Caloric Restriction Combined with Immobilization as Translational Model for Sarcopenia Expressing Key-Pathways of Human Pathology.

The prevalence of sarcopenia is increasing while it is often challenging, expensive and time-consuming to test the effectiveness of interventions against sarcopenia. Translational mouse models that adequately mimic underlying physiological pathways could accelerate research but are scarce. Here, we investigated the translational value of three potential mouse models for sarcopenia, namely partial immobilized (to mimic sedentary lifestyle), caloric restricted (CR; to mimic malnutrition) and a combination (immobilized & CR) model. C57BL/6J mice were calorically restricted (-40%) and/or one hindleg was immobilized for two weeks to induce loss of muscle mass and function. Muscle parameters were compared to those of young control (4 months) and old reference mice (21 months). Transcriptome analysis of quadriceps muscle was performed to identify underlying pathways and were compared with those being expressed in aged human vastus lateralis muscle-biopsies using a meta-analysis of five different human studies. Caloric restriction induced overall loss of lean body mass (-15%, p<0.001), whereas immobilization decreased muscle strength (-28%, p<0.001) and muscle mass of hindleg muscles specifically (on average -25%, p<0.001). The proportion of slow myofibers increased with aging in mice (+5%, p<0.05), and this was not recapitulated by the CR and/or immobilization models. The diameter of fast myofibers decreased with aging (-7%, p<0.05), and this was mimicked by all models. Transcriptome analysis revealed that the combination of CR and immobilization recapitulated more pathways characteristic for human muscle-aging (73%) than naturally aged (21 months old) mice (45%). In conclusion, the combination model exhibits loss of both muscle mass (due to CR) and function (due to immobilization) and has a remarkable similarity with pathways underlying human sarcopenia. These findings underline that external factors such as sedentary behavior and malnutrition are key elements of a translational mouse model and favor the combination model as a rapid model for testing the treatments against sarcopenia.

Translational characterization of the temporal dynamics of metabolic dysfunctions in liver, adipose tissue and the gut during diet-induced NASH development in Ldlr-/-.Leiden mice.

Background: NAFLD progression, from steatosis to inflammation and fibrosis, results from an interplay of intra- and extrahepatic mechanisms. Disease drivers likely include signals from white adipose tissue (WAT) and gut. However, the temporal dynamics of disease development remain poorly understood. Methods: High-fat-diet (HFD)-fed Ldlr-/-.Leiden mice were compared to chow-fed controls. At t = 0, 8, 16, 28 and 38w mice were euthanized, and liver, WAT depots and gut were analyzed biochemically, histologically and by lipidomics and transcriptomics together with circulating factors to investigate the sequence of pathogenic events and organ cross-talk during NAFLD development. Results: HFD-induced obesity was associated with an increase in visceral fat, plasma lipids and hyperinsulinemia at t = 8w, along with increased liver steatosis and circulating liver damage biomarkers. In parallel, upstream regulator analysis predicted that lipid catabolism regulators were deactivated and lipid synthesis regulators were activated. Subsequently, hepatocyte hypertrophy, oxidative stress and hepatic inflammation developed. Hepatic collagen accumulated from t = 16 w and became pronounced at t = 28-38 w. Epididymal WAT was maximally hypertrophic from t = 8 w, which coincided with inflammation development. Mesenteric and subcutaneous WAT hypertrophy developed slower and did not appear to reach a maximum, with minimal inflammation. In gut, HFD significantly increased permeability, induced a shift in microbiota composition from t = 8 w and changed circulating gut-derived metabolites. Conclusion: HFD-fed Ldlr-/-.Leiden mice develop obesity, dyslipidemia and insulin resistance, essentially as observed in obese NAFLD patients, underlining their translational value. We demonstrate that marked epididymal-WAT inflammation, and gut permeability and dysbiosis precede the development of NAFLD stressing the importance of a multiple-organ approach in the prevention and treatment of NAFLD.

The Human Milk Oligosaccharide 2'-Fucosyllactose Alleviates Liver Steatosis, ER Stress and Insulin Resistance by Reducing Hepatic Diacylglycerols and Improved Gut Permeability in Obese Ldlr-/-.Leiden Mice.

Non-alcoholic fatty liver disease (NAFLD) is a complex multifactorial disorder that is associated with gut dysbiosis, enhanced gut permeability, adiposity and insulin resistance. Prebiotics such as human milk oligosaccharide 2'-fucosyllactose are thought to primarily improve gut health and it is uncertain whether they would affect more distant organs. This study investigates whether 2'-fucosyllactose can alleviate NAFLD development in manifest obesity. Obese hyperinsulinemic Ldlr-/-.Leiden mice, after an 8 week run-in on a high-fat diet (HFD), were treated with 2'-fucosyllactose by oral gavage until week 28 and compared to HFD-vehicle controls. 2'-fucosyllactose did not affect food intake, body weight, total fat mass or plasma lipids. 2'-fucosyllactose altered the fecal microbiota composition which was paralleled by a suppression of HFD-induced gut permeability at t = 12 weeks. 2'-fucosyllactose significantly attenuated the development of NAFLD by reducing microvesicular steatosis. These hepatoprotective effects were supported by upstream regulator analyses showing that 2'-fucosyllactose activated ACOX1 (involved in lipid catabolism), while deactivating SREBF1 (involved in lipogenesis). Furthermore, 2'-fucosyllactose suppressed ATF4, ATF6, ERN1, and NUPR1 all of which participate in endoplasmic reticulum stress. 2'-fucosyllactose reduced fasting insulin concentrations and HOMA-IR, which was corroborated by decreased intrahepatic diacylglycerols. In conclusion, long-term supplementation with 2'-fucosyllactose can counteract the detrimental effects of HFD on gut dysbiosis and gut permeability and attenuates the development of liver steatosis. The observed reduction in intrahepatic diacylglycerols provides a mechanistic rationale for the improvement of hyperinsulinemia and supports the use of 2'-fucosyllactose to correct dysmetabolism and insulin resistance.

A Translational Mouse Model for NASH with Advanced Fibrosis and Atherosclerosis Expressing Key Pathways of Human Pathology.

Non-alcoholic steatohepatitis (NASH) is a fast-growing liver disorder that is associated with an increased incidence of cardiovascular disease and type 2 diabetes. Animal models adequately mimicking this condition are scarce. We herein investigate whether Ldlr-/-. Leiden mice on different high-fat diets represent a suitable NASH model. Ldlr-/-. Leiden mice were fed a healthy chow diet or fed a high-fat diet (HFD) containing lard or a fast food diet (FFD) containing milk fat. Additionally, the response to treatment with obeticholic acid (OCA) was evaluated. Both high-fat diets induced obesity, hyperlipidemia, hyperinsulinemia, and increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Mice on both diets developed progressive macro- and microvesicular steatosis, hepatic inflammation, and fibrosis, along with atherosclerosis. HFD induced more severe hyperinsulinemia, while FFD induced more severe hepatic inflammation with advanced (F3) bridging fibrosis, as well as more severe atherosclerosis. OCA treatment significantly reduced hepatic inflammation and fibrosis, and it did not affect atherosclerosis. Hepatic transcriptome analysis was compared with human NASH and illustrated similarity. The present study defines a translational model of NASH with progressive liver fibrosis and simultaneous atherosclerosis development. By adaptation of the fat content of the diet, either insulin resistance (HFD) or hepatic inflammation and fibrosis (FFD) can be aggravated.

A novel nutritional supplement prevents muscle loss and accelerates muscle mass recovery in caloric-restricted mice.

BACKGROUND: Muscle atrophy is defined as decreased muscle mass, associated with aging as well as with various chronic diseases and is a fundamental cause of frailty, functional decline and disability. Frailty represents a huge potential public health issue worldwide with high impact on healthcare costs. A major clinical issue is therefore to devise new strategies preventing muscle atrophy. In this study, we tested the efficacy of Vital01, a novel oral nutritional supplement (ONS), on body weight and muscle mass using a caloric restriction-induced mouse model for muscle atrophy. METHODS: Mice were calorically restricted for 2 weeks to induce muscle atrophy: one control group received 60% kcal of the normal chow diet and one intervention group received 30% kcal chow and 30 kcal% Vital01. The effects on body weight, lean body mass, muscle histology and transcriptome were assessed. In addition, the effects of Vital01, in mice with established muscle atrophy, were assessed and compared to a standard ONS. To this end, mice were first calorically restricted on a 60% kcal chow diet and then refed with either 100 kcal% chow, a mix of Vital01 (receiving 60% kcal chow and 40 kcal% Vital01) or with a mix of standard, widely prescribed ONS (receiving 60 kcal% chow and 40 kcal% Fortisip Compact). RESULTS: Vital01 attenuated weight loss (-15% weight loss for Vital01 vs. -25% for control group, p < 0.01) and loss of muscle mass (Vital01 with -13%, -12% and -18%, respectively, for gastrocnemius, quadriceps and tibialis vs. 25%, -23% and -28%, respectively, for control group, all p < 0.05) and also restored body weight, fat and muscle mass more efficiently when compared to Fortisip Compact. As assessed by transcriptome analysis and Western blotting of key proteins (e.g. phospoAKT, mTOR and S6K), Vital01 attenuated the catabolic and anabolic signaling pathways induced by caloric restriction and modulated inflammatory and mitochondrial pathways. In addition, Vital01 affected pathways related to matrix proteins/collagens homeostasis and tended to reduce caloric restriction-induced collagen fiber density in the quadriceps (with -27%, p = 0.051). CONCLUSIONS: We demonstrate that Vital01 preserves muscle mass in a calorically restricted mouse model for muscle atrophy. Vital01 had preventive effects when administered during development of muscle atrophy. Furthermore, when administered in a therapeutic setting to mice with established muscle atrophy, Vital01 rapidly restored body weight and accelerated the recurrence of fat and lean body mass more efficiently than Fortisip Compact. Bioinformatics analysis of gene expression data identified regulatory pathways that were specifically influenced by Vital01 in muscle.

Diet-Independent Correlations between Bacteria and Dysfunction of Gut, Adipose Tissue, and Liver: A Comprehensive Microbiota Analysis in Feces and Mucosa of the Ileum and Colon in Obese Mice with NAFLD.

Development of non-alcoholic fatty liver disease (NAFLD) is linked to obesity, adipose tissue inflammation, and gut dysfunction, all of which depend on diet. So far, studies have mainly focused on diet-related fecal microbiota changes, but other compartments may be more informative on host health. We present a first systematic analysis of microbiota changes in the ileum and colon using multiple diets and investigating both fecal and mucosal samples. Ldlr-/-.Leiden mice received one of three different energy-dense (ED)-diets (n = 15/group) for 15 weeks. All of the ED diets induced obesity and metabolic risk factors, altered short-chain fatty acids (SCFA), and increased gut permeability and NAFLD to various extents. ED diets reduced the diversity of high-abundant bacteria and increased the diversity of low-abundant bacteria in all of the gut compartments. The ED groups showed highly variable, partially overlapping microbiota compositions that differed significantly from chow. Correlation analyses demonstrated that (1) specific groups of bacteria correlate with metabolic risk factors, organ dysfunction, and NAFLD endpoints, (2) colon mucosa had greater predictive value than other compartments, (3) correlating bacteria differed per compartment, and (4) some bacteria correlated with plasma SCFA levels. In conclusion, this comprehensive microbiota analysis demonstrates correlations between the microbiota and dysfunctions of gut, adipose tissue, and liver, independent of a specific disease-inducing diet.

The type of collagen cross-link determines the reversibility of experimental skin fibrosis.

Fibrotic processes in humans are characterised by an excessive accumulation of collagen containing increased levels of hydroxyallysine-derived cross-links. The occurrence of these cross-links appears to be an important criterion in assessing the irreversibility of fibrosis. We hypothesise that increased hydroxyallysine cross-linking results in a collagenous matrix that is less susceptible to proteolytic degradation and therefore the collagen deposition is no longer reversible. In this report, we show that collagen matrices with increased hydroxyallysine cross-link levels were less susceptible to matrix metalloproteinase 1 degradation than are collagen matrices containing low hydroxyallysine levels. These data indicate that the type of collagen cross-link influences collagen catabolism. In vivo evidence for the importance of the cross-linking type in determining the reversibility of the fibrotic process was found using the bleomycin-induced skin fibrosis mouse model. The analysis of the accumulated collagen in the fibrotic skin of bleomycin-treated mice did not reveal an increase in hydroxyallysine cross-link levels. In concurrence with our hypothesis, the collagen accumulation resolved in time when the mice were no longer receiving bleomycin treatment, showing the reversibility of the fibrosis. In conclusion, our data indicate that the type of collagen cross-linking is an important factor in determining whether the outcome of the fibrotic process is reversible or not.

Pharmacogenetic heterogeneity of transgene expression in muscle and tumours.

BACKGROUND: Recombinant adenoviruses are employed to deliver a therapeutic transgene in the liver, muscle or tumour tissue. However, to rationalise this delivery approach, the factors of variation between individuals need to be identified. It is assumed that differences between inbred strains of laboratory animals are considered to reflect differences between patients. Previously we showed that transgene expression in the liver of different rat strains was dependent on the transcription efficiency of the transgene. In the present paper we investigated if transfection of muscle and tumour tissue were also subject to such variations. METHODS: Variation, in transgene expression, after intramuscular gene delivery was determined in different rodent strains and gene expression in tumours was investigated in different human and rodent cell lines as well as in subcutaneously implanted rodent tumours. The molecular mechanisms involved in transgene expression were dissected using an adenovirus encoding luciferase. The luciferase activity, the viral DNA copies and the luciferase transcripts were assessed in cultured cells as well as in the tissues. RESULTS: Large differences of luciferase activity, up to 2 logs, were observed between different rodent strains after intramuscular injection of Ad Luciferase. This inter-strain variation of transgene expression was due to a difference in transcription efficiency. The transgene expression level in tumour cell lines of different tissue origin could be explained largely by the difference of infectibility to the adenovirus. In contrast, the main step responsible for luciferase activity variation, between six human breast cancer cell lines with similar phenotype, was at the transcriptional level. CONCLUSION: Difference in transcriptional efficiency in muscles as observed between different inbred strains and between human breast cancer cell lines may be expected to occur between individual patients. This might have important consequences for clinical gene therapy. The variation between tumour types and tissues within a species are mainly at the levels of infectivity.

Genetic heterogeneity in response to adenovirus gene therapy.

BACKGROUND: After intravenous delivery of the adenoviral vector into rats or mice, 95-99% of the encoded protein is produced in the hepatocytes. We observed, as have others, that the early expression levels of the vector encoded protein vary, greatly, within a species, from one animal strain to another. This study was initiated to determine the molecular mechanism causing the difference: hepatic transfection, transcription or translation. For this purpose different doses of Ad5 luciferase and Ad5 LacZ were intravenously injected into Brown Norway rats and Wag/Rij rats, two strains that differ by a factor of 10 in encoded protein levels. The proportion of LacZ positive hepatocytes, the adenoviral DNA, specific transgenic RNA and luciferase protein were compared in the two strains. RESULTS: The number of transduced hepatocytes and the amounts of Ad5 DNA in the livers was similar in both strains, whereas the Brown Norway rats produced 8 to 10 times more of both vector encoded proteins and of transgene mRNA than the Wag/Rij rats. CONCLUSIONS: It is concluded that the difference between strains in vector encoded protein expression is due to different transcriptional events. No evidence was obtained to suggest that the differences are related to liver damage influenced by vector toxicity or immune reactions.

A comparison of efficacy and toxicity between electroporation and adenoviral gene transfer.

BACKGROUND: Electroporation of skeletal muscle after injection of naked DNA was shown by others to increase transgene expression. Information regarding tissue damage caused by electroporation is conflicting. It is also not well known how plasmid electroporation compares with transfection by adenoviral vectors. To investigate these questions the most used protocol for muscle electroporation was used, i.e. 8 pulses of 200 V/cm and 20 ms at a frequency of 1 Hz. RESULTS: Intra-muscular DNA transfer of pLuciferase was increased by 2 logs after electroporation, confirming data described by others. However, the blood levels of the encoded protein were still lower than those obtained after injection of first generation adenoviral vectors. Also, the electroporation procedure, on its own, caused severe muscle damage consisting of rhabdomyolysis and infiltration, whereas the adenoviral vectors caused only a slight infiltration. As damage of targeted tissue may be an advantage in the case of tumour transfection, we also compared the two transfection methods in tumour tissue. In case of poorly permissive tumours, adenoviral vectors cannot transfect more than 2% of the tumour tissue without inducing significant liver damage. In contrast, the electroporation seems to offer a wider therapeutic window since it does not cause any systemic toxicity and still induce's significant transfection. CONCLUSIONS: Plasmid electroporation of the muscle induce severe local damage and is of no advantage over adenoviral vectors for obtaining high blood levels of a vector encoded protein. In contrast, electroporation of tumours might be safer than adenoviral gene transfer.

Adenoviral gene transfer of angiostatic ATF-BPTI inhibits tumour growth.

BACKGROUND: The outgrowth of new vessels--angiogenesis--in the tumour mass is considered to be a limiting factor of tumour growth. To inhibit the matrix lysis that is part of the tumour angiogenesis, we employed the chimeric protein mhATF-BPTI, composed of the receptor binding part of the urokinase (ATF) linked to an inhibitor of plasmin (BPTI). METHODS: For delivery, recombinant adenovirus encoding the transgene of interest was injected intravenously or locally into the tumour. The anti tumour effect of this compound was compared to that of human endostatin and of mhATF alone in two different rat bronchial carcinomas growing either as subcutaneous implants or as metastases. RESULTS: Significant inhibition of the tumour growth and decrease of the number of lung metastasis was achieved when the concentration of mhATF-BPTI at the tumour site was above 400 of ng/g tissue. This concentration could be achieved via production by the liver, only if permissive to the recombinant adenovirus. When the tumour cells could be transduced, local delivery of the vector was enough to obtain a response. In the case of metastasis, the capacity of the lung tissue to concentrate the encoded protein was essential to reach the required therapeutic levels. Further, endostatin or mhATF could not reproduce the effects of mhATF-BPTI, at similar concentrations (mhATF) and even at 10-fold higher concentration (endostatin). CONCLUSION: The ATF-BPTI was shown to inhibit tumour growth of different rat lung tumours when critical concentration was reached. In these tumour models, endostatin or ATF induce almost no tumour response.