Delayed effects of radiation in adipose tissue reflect progenitor damage and not cellular senescence.

The pathogenesis of many age-related diseases is linked to cellular senescence, a state of inflammation-inducing, irreversible cell cycle arrest. The consequences and mechanisms of age-associated cellular senescence are often studied using in vivo models of radiation exposure. However, it is unknown whether radiation induces persistent senescence, like that observed in ageing. We performed analogous studies in mice and monkeys, where young mice and rhesus macaques received sub-lethal doses of ionizing radiation and were observed for ~ 15% of their expected lifespan. Assessments of 8-hydroxy-2' -deoxyguanosine (8-OHdG), senescence-associated beta-galactosidase (SAß-gal), and p16Ink4a and p21 were performed on mitotic and post-mitotic tissues - liver and adipose tissue - 6 months and 3 years post-exposure for the mice and monkeys, respectively. No elevations in 8-OHdG, SA-ßgal staining, or p16 Ink4a or p21 gene or protein expression were found in mouse and monkey liver or adipose tissue compared to control animals. Despite no evidence of senescence, progenitor cell dysfunction persisted after radiation exposure, as indicated by lower in situ CD34+ adipose cells (p = 0.03), and deficient adipose stromal vascular cell proliferation (p < 0.05) and differentiation (p = 0.04) ex vivo. Our investigation cautions that employing radiation to study senescence-related processes should be limited to the acute post-exposure period and that stem cell damage likely underpins the dysfunction associated with delayed effects of radiation.

PET Imaging of [11C]MPC-6827, a Microtubule-Based Radiotracer in Non-Human Primate Brains.

Dysregulation of microtubules is commonly associated with several psychiatric and neurological disorders, including addiction and Alzheimer's disease. Imaging of microtubules in vivo using positron emission tomography (PET) could provide valuable information on their role in the development of disease pathogenesis and aid in improving therapeutic regimens. We developed [11C]MPC-6827, the first brain-penetrating PET radiotracer to image microtubules in vivo in the mouse brain. The aim of the present study was to assess the reproducibility of [11C]MPC-6827 PET imaging in non-human primate brains. Two dynamic 0-120 min PET/CT imaging scans were performed in each of four healthy male cynomolgus monkeys approximately one week apart. Time activity curves (TACs) and standard uptake values (SUVs) were determined for whole brains and specific regions of the brains and compared between the "test" and "retest" data. [11C]MPC-6827 showed excellent brain uptake with good pharmacokinetics in non-human primate brains, with significant correlation between the test and retest scan data (r = 0.77, p = 0.023). These initial evaluations demonstrate the high translational potential of [11C]MPC-6827 to image microtubules in the brain in vivo in monkey models of neurological and psychiatric diseases.

Transcriptomic Analysis of Cell-free Fetal RNA in the Amniotic Fluid of Vervet Monkeys (Chlorocebus sabaeus).

NHP are important translational models for understanding the genomic underpinnings of growth, development, fetal programming, and predisposition to disease, with potential for the development of early health biomarkers. Understanding how prenatal gene expression is linked to pre- and postnatal health and development requires methods for assessing the fetal transcriptome. Here we used RNAseq methodology to analyze the expression of cell-free fetal RNA in the amniotic fluid supernatant (AFS) of vervet monkeys. Despite the naturally high level of degradation of free-floating RNA, we detected more than 10,000 gene transcripts in vervet AFS. The most highly expressed genes were H19, IGF2, and TPT1, which are involved in embryonic growth and glycemic health. We noted global similarities in expression profiles between vervets and humans, with genes involved in embryonic growth and glycemic health among the genes most highly expressed in AFS. Our study demonstrates both the feasibility and usefulness of prenatal transcriptomic profiles, by using amniocentesis procedures to obtain AFS and cell-free fetal RNA from pregnant vervets.

Whole Body Irradiation Induces Diabetes and Adipose Insulin Resistance in Nonhuman Primates.

PURPOSE: Diabetes mellitus is a delayed effect of radiation exposure in human and nonhuman primates. Diabetes mellitus is characterized by peripheral tissue insulin resistance, and as a result, irradiation exposure may cause important changes in insulin-sensitive tissues such as muscle and adipose. METHODS AND MATERIALS: We prospectively investigated changes in response to irradiation (4 Gy whole body exposure) in 16 male rhesus macaques. We evaluated changes in body composition and glycemic control for 2 years. Insulin responsiveness, lipolysis, inflammation, and fibrosis were evaluated at study end. RESULTS: Irradiated animals accumulate less fat and significantly increased percent glycation of hemoglobin A1c over time, such that 40% of irradiated monkeys had values that define them as diabetic at 2 years. Subcutaneous (SQ) adipose tissue was insulin resistant, as evidenced by reduced phosphorylation of the insulin receptor substrate-1 in response to insulin challenge and had increased basal lipolysis despite comparable insulin exposures to control animals. Irradiated SQ adipose tissue had more macrophage infiltration and adipocytes were larger. The observed hypertrophy was associated with decreased glycemic control and macrophage infiltration correlated with decreased adiponectin, signifying that inflammation is associated with worsening health. No evidence of SQ adipose fibrosis was detected. CONCLUSIONS: Our study is the first to prospectively illustrate that sublethal irradiation exposures directly propagate metabolic disease in the absence of obesity in nonhuman primates and implicate SQ adipose dysfunction as a target tissue.

Biomarkers of leaky gut are related to inflammation and reduced physical function in older adults with cardiometabolic disease and mobility limitations.

Intestinal barrier dysfunction is hypothesized to be a contributing determinant of two prominent characteristics of aging: inflammation and decline in physical function. A relationship between microbial translocation (MT), or their biomarkers (lipopolysaccharide binding protein-1 [LBP-1], soluble cluster of differentiation [sCD]-14), and physical function has been reported in healthy older adults, rats, and invertebrates. However, it is not known whether the existence of comorbidities, or clinical interventions intended to reduce comorbidities through weight loss or exercise, alters this connection. We measured inflammation, MT, and physical function in 288 overweight/obese older patients with cardiometabolic disease and self-reported mobility limitations who were enrolled in a weight loss and lifestyle intervention study. At baseline, inflammatory cytokines and LBP-1 were positively correlated after adjustment for age, gender, and body mass index. A higher LBP-1 was significantly associated with poorer physical functional after covariate adjustment. Further, even when IL-6 levels were included in the models, 400-m walk time (p = 0.003), short physical performance battery (p = 0.07), and IL-8 (p < 0.001) remained positively associated with LBP-1. Lifestyle interventions improved body mass and some functional measures; however, MT and inflammation were unchanged. MT is reliably related to inflammation, and to poorer physical function in older adults with comorbid conditions. Intestinal barrier function did not appear to improve as a result of intervention assignment, suggesting alternative strategies are needed to target this pro-inflammatory pathway in aging.

A Low Iron Diet Protects from Steatohepatitis in a Mouse Model.

High tissue iron levels are a risk factor for multiple chronic diseases including type 2 diabetes mellitus (T2DM) and non-alcoholic fatty liver disease (NAFLD). To investigate causal relationships and underlying mechanisms, we used an established NAFLD model-mice fed a high fat diet with supplemental fructose in the water ("fast food", FF). Iron did not affect excess hepatic triglyceride accumulation in the mice on FF, and FF did not affect iron accumulation compared to normal chow. Mice on low iron are protected from worsening of markers for non-alcoholic steatohepatitis (NASH), including serum transaminases and fibrotic gene transcript levels. These occurred prior to the onset of significant insulin resistance or changes in adipokines. Transcriptome sequencing revealed the major effects of iron to be on signaling by the transforming growth factor beta (TGF-ß) pathway, a known mechanistic factor in NASH. High iron increased fibrotic gene expression in vitro, demonstrating that the effect of dietary iron on NASH is direct. Conclusion: A lower tissue iron level prevents accelerated progression of NAFLD to NASH, suggesting a possible therapeutic strategy in humans with the disease.

Microbial translocation into amniotic fluid of vervet monkeys is common and unrelated to adverse infant outcomes.

Amniotic fluid was collected from pregnant female African green monkeys (n = 20). Analyses indicate microbial translocation into amniotic fluid during pregnancy is typical, and microbial load reduces across gestation. Microbial translocation does not relate to infant outcome or maternal factors. Lastly, we demonstrate that sample contamination is easily introduced and detectable.

Greater Microbial Translocation and Vulnerability to Metabolic Disease in Healthy Aged Female Monkeys.

Monkeys demonstrate gastrointestinal barrier dysfunction (leaky gut) as evidenced by higher biomarkers of microbial translocation (MT) and inflammation with ageing despite equivalent health status, and lifelong diet and environmental conditions. We evaluated colonic structural, microbiomic and functional changes in old female vervet monkeys (Chlorocebus aethiops sabeus) and how age-related leaky gut alters responses to Western diet. We additionally assessed serum bovine immunoglobulin therapy to lower MT burden. MT was increased in old monkeys despite comparable histological appearance of the ascending colon. Microbiome profiles from 16S sequencing did not show large differences by age grouping, but there was evidence for higher mucosal bacterial loads using qPCR. Innate immune responses were increased in old monkeys consistent with higher MT burdens. Western diet challenge led to elevations in glycemic and hepatic biochemistry values only in old monkeys, and immunoglobulin therapy was not effective in reducing MT markers or improving metabolic health. We interpret these findings to suggest that ageing may lead to lower control over colonization at the mucosal surface, and reduced clearance of pathogens resulting in MT and inflammation. Leaky gut in ageing, which is not readily rescued by innate immune support with immunoglobulin, primes the liver for negative consequences of high fat, high sugar diets.

Alfalfa-derived HSP70 administered intranasally improves insulin sensitivity in mice.

Heat shock protein (HSP) 70 is an abundant cytosolic chaperone protein that is deficient in insulin-sensitive tissues in diabetes and unhealthy aging, and is considered a longevity target. It is also protective in neurological disease models. Using HSP70 purified from alfalfa and administered as an intranasal solution, we tested in whether the administration of Hsp70 to diet-induced diabetic mice would improve insulin sensitivity. Both the 10 and 40 µg given three times per week for 26 days significantly improved the response to insulin. The HSP70 was found to pass into the olfactory bulbs within 4-6 hours of a single dose. These results suggest that a relatively inexpensive, plentiful source of HSP70 administered in a simple, non-invasive manner, has therapeutic potential in diabetes.

Regulators of mitochondrial quality control differ in subcutaneous fat of metabolically healthy and unhealthy obese monkeys.

OBJECTIVE: Obesity exists with and without accompanying cardiometabolic disease, termed metabolically unhealthy obesity (MUO) and healthy obesity (MHO), respectively. Underlying differences in the ability of subcutaneous (SQ) fat to respond to nutrient excess are emerging as a key pathway. This study aimed to document the first spontaneous animal model of MHO and MUO and differences in SQ adipose tissue. METHODS: Vervet monkeys (Chlorocebus aethiops; N = 171) were screened for metabolic syndrome. A subset of MHO and MUO monkeys (n = 6/group) had SQ fat biopsies collected for histological evaluations and examination of key mitochondrial proteins. RESULTS: Obesity was seen in 20% of monkeys, and within this population, 31% were healthy, which mirrors human prevalence estimates. MUO monkeys had more than 60% lower adiponectin concentrations despite similar fat cell size, uncoupling protein 3, and activated macrophage abundance. However, alternatively activated/anti-inflammatory macrophages were 70% lower. Deficiencies of 50% or more in mitochondrial quality control regulators and selected mitochondrial fission and fusion markers were observed in the SQ fat of MUO monkeys despite comparable mitochondrial content. CONCLUSIONS: A novel and translatable spontaneously obese animal model of MHO and MUO, occurring independently of dietary factors, was characterized. Differences in mitochondrial quality and inflammatory cell populations of subcutaneous fat may underpin divergent metabolic health.

Fatty liver promotes fibrosis in monkeys consuming high fructose.

OBJECTIVE: Nonalcoholic fatty liver diseases (NAFLD) are related to development of liver fibrosis which currently has few therapeutic options. Rodent models of NAFLD inadequately model the fibrotic aspects of the disease and fail to demonstrate the spectrum of cardiometabolic diseases without genetic manipulation. This study aimed to document a monkey model of fatty liver and fibrosis, which naturally develop cardiometabolic disease pathophysiologies. METHODS: Twenty-seven cynomolgus monkeys (Macaca fascicularis) fed diets either low or high in simple carbohydrates, supplied as fructose [control and high-fructose diet (HRr)], on low-fat, cholesterol-free background were studied. The HFr was consumed for up to 7 years, and liver tissue was histologically evaluated for fat and fibrosis extent. RESULTS: The HFr diet increased steatosis, and its extent was related to duration of fructose exposure. Lipid droplet size also increased with HFr duration; however, compared with control, the lipid droplets were smaller on average. Fibrosis extent was significantly greater with fructose feeding and was predicted by fructose exposure, extent of fatty liver, and age. CONCLUSIONS: These data are the first to demonstrate that high-carbohydrate diets alone can generate both liver fat and fibrosis and thus allow further study of mechanisms and therapeutic options in the translational animal model.

Microbial translocation and skeletal muscle in young and old vervet monkeys.

Intestinal barrier dysfunction leads to microbial translocation (MT) and inflammation in vertebrate and invertebrate animal models. Age is recently recognized as a factor leading to MT, and in some human and animal model studies, MT was associated with physical function. We evaluated sarcopenia, inflammation, MT biomarkers, and muscle insulin sensitivity in healthy female vervet monkeys (6-27 years old). Monkeys were fed consistent diets and had large and varied environments to facilitate physical activity, and stable social conditions. Aging led to sarcopenia as indicated by reduced walking speeds and muscle mass, but general metabolic health was similar in older monkeys (n = 25) as compared to younger ones (n = 26). When older monkeys were physically active, their MT burden approximated that in young monkeys; however, when older monkeys were sedentary, MT burden was dramatically increased. MT levels were positively associated with inflammatory burden and negatively associated with skeletal muscle insulin sensitivity. Time spent being active was positively associated with insulin sensitivity as expected, but this relationship was specifically modified by the individual monkey's MT, not inflammatory burden. Our data supports clinical observations that MT interacts with physical function as a factor in healthy aging.

Type 2 Diabetes is a Delayed Late Effect of Whole-Body Irradiation in Nonhuman Primates.

One newly recognized consequence of radiation exposure may be the delayed development of diabetes and metabolic disease. We document the development of type 2 diabetes in a unique nonhuman primate cohort of monkeys that were whole-body irradiated with high doses (6.5-8.4 Gy) 5-9 years earlier. We report here a higher prevalence of type 2 diabetes in irradiated monkeys compared to age-matched nonirradiated monkeys. These irradiated diabetic primates demonstrate insulin resistance and hypertriglyceridemia, however, they lack the typical obese presentation of primate midlife diabetogenesis. Surprisingly, body composition analyses by computed tomography indicated that prior irradiation led to a specific loss of visceral fat mass. Prior irradiation led to reductions in insulin signaling effectiveness in skeletal muscle and higher monocyte chemoattractant protein 1 levels, indicative of increased inflammation. However, there was an absence of large defects in pancreatic function with radiation exposure, which has been documented previously in animal and human studies. Monkeys that remained healthy and did not become diabetic in the years after irradiation were significantly leaner and smaller, and were generally smaller and younger at the time of exposure. Irradiation also resulted in smaller stature in both diabetic and nondiabetic monkeys, compared to nonirradiated age-matched controls. Our study demonstrates that diabetogenesis postirradiation is not a consequence of disrupted adipose accumulation (generalized or in ectopic depots), nor generalized pancreatic failure, but suggests that peripheral tissues such as the musculature are impaired in their response to insulin exposure. Ongoing inflammation in these animals appears to be a consequence of radiation exposure and can interfere with insulin signaling. The reasons that some animals remain protected from diabetes as a late effect of irradiation are not clear, but may be related to body size. The translational relevance for these results suggest that muscle may be an important and underappreciated target organ for the delayed late effect of whole-body irradiation, leading to increased risk of insulin resistance and diabetes development.