Alessandra Warren

The Ratio of Macronutrients, Not Caloric Intake, Dictates Cardiometabolic Health, Aging, and Longevity in Ad Libitum-Fed Mice

The fundamental questions of what represents a macronutritionally balanced diet and how this maintains health and longevity remain unanswered. Here, the Geometric Framework, a state-space nutritional modeling method, was used to measure interactive effects of dietary energy, protein, fat, and carbohydrate on food intake, cardiometabolic phenotype, and longevity in mice fed one of 25 diets ad libitum. Food intake was regulated primarily by protein and carbohydrate content. Longevity and health were optimized when protein was replaced with carbohydrate to limit compensatory feeding for protein and suppress protein intake. These consequences are associated with hepatic mammalian target of rapamycin (mTOR) activation and mitochondrial function and, in turn, related to circulating branched-chain amino acids and glucose. Calorie restriction achieved by high-protein diets or dietary dilution had no beneficial effects on lifespan. The results suggest that longevity can be extended in ad libitum-fed animals by manipulating the ratio of macronutrients to inhibit mTOR activation.

T lymphocytes interact with hepatocytes through fenestrations in murine liver sinusoidal endothelial cells

The liver has an established ability to induce tolerance. Recent evidence indicates that this unique property might be related to its distinctive architecture allowing T cells to be activated in situ independently of lymphoid tissues. Unlike lymph node–activated T cells, liver‐activated T cells are short‐lived, a mechanism that might contribute to the “liver tolerance effect.” Although the potential role of hepatocytes as tolerogenic antigen‐presenting cells has been demonstrated, the question as to whether these cells are able to interact with CD8+ T cells in physiological settings remains controversial. Contradicting the immunological dogma stating that naïve T lymphocytes are prevented from interacting with parenchymal cells within non‐lymphoid organs by an impenetrable endothelial barrier, we show here that the unique morphology of the liver sinusoidal endothelial cell (LSEC) permits interactions between lymphocytes and hepatocytes. Using electron microscopy, we demonstrate that liver resident lymphocytes as well as circulating naïve CD8+ T cells make direct contact with hepatocytes through cytoplasmic extensions penetrating the endothelial fenestrations that perforate the LSECs. Furthermore, the expression of molecules required for primary T cell activation, MHC class I and ICAM‐1, is polarized on hepatocytes to the perisinusoidal cell membrane, thus maximizing the opportunity for interactions with circulating lymphocytes. In conclusion, this study has identified, at the ultrastructural level, a unique type of interaction between naïve T lymphocytes and liver parenchymal cells in vivo. These results hold implications for the pathogenesis of viral hepatitis in which hepatocytes may represent the main antigen‐presenting cell, and for the development of immune tolerance as lymphocytes pass through the liver. (HEPATOLOGY 2006;44:1182–1190.)

Age-related pseudocapillarization of the human liver.

Age‐related changes in liver function are important because they may promote susceptibility to adverse drug reactions, neurotoxicity, atherosclerosis, and other important diseases in older people. Age‐related changes in the rat hepatic sinusoidal endothelium, termed pseudocapillarization, have been described recently and these may contribute to hepatic impairment. The present study has examined surgical and post‐mortem specimens with immunohistochemistry and transmission electron microscopy to determine whether pseudocapillarization also occurs in older humans. The age of the subject, independent of systemic disease or hepatic pathology in surgical and post‐mortem samples of human liver, was associated with increased peri‐sinusoidal expression of von Willebrands factor, collagen I, collagen IV, and staining with Massons trichrome. Electron microscopy revealed significant age‐related thickening of the sinusoidal endothelium (young 165 ± 17 nm, middle age 222 ± 11 nm, older 289 ± 9 nm, p < 0.001) with loss of fenestrations (young 7.7 ± 0.7 per 10 µm, middle age 3.6 ± 0.5 per 10 µm, older 1.5 ± 0.4 per 10 µm, p < 0.001), and age‐related deposition of basal lamina and collagen. In conclusion, ageing in humans is associated with morphological changes in the sinusoidal endothelium and space of Disse which are presumptively related to the ageing process and potentially represent an important link between the ageing process and disease susceptibility. Copyright

A High-Fat Diet and NAD+ Activate Sirt1 to Rescue Premature Aging in Cockayne Syndrome

Cockayne syndrome (CS) is an accelerated aging disorder characterized by progressive neurodegeneration caused by mutations in genes encoding the DNA repair proteins CS group A or B (CSA or CSB). Since dietary interventions can alter neurodegenerative processes, Csb(m/m) mice were given a high-fat, caloric-restricted, or resveratrol-supplemented diet. High-fat feeding rescued the metabolic, transcriptomic, and behavioral phenotypes of Csb(m/m) mice. Furthermore, premature aging in CS mice, nematodes, and human cells results from aberrant PARP activation due to deficient DNA repair leading to decreased SIRT1 activity and mitochondrial dysfunction. Notably, β-hydroxybutyrate levels are increased by the high-fat diet, and β-hydroxybutyrate, PARP inhibition, or NAD(+) supplementation can activate SIRT1 and rescue CS-associated phenotypes. Mechanistically, CSB can displace activated PARP1 from damaged DNA to limit its activity. This study connects two emerging longevity metabolites, β-hydroxybutyrate and NAD(+), through the deacetylase SIRT1 and suggests possible interventions for CS.

Old Age and the Hepatic Sinusoid

Morphological changes in the hepatic sinusoid with old age are increasingly recognized. These include thickening and defenestration of the liver sinusoidal endothelial cell, sporadic deposition of collagen and basal lamina in the extracellular space of Disse, and increased numbers of fat engorged, nonactivated stellate cells. In addition, there is endothelial up‐regulation of von Willebrand factor and ICAM‐1 with reduced expression of caveolin‐1. These changes have been termed age‐related pseudocapillarization. The effects of old age on Kupffer cells are inconsistent, but impaired responsiveness is likely. There are functional implications of these aging changes in the hepatic sinusoid. There is reduced sinusoidal perfusion, which will impair the hepatic clearance of highly extracted substrates. Blood clearance of a variety of waste macromolecules takes place in liver sinusoidal endothelial cells (LSECs). Previous studies indicated either that aging had no effect, or reduced the endocytic capacity of LSECs. However, a recent study in mice showed reduced endocytosis in pericentral regions of the liver lobules. Reduced endocytosis may increase systemic exposure to potential harmful waste macromolecules such as advanced glycation end products Loss of fenestrations leads to impaired transfer of lipoproteins from blood to hepatocytes. This provides a mechanism for impaired chylomicron remnant clearance and postprandial hyperlipidemia associated with old age. Given the extensive range of substrates metabolized by the liver, age‐related changes in the hepatic sinusoid and microcirculation have important systemic implications for aging and age‐related diseases. Anat Rec, 291:672–683, 2008.

Hepatocyte entry leads to degradation of autoreactive CD8 T cells

Although most self-reactive T cells are eliminated in the thymus, mechanisms to inactivate or control T cells specific for extrathymic antigens are required and exist in the periphery. By investigating the site in which autoreactive T cells are tolerized, we identify a unique mechanism of peripheral deletion in which naïve autoreactive CD8 T cells are rapidly eliminated in the liver after intrahepatic activation. T cells actively invade hepatocytes, enter endosomal/lysosomal compartments, and are degraded. Blockade of this process leads to accumulation of autoreactive CD8 T cells in the liver and breach of tolerance, with the development of autoimmune hepatitis. Cell into cell invasion, or emperipolesis, is a long-observed phenomenon for which a physiological role has not been previously demonstrated. We propose that this “suicidal emperipolesis” is a unique mechanism of autoreactive T-cell deletion, a process critical for the maintenance of tolerance.

Hepatic sinusoidal pseudocapillarization with aging in the non-human primate

BACKGROUND/AIMS Age-related changes in the hepatic sinusoid termed pseudocapillarization have been reported in the rat and human and have implications for disease susceptibility in old age. In this study, we investigated whether similar changes occur in the livers of old baboons and thus represent a widespread aging change. METHODS Liver tissue from five young baboons (5.4+/-0.5yrs) and five old baboons (21.8+/-0.7yrs) was compared by transmission electron microscopy, scanning electron microscopy and immunohistochemistry. RESULTS The thickness of the sinusoidal endothelium was increased in old baboons (130+/-8 nm versus 186+/-9 nm, P<0.001) and the frequency of endothelial fenestrae decreased, with the porosity declining from 4.2+/-0.5% to 2.4+/-0.4% (P=0.006). The expression of laminin and von Willebrands factor was more extensive in old baboons. Novel perisinusoidal ring-shaped cells, probably fat-engorged stellate cells, were prominent in the old baboons. CONCLUSIONS Pseudocapillarization is a significant age-related change in the baboon liver. Aging in baboons is associated with a novel aging change in the stellate cell not reported in other species. Hepatic pseudocapillarization is a widespread aging liver change found in several species including humans and other non-human primates.

Activation of Thermogenesis in Brown Adipose Tissue and Dysregulated Lipid Metabolism Associated with Cancer Cachexia in Mice

Cancer cachexia/anorexia is a complex syndrome that involves profound metabolic imbalances and is directly implicated as a cause of death in at least 20% to 30% of all cancers. Brown adipose tissue (BAT) plays a key role in thermogenesis and energy balance and potentially contributes to the physiologic perturbations associated with cachexia. In this study, we investigated the impact of cachexia-inducing colorectal tumor on BAT in mice. We found that brown adipocytes were smaller and exhibited profound delipidation in cachectic tumor-bearing mice. Diurnal expression profiling of key regulators of lipid accumulation and fatty acid β-oxidation and their corresponding target genes revealed dramatic molecular changes indicative of active BAT. Increased Ucp1, Pbe, and Cpt1α expression at specific points coincided with higher BAT temperatures during the dark cycle, suggestive of a temporal stimulation of thermogenesis in cachexia. These changes persisted when cachectic mice were acclimatized to 28°C confirming inappropriate stimulation of BAT despite thermoneutrality. Evidence of inflammatory signaling also was observed in the BAT as an energetically wasteful and maladaptive response to anorexia during the development of cachexia.

Hepatic pseudocapillarization in aged mice

Age-related changes in the hepatic sinusoid of the rat, human and baboons called pseudocapillarization have been discovered and are important because they are considered to be implicated in the pathogenesis of some age-related diseases. In this study, we investigated whether similar changes occur in the livers of old mice. Livers of young (3-4 months) and old (20-24 months) mice were perfusion-fixed and studied using electron microscopy and immunohistochemistry. The thickness of the sinusoidal endothelium was increased in old mice (154+/-4 versus 244+/-8 nm, P<0.001). There was a reduction in fenestrations within the endothelium (porosity decreased from 4.1+/-0.3 to 2.2+/-0.2%, P<0.001). There was perisinusoidal staining with Sirius red in old mice, however, expression of laminin and von Willebrands factor was similar in young and old mice. Novel perisinusoidal fat-engorged stellate cells were found extensively in the old mice. This study confirmed that pseudocapillarization is a widespread aging change in the liver, now documented in several species including the mouse. Mice are an appropriate animal model for studying aging and the hepatic sinusoid.

Defining the Nutritional and Metabolic Context of FGF21 Using the Geometric Framework

Fibroblast growth factor 21 (FGF21) is the first known endocrine signal activated by protein restriction. Although FGF21 is robustly elevated in low-protein environments, increased FGF21 is also seen in various other contexts such as fasting, overfeeding, ketogenic diets, and high-carbohydrate diets, leaving its nutritional context and physiological role unresolved and controversial. Here, we use the Geometric Framework, a nutritional modeling platform, to help reconcile these apparently conflicting findings in mice confined to one of 25 diets that varied in protein, carbohydrate, and fat content. We show that FGF21 was elevated under low protein intakes and maximally when low protein was coupled with high carbohydrate intakes. Our results explain how elevation of FGF21 occurs both under starvation and hyperphagia, and show that the metabolic outcomes associated with elevated FGF21 depend on the nutritional context, differing according to whether the animal is in a state of under- or overfeeding.