Dmitri Svistounov

Osteoblasts mediate the adverse effects of glucocorticoids on fuel metabolism

Long-term glucocorticoid treatment is associated with numerous adverse outcomes, including weight gain, insulin resistance, and diabetes; however, the pathogenesis of these side effects remains obscure. Glucocorticoids also suppress osteoblast function, including osteocalcin synthesis. Osteocalcin is an osteoblast-specific peptide that is reported to be involved in normal murine fuel metabolism. We now demonstrate that osteoblasts play a pivotal role in the pathogenesis of glucocorticoid-induced dysmetabolism. Osteoblast-targeted disruption of glucocorticoid signaling significantly attenuated the suppression of osteocalcin synthesis and prevented the development of insulin resistance, glucose intolerance, and abnormal weight gain in corticosterone-treated mice. Nearly identical effects were observed in glucocorticoid-treated animals following heterotopic (hepatic) expression of both carboxylated and uncarboxylated osteocalcin through gene therapy, which additionally led to a reduction in hepatic lipid deposition and improved phosphorylation of the insulin receptor. These data suggest that the effects of exogenous high-dose glucocorticoids on insulin target tissues and systemic energy metabolism are mediated, at least in part, through the skeleton.

Age-related changes in the hepatic microcirculation in mice.

Aging of the liver is associated with impaired metabolism of drugs, adverse drug interactions, and susceptibility to toxins. Since reduced hepatic blood flow is suspected to contribute this impairment, we examined age-related alterations in hepatic microcirculation. Livers of C57Bl/6 mice were examined at 0.8 (pre-pubertal), 3 (young adult), 14 (middle-aged), and 27 (senescent) months of age using in vivo and electron microscopic methods. The results demonstrated a 14% reduction in the numbers of perfused sinusoids between 0.8 and 27 month mice associated with 35% reduction in sinusoidal blood flow. This was accompanied by an inflammatory response evidenced by a fivefold increase in leukocyte adhesion in 27 month mice, up-regulated expression of ICAM-1, and increases in intrahepatic macrophages. Sinusoidal diameter decreased 6-10%. Liver sinusoidal endothelial cell (LSEC) dysfunction was seen as early as 14 months when there was a threefold increase in the numbers of swollen LSEC. The endocytotic capacity of LSEC also was found to be reduced in older animals. The sinusoidal endothelium in 27 month old mice exhibited pseudocapillarization. In conclusion, the results suggest that leukocyte accumulation in the sinusoids and narrowing of sinusoidal lumens due to pseudocapillarization and dysfunction of LSEC reduce sinusoidal blood flow in aged livers.

Role of liver sinusoidal endothelial cells and stabilins in elimination of oxidized low-density lipoproteins

Atherogenesis is associated with elevated levels of low-density lipoprotein (LDL) and its oxidized form (oxLDL) in the blood. The liver is an important scavenger organ for circulating oxLDLs. The present study aimed to examine endocytosis of mildly oxLDL (the major circulating form of oxLDLs) in liver sinusoidal endothelial cells (LSECs) and the involvement of the scavenger receptors stabilin-1 and stabilin-2 in this process. Freshly isolated LSECs, Kupffer cells (KCs), and stabilin-1- and stabilin-2-transfected human embryonic kidney cells were incubated with fluorescently labeled or radiolabeled oxLDLs [oxidized for 3 h (oxLDL(3)), 6 h, or 24 h (oxLDL(24))] to measure endocytosis. The intracellular localization of oxLDLs and stabilins in LSECs was examined by immunofluorescence and immunogold electron microscopy. Whereas oxLDL(24) was endocytosed both by LSECs and KCs, oxLDL(3) (mildly oxLDL) was taken up by LSECs only. The LSEC uptake of oxLDLs was significantly inhibited by the scavenger receptor ligand formaldehyde-treated serum albumin. Uptake of all modified LDLs was high in stabilin-1-transfected cells, whereas stabilin-2-transfected cells preferentially took up oxLDL(24), suggesting that stabilin-1 is a more important receptor for mildly oxLDLs than stabilin-2. Double immunogold labeling experiments in LSECs indicated interactions of stabilin-1 and stabilin-2 with oxLDL(3) on the cell surface, in coated pits, and endocytic vesicles. LSECs but not KCs endocytosed mildly oxLDL. Both stabilin-1 and stabilin-2 were involved in the LSEC endocytosis of oxLDLs, but experiments with stabilin-transfected cells pointed to stabilin-1 as the most important receptor for mildly oxLDL.

The Relationship between Fenestrations, Sieve Plates and Rafts in Liver Sinusoidal Endothelial Cells

Fenestrations are transcellular pores in endothelial cells that facilitate transfer of substrates between blood and the extravascular compartment. In order to understand the regulation and formation of fenestrations, the relationship between membrane rafts and fenestrations was investigated in liver sinusoidal endothelial cells where fenestrations are grouped into sieve plates. Three dimensional structured illumination microscopy, scanning electron microscopy, internal reflectance fluorescence microscopy and two-photon fluorescence microscopy were used to study liver sinusoidal endothelial cells isolated from mice. There was an inverse distribution between sieve plates and membrane rafts visualized by structured illumination microscopy and the fluorescent raft stain, Bodipy FL C5 ganglioside GM1. 7-ketocholesterol and/or cytochalasin D increased both fenestrations and lipid-disordered membrane, while Triton X-100 decreased both fenestrations and lipid-disordered membrane. The effects of cytochalasin D on fenestrations were abrogated by co-administration of Triton X-100, suggesting that actin disruption increases fenestrations by its effects on membrane rafts. Vascular endothelial growth factor (VEGF) depleted lipid-ordered membrane and increased fenestrations. The results are consistent with a sieve-raft interaction, where fenestrations form in non-raft lipid-disordered regions of endothelial cells once the membrane-stabilizing effects of actin cytoskeleton and membrane rafts are diminished.

Liver Aging and Pseudocapillarization in a Werner Syndrome Mouse Model

Werner syndrome is a progeric syndrome characterized by premature atherosclerosis, diabetes, cancer, and death in humans. The knockout mouse model created by deletion of the RecQ helicase domain of the mouse Wrn homologue gene (Wrn(∆hel/∆hel)) is of great interest because it develops atherosclerosis and hypertriglyceridemia, conditions associated with aging liver and sinusoidal changes. Here, we show that Wrn(∆hel/∆hel) mice exhibit increased extracellular matrix, defenestration, decreased fenestration diameter, and changes in markers of liver sinusoidal endothelial cell inflammation, consistent with age-related pseudocapilliarization. In addition, hepatocytes are larger, have increased lipofuscin deposition, more frequent nuclear morphological anomalies, decreased mitochondria number, and increased mitochondrial diameter compared to wild-type mice. The Wrn(∆hel/∆hel) mice also have altered mitochondrial function and altered nuclei. Microarray data revealed that the Wrn(∆hel/∆hel) genotype does not affect the expression of many genes within the isolated hepatocytes or liver sinusoidal endothelial cells. This study reveals that Wrn(∆hel/∆hel) mice have accelerated typical age-related liver changes including pseudocapillarization. This confirms that pseudocapillarization of the liver sinusoid is a consistent feature of various aging models. Moreover, it implies that DNA repair may be implicated in normal aging changes in the liver.

Hepatic disposal of advanced glycation end products during maturation and aging

UNLABELLED Aging is characterized by progressive loss of metabolic and biochemical functions and accumulation of metabolic by-products, including advanced glycation end products (AGEs), which are observed in several pathological conditions. A number of waste macromolecules, including AGEs are taken up from the circulation by endocytosis mainly into liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs). However, AGEs still accumulate in different tissues with aging, despite the presence of this clearance mechanism. The aim of the present study was to determine whether the efficiency of LSECs and KCs for disposal of AGEs changes through aging. RESULTS After intravenous administration of (14)C-AGE-albumin in pre-pubertal, young adult, middle aged and old mice, more than 90% of total recovered (14)C-AGE was liver associated, irrespective of age. LSECs and KCs represented the main site of uptake. A fraction of the (14)C-AGE degradation products ((14)C-AGE-DPs) was stored for months in the lysosomes of these cells after uptake. The overall rate of elimination of (14)C-AGE-DPs from the liver was markedly faster in pre-pubertal than in all post-pubertal age groups. The ability to eliminate (14)C-AGE-DPs decreased to similar extents after puberty in LSECs and KCs. A rapid early removal phase was characteristic for all age groups except the old group, where this phase was absent. CONCLUSIONS Removal of AGE-DPs from the liver scavenger cells is a very slow process that changes with age. The ability of these cells to dispose of AGEs declines after puberty. Decreased AGE removal efficiency early in life may lead to AGE accumulation.

Dietary macronutrients and the aging liver sinusoidal endothelial cell

Fenestrations are pores within the liver sinusoidal endothelial cells (LSECs) that line the sinusoids of the highly vascularized liver. Fenestrations facilitate the transfer of substrates between blood and hepatocytes. With pseudocapillarization of the hepatic sinusoid in old age, there is a loss of fenestrations. LSECs are uniquely exposed to gut-derived dietary and microbial substrates delivered by the portal circulation to the liver. Here we studied the effect of 25 diets varying in content of macronutrients and energy on LSEC fenestrations using the Geometric Framework method in a large cohort of mice aged 15 mo. Macronutrient distribution rather than total food or energy intake was associated with changes in fenestrations. Porosity and frequency were inversely associated with dietary fat intake, while fenestration diameter was inversely associated with protein or carbohydrate intake. Fenestrations were also linked to diet-induced changes in gut microbiome, with increased fenestrations associated with higher abundance of Firmicutes and reduced abundance of Bacteroidetes Diet-induced changes in levels of several fatty acids (C16:0, C19:0, and C20:4) were also significantly inversely associated with fenestrations, suggesting a link between dietary fat and modulation of lipid rafts in the LSECs. Diet influences fenestrations and these data reflect both the key role of the LSECs in clearing gut-derived molecules from the vascular circulation and the impact these molecules have on LSEC morphology.

Liver Sinusoidal Endothelial Cells and Regulation of Blood Lipoproteins

Dmitri Svistounov1, Svetlana N. Zykova1,2, Victoria C. Cogger1, Alessandra Warren1, Aisling C. McMahon1, Robin Fraser 3 and David G. Le Couteur1 1Centre for Education and Research on Ageing and ANZAC Research Institute, University of Sydney and Concord RG Hospital, Sydney, 2Department of Nephrology, University Hospital of Northern Norway, Tromso, 3University of Otago, Christchurch, 1Australia 2Norway 3New Zealand

Pseudocapillarization and the Aging Liver

Old age is associated with changes in the cells of the hepatic sinusoid. The liver sinusoidal endothelial cell undergoes pseudocapillarization characterized by defenestration, thickening, and altered expression of endothelial and extracellular matrix antigens. Pseudocapillarization contributes to age-related dyslipidemia and reduction in hepatic perfusion and might also have a role in age-related changes in drug metabolism and susceptibility to autoimmune disease. Old age is also associated with impaired endocytosis activity by liver endothelial cells. With respect to the other cells of the hepatic sinusoid and aging, there are increased numbers of activated Kupffer cells but they respond less well to stimuli. Stellate cells become engorged with fat and do not appear to be activated. Such aging changes in the cells of the hepatic sinusoid are likely to impact on overall hepatic function.

Mechanisms and Implications of Age-Related Changes in the Liver

As the worlds population ages in unprecedented numbers and proportions, management of the basic health needs of an older population is a key challenge [1]. Older age is unquestionably associated with increased vulnerability and susceptibility to disease and disability [2]. Ageing is the major independent risk factor for most diseases of the Western world including atherosclerosis, cancer, and arthritis as well as for the prototypical aging diseases such as dementia and osteoporosis [3]. Despite this, our understanding of how old age predisposes us to disease remains rudimentary [4]. A greater understanding of the ageing process will provide insight into the underlying causes of many diseases and open up new avenues for prevention and therapy. Our research into the underlying causes of ageing led us to examine the liver architecture in great detail. The age-related decrease in liver function is substantial and very relevant for systemic exposure to substrates implicated in disease pathogenesis and ageing. In the past, the liver was considered to be relatively unaffected by ageing and age-related diseases. Functional changes, particularly related to impaired drug metabolism (which is reduced by 40–50% in old age), were attributed only to age-related reduction in blood flow and liver mass [5]. However, as such mechanisms are unable to fully explain age-related impairment of hepatic function and the systemic effects of these changes, much greater investigation into other hepatic factors such as liver blood vessel ultrastructure, immune function, and gene and protein expression is warranted. This special issue brings together many of the current areas of research in the liver and ageing. It details findings on the systemic role of the liver in health, disease, and treatment options in the setting of old age. The first paper in this issue addresses the very important area of DNA repair in ageing in the liver. M. Lebel et al. have written an eloquent review that thoroughly explores the role of genetic instability in age-related loss of liver function. The authors postulate that this is an area of great promise in understanding and combating diminished liver function with age. S. J. Mitchell et al. question the evidence base for the current prescribing guidelines for the common analgesic paracetamol in older people in their manuscript, which examines how poorly understood pharmacodynamics and pharmacokinetics are in the older person. Better understanding is required to reduce the risks both of underdosing this important analgesic and of causing accidental hepatotoxicity. D. L. Schmucker and H. Sanchez investigate the impaired liver regeneration seen in older people and animal models and conclude that the regenerative capacity of older liver is not impaired, rather the rate of regeneration is reduced. This conclusion has very important implications for the contentious issue of the use of donor livers from older people in liver transplantation. A. Warren et al. present an ultrastructural study of the liver in old age, with particular focus on the hepatic stellate cell, a cell synonymous with fibrotic liver changes in disease. The study shows for the first time that, while there is lipid engorgement of the cells with ageing, there is no activation. Smooth muscle actin expression, the hallmark of the hepatic stellate cell dedifferentiation into a fibroblast, is seen in many chronic liver diseases but not in old age. This finding yet again shows that ageing changes in the liver are distinct from the pathological processes seen in disease states. X. He et al. address the important issue of cancer therapy in the older person and how liver-related changes drastically affect the efficacy and toxicity of chemotherapy agents. Due to comorbidities and poor functional status, older people have generally been omitted from drug therapy trials in this area. The authors argue that the key to increasing treatment efficacy and decreasing toxicity in this group is tailoring treatment specifically to individuals with their age and comorbidities foremost in their treatment plan. Finally the paper by L. Gan et al. examines nonalcoholic fatty liver disease (NAFLD) in ageing. NAFLD is the most common liver disease today and most people diagnosed with NAFLD are aged over 60 years. The authors point out that “Advanced age is associated with disease severity and fibrosis progression; a relatively high proportion of individuals with progressive forms of NAFLD develop cirrhosis by the time they are in their 70s or beyond, although more data are required on the exact risks.” The paper also presents some management strategies for older people with NAFLD. These articles present the state of knowledge of many aspects of the liver in ageing. We would suggest that they put forward a compelling argument that a thorough understanding of how the liver changes with age is important in disease prevention, modulation, and treatment in the setting of the older person. Victoria C. Cogger Dmitri Svistounov Sarah N. Hilmer