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.