Evaluation of the potential of organ glucose metabolism by 18F-FDG accumulation with insulin loading in super-aged mice compared with young normal mice

It is important to determine the functional changes of organs that occur as a result of aging, the understanding of which may lead to the maintenance of a healthy life. Glucose metabolism in healthy bodies is one of the potential markers used to evaluate the changes of organ function. Thus, information about normal organ glucose metabolism may help to understand the functional changes of organs. [18F]-Fluoro-2-deoxy-2-D-glucose (18F-FDG), a glucose analog, has been used to measure glucose metabolism in various elds, such as basic medical research and drug discovery. However, glucose metabolism changes in super-aged animals have not yet been fully claried. The aim of this study is to evaluate changes in glucose metabolism in organs and brain regions by measuring 18F-FDG accumulation and 18F-FDG autoradiography with insulin loading in super-aged and young wild-type mice. In control groups, the levels of 18F-FDG accumulation in the blood, plasma, muscle, lungs, spleen, pancreas, testes, stomach, small intestine, kidneys, liver, brain, and brain regions, namely, the cortex, striatum, thalamus, and hippocampus, were all signicantly higher in the super-aged mice. After insulin loading, the 18F-FDG accumulation levels showed negative changes in the pancreas and kidneys, as well as in the cortex, striatum, thalamus, and hippocampus in the super-aged mice, whereas positive changes were observed in those in the young mice. These results demonstrate that insulin loading decreases effect on 18F-FDG accumulation levels in some organs of the super-aged mice. Therefore, aging can increase insulin resistance and lead to systemic glucose metabolism dysfunction. 4 expression and translocation 49 . In this study, 18 F-FDG accumulation showed positive changes in the cortex, striatum, thalamus, and hippocampus in the young mice, whereas negative changes were observed in those in the super-aged mice after insulin loading. These indicate that the insulin sensitivity of these brain regions might gradually decrease with age and lead to age-related brain diseases such as AD and PD. The characteristics of glucose metabolism in the localized brain regions of these super-aged mice can be used to develop therapeutic models for age-related brain diseases.


Introduction
With a globally aging population, the health issues caused by aging and age-related diseases have become inevitable challenges for all countries. Understanding the functional changes of organs that occur as a result of aging is essential to prevent these age-related diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), dementia, stroke, peripheral neuropathy, macular degeneration, cataracts, senile deafness, diabetes mellitus, osteoporosis, osteoarthritis, atherosclerosis, prostatic hyperplasia, and even cancer 1 . In particular, the metabolism of glucose as an energy source has been regarded as a potential indicator for these disorders.
Under tight hormonal control by insulin, glucose homeostasis is maintained by a balance among glucose ingestion, utilization, and production 2 . As age advances, glucose homeostasis tends to gradually become disrupted, giving rise to type 2 diabetes (T2D) and cardiovascular diseases [3][4][5] . Insulin resistance is one of the major mechanisms underlying abnormal glucose tolerance 6 . Recognition of abnormal glucose metabolism in the elderly is important in implementing age-appropriate preventive and therapeutic strategies. It is widely accepted that aging is accompanied by an increase in insulin resistance 7,8 . This age-related insulin resistance has been variously attributed to several factors, including mitochondrial dysfunctions, reduced lean muscle mass and elevated adiposity, hormonal changes, increased oxidative stress, in ammation, and reduced physical activity [9][10][11][12][13][14] . However, these studies do not provide insights into organ-speci c differences in insulin resistance.
The glucose analog [ 18 F]-Fluoro-2-deoxy-2-D-glucose ( 18 F-FDG), a molecular imaging probe, is widely used in nuclear medicine for evaluating tissue glucose utilization and glucose metabolism [15][16][17] . Although 18 F-FDG accumulation has been investigated in various elds, such as basic medical research and drug discovery, changes in glucose metabolism evaluated using 18 F-FDG in super-aged animals have not yet been fully clari ed. Determining whether 18 F-FDG distribution is associated with aging could provide insight into metabolic changes and help to prevent age-related diseases.
Therefore, in this study, we attempted to clarify the changes in glucose metabolism that occur with aging by comparing 18 F-FDG accumulation levels after insulin loading in super-aged and young wild-type mice.

Results
Organ 18 F-FDG accumulation experiment in control groups The body weight and blood glucose level were determined in young and super-aged mice groups ( Table   1). The levels of organ 18 F-FDG accumulation were determined in young and super-aged mice groups ( Table 2). The body weight of the super-aged mice was signi cantly higher than that of the young mice (Table 1, Fig. 1a). The blood glucose concentration was not signi cantly different between two groups ( Table 1, Fig. 1b). Apart from the white adipose tissue, brown adipose tissue, large intestine, and heart, the levels of 18 F-FDG uptake in the blood, plasma, muscle, lungs, spleen, pancreas, testes, stomach, small intestine, kidneys, liver and brain were all signi cantly higher in the super-aged mice than in the young mice ( Table 2, Fig. 1c).

Changes in 18 F-FDG accumulation level in organs
In the young mice group, the levels of 18 F-FDG uptake in the muscle, heart, pancreas, white adipose tissue, stomach, kidneys, brown adipose tissue, and brain signi cantly increased after insulin loading. In contrast, the levels of 18 F-FDG uptake in the blood, plasma, spleen, testes, large intestine, and liver signi cantly decreased after insulin loading ( Table 2, Fig. 2). On the other hand, in the super-aged mice group, the levels of 18 F-FDG uptake in the muscle, heart, white adipose tissue, stomach, and brown adipose tissue signi cantly increased after insulin loading. However, the levels of 18 F-FDG uptake in blood, plasma, spleen, testes, and liver were signi cantly decreased after insulin loading ( Table 2, Fig. 3).
Compared with the young mice group, the levels of 18 F-FDG uptake in the pancreas, kidneys and brain did not exhibit observable changes after insulin loading in the super-aged mice group. In contrast, those in the lungs and small intestine signi cantly decreased after insulin loading (Table 2, Fig. 3).
The body weight showed no signi cant difference between control and insulin-loaded groups in both young and super-aged mice ( Table 1, Fig. 4a). When the blood glucose concentration decreased and displayed as "low" after insulin loading, we de ned this blood glucose concentration as 20 mmol/dl which is the detection limit of blood glucose meter. After insulin loading, the blood glucose concentration signi cantly decreased in both young and super-aged mice groups (Table 1, Fig. 4b). The rate of 18 F-FDG uptake level change in the control and insulin-loaded groups was assessed according to the following formula: ([mean level of 18 F-FDG uptake in insulin-loaded group -mean level of 18 F-FDG uptake in control group] / mean level of 18 F-FDG uptake in control group) × 100%. In these mice, the rates of change in the muscle, heart, white adipose tissue, stomach, brown adipose tissue, and brain showed positive changes, whereas those in the blood, plasma, lungs, spleen, testes, small intestine, large intestine, and liver showed negative changes (Fig. 4c). Moreover, the rates of change in the pancreas and kidneys showed positive changes in the young mice but negative changes in the super-aged mice (Fig. 4c). Regarding these positive changes, the rate of change in the muscle in the young mice was higher than that in the super-aged mice (223.3% vs 107.9%), as well as brain (31.6%vs 13.0%) (Fig. 4c).

Brain 18 F-FDG autoradiographic experiment in control groups
The body weight and blood glucose concentration were determined in the young and super-aged mice ( Table 3). The levels of 18 F-FDG uptake in brain regions were determined in the young and super-aged mice ( Table 4). The body weight of the super-aged mice was signi cantly higher than that of the young mice (P < 0.0001) ( Table 3, Fig. 5a). The blood glucose concentration was not signi cantly different between the two groups ( Table 3, Fig. 5b). The levels of 18 F-FDG accumulation in the cortex, striatum, thalamus, and hippocampus were all signi cantly higher in the super-aged mice than in the young mice (P < 0.001) ( Table 4, Fig. 5c).

Changes in 18 F-FDG accumulation level in brain regions
The levels of 18 F-FDG accumulation in brain regions in the insulin-loaded young and super-aged mice were determined and compared with those in the control young and super-aged groups (Table 4). In the brain, the levels of 18 F-FDG accumulation in the cortex, striatum, and hippocampus signi cantly increased after insulin loading in the young group (Table 4, Fig. 6a). Compared with the young group, the levels of 18 F-FDG accumulation in the striatum, thalamus and hippocampus did not show observable changes after insulin loading in the super-aged group (Table 4, Fig. 6b). In contrast, the level of 18 F-FDG accumulation in the cortex signi cantly decreased after insulin loading in the super-aged group (Table 4, The body weight showed no signi cant difference between control and insulin-loaded groups in both young and super-aged mice ( Table 3, Fig. 7a). When the blood glucose concentration decreased and displayed as "low" after insulin loading, we de ned this blood glucose concentration as 20 mmol/dl which is the detection limit of blood glucose meter. After insulin loading, the blood glucose concentration signi cantly decreased in both young and super-aged groups ( Table 3, Fig. 7b). The rate of 18 F-FDG accumulation level change in the control and insulin-loaded groups was assessed according to the following formula: ([mean level of 18 F-FDG accumulation in insulin loaded group -mean level of 18 F-FDG accumulation in control group] / mean level of 18 F-FDG accumulation in control group) × 100%. The 18 F-FDG accumulation level showed positive changes in the cortex, striatum, thalamus, and hippocampus in the young group, whereas negative changes were observed in those in super-aged group (Fig. 7c).

Discussion
Alterations in glucose homeostasis are enhanced with age and can be linked to T2D, cardiocerebrovascular injury, and other age-related diseases 18 . To clarify the changes in glucose metabolism with aging, we examined and compared the body weight, blood glucose concentration, and 18 F-FDG accumulation level in each organ between the super-aged and young mice.
In this study, the body weight of the super-aged mice was markedly higher than that of the young mice.
Previous studies have indicated that the progressive decline in insulin action with age can be attributed largely to gradual increases in the degree of relative obesity and the number of sites of fat deposition. In addition, insulin resistance is associated with sarcopenia and an accompanying relative increase in fat mass 19,20 . In a longitudinal study of over 4,500 healthy individuals, Lindstrom and Tuomilehto 21 and Salmon 22 showed that the likelihood of patients aged 55-64 years developing drug-treated diabetes was roughly equivalent to patients with a BMI > 30. Since muscle is a crucial variable in determining the e cacy of glucose uptake, a decrease in muscle mass leads to a simultaneous decrease in glucose disposal rate 23,24 . Although the mechanisms underlying the link between insulin resistance and fat deposition have not yet been fully elucidated, it has been suggested that because of both an increased level of the β-adrenoceptor pathway and a reduced α2-adrenoceptor component level, visceral adipose tissue shows a high lipolytic response to catecholamine, exposing the liver to high free fatty acid concentrations, which in turn induces insulin resistance [24][25][26][27] . In this study, after insulin loading, the muscle 18 F-FDG uptake rate of super-aged mice was signi cantly lower than that of young mice (107.9% vs 223.3%). In super-aged mice, the increase in adipose tissue weight may lead to insulin resistance to decrease glucose uptake level in the muscle.
In all the insulin-loaded groups, the levels of 18 F-FDG uptake in the muscle, heart, white adipose tissue, stomach, and brown adipose tissue were signi cantly higher than in all the control groups. Glucose transporter type 4 (Glut-4), which is an insulin-regulated glucose transporter, is primarily expressed in several tissues, including adipocytes, as well as the skeletal and cardiac muscles 28,29 . An excessively high blood insulin concentration leads to a series of signal cascades, including the autophosphorylation of the insulin cell surface receptor, the activation of receptor tyrosine kinase, the tyrosine phosphorylation of insulin receptor substrates 1 and 2, the activation of phosphatidylinositol 3-kinase, and the activation of protein kinase B and its downstream mediator AS160. These signal cascades eventually induce the translocation of a large quantity of Glut-4 from intracellular vesicles to the plasma membrane 30,31 . Thus, hyperinsulinemia markedly increases the level of glucose uptake in adipocytes and the skeletal and cardiac muscles.
In all the insulin-loaded groups, the levels of 18 F-FDG uptake in the blood, plasma, spleen, testes, and liver were signi cantly lower than those in all the control groups. Previous studies ascribed the reduced 18 F-FDG uptake in tumors and in ammatory lesions with insulin-induced hypoglycemia to the effects of insulin, which shifts 18 F-FDG from the original area to insulin-sensitive organs 32,33 . This insulin effect may also explain the reduced 18 F-FDG accumulation in insulin-insensitive organs with insulin-induced hypoglycemia.
In the control groups, excluding the heart, white adipose tissue, brown adipose tissue, and large intestine, the levels of 18 F-FDG accumulation in the blood, plasma, muscle, lungs, spleen, pancreas, testes, stomach, small intestine, kidneys, liver, and brain were all signi cantly higher in the super-aged mice than that in the young mice. However, the blood glucose concentration was not signi cantly different between the young and super-aged control groups. Hyperinsulinemia has been implicated in the progression of obesity, insulin resistance, and T2D. Elevated insulin levels can be a cause and consequence of obesity and insulin resistance [34][35][36] . Hyperinsulinemia, which is inevitably associated with insulin resistance, also appears to negatively affect kidney function via the induction of glomerular hyper ltration and increase in vascular permeability 37 . In this study, the levels of 18 F-FDG uptake in the kidneys in the young insulinloaded group were signi cantly higher than those in the young control group. Compared with the young group, the levels of 18 F-FDG uptake in kidneys in the super-aged group did not markedly change after insulin loading. Moreover, the levels of 18 F-FDG uptake in the kidneys showed positive changes in the young mice, but negative changes were observed in the super-aged mice after insulin loading. As mentioned above, insulin-induced hypoglycemia shifts 18 F-FDG from the original area to insulin-sensitive organs 32,33 . This insulin effect may also explain the reduced 18 F-FDG uptake in the kidneys in the superaged mice, because the decreased insulin sensitivity of the kidneys leads to the shift of 18 F-FDG to insulin-sensitive organs. These ndings suggest that there were obstacles in glucose metabolism in the kidneys in super-aged mice. On the other hand, this insulin resistance in old mice negatively affects kidney function, which leads to the excretion disorders of 18 F-FDG, and subsequently to higher levels of 18 F-FDG accumulation in organs of elderly mice. Kidney dysfunction is a major cause of morbidity and mortality, whose prevalence is rising worldwide mainly because of the aging of populations 38 . However, the epidemics of abnormalities associated with insulin resistance 39 might play a role in the increase in the prevalence of kidney dysfunction 40 . Insulin receptor substrate 1 plays a key role in insulin signaling and action in several organs including the kidneys 41 . Among the genes involved in the insulin signaling pathway is the gene encoding transmembrane glycoprotein ectonucleotide pyrophosphatase phosphodiesterase 1, which binds to and inhibits the insulin receptor and subsequent downstream insulin signaling and action in both cultured cells and animal models 42 . We consider that aging could induce insulin resistance through abnormal-insulin-signaling-related genes, and ultimately lead to glucose intolerance and kidney dysfunction.
In addition, after insulin loading, the brain glucose uptake rate in old mice was signi cantly lower than that in young mice (13.0% vs 31.6%). To more speci cally evaluate the changes in glucose metabolism in the brain, we carried out 18 F-FDG autoradiography to evaluate the 18 F-FDG accumulation in brain regions after insulin loading in super-aged and young wild-type mice. We found that the levels of 18 F-FDG accumulation in the cortex, striatum, thalamus, and hippocampus were all signi cantly higher in the super-aged mice than in the young mice. However, the blood glucose concentration was not signi cantly different between the young and super-aged groups. This is consistent with the nding that the levels of 18 F-FDG uptake in the brain were signi cantly higher in the super-aged mice that in the young mice in the study on the 18 F-FDG distribution in organs. In the young mice group, the levels of 18 F-FDG accumulation in the cortex, striatum, and hippocampus signi cantly increased after insulin loading. Compared with the young group, the levels of 18 F-FDG accumulation in the striatum, thalamus and hippocampus in the super-aged group did not markedly change after insulin loading. In contrast, the level of 18 F-FDG accumulation in the cortex signi cantly decreased after insulin loading in the super-aged group. Aging is associated with reductions in the levels of both insulin and its receptor in the brain, which may even cause the brain to be in the state of insulin resistance [43][44][45][46] . Cholerton et al. 44 indicated that chronic high levels of insulin and insulin resistance may exert a negative effect on several body systems, including the central nervous system, for some time prior to the onset of diabetes. There is increasing support to the idea that such early insulin abnormalities may be associated with the initiation of the cascade of the AD pathology in some individuals, years or even decades before the rst clinical dementia symptoms are manifest 44 . Bingham et al. 16 demonstrated an enhanced cerebral glucose metabolism that was particularly pronounced in the cortex following the administration of a low dose of insulin. The basis for brain-region-speci c insulin effects on glucose metabolism may be attributable to the distribution of GLUTs 47,48 . Insulin-sensitive GLUTs 4 and 8 are selectively distributed in the brain, and insulin increases the levels of brain GLUT 4 expression and translocation 49 . In this study, 18 F-FDG accumulation showed positive changes in the cortex, striatum, thalamus, and hippocampus in the young mice, whereas negative changes were observed in those in the super-aged mice after insulin loading. These indicate that the insulin sensitivity of these brain regions might gradually decrease with age and lead to age-related brain diseases such as AD and PD. The characteristics of glucose metabolism in the localized brain regions of these super-aged mice can be used to develop therapeutic models for age-related brain diseases.

Conclusions
In summary, we demonstrated that aging can induce insulin resistance and lead to dysfunction of systemic glucose metabolism. Insulin resistance could affect glucose metabolism and eventually cause age-related diseases. On the basis of ndings, ameliorating this dysfunction may be a good preventive and therapeutic strategy for age-related diseases.

Preparation of animal models
The entire experimental protocols were approved by the Laboratory Animal Care and Use Committee of Fukushima Medical University (approval number 30021) and performed in accordance with the Guidelines for Animal Experiments at Fukushima Medical University. Eight-week-old and 96-week-old male C57BL/6J mice were purchased from Charles River Laboratories Japan, Inc. (Yokohama, Japan) and maintained in a speci c-pathogen-free animal experiment facility. The room temperature was maintained between 23 and 25°C, and the relative humidity was maintained between 45 and 55%. The institutional laboratory housing provided a 12-h light/dark cycle and met all the criteria of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International (http://www.aaalac.org/) 50 . All animals were fasted overnight and then divided into four subgroups: young control group, super-aged control group, young insulin-loaded group, and super-aged insulinloaded group.

Organ 18 F-FDG accumulation study
Eight-week-old and 96-week-old male mice (n = 5, each group) were assigned to the control and insulinloaded groups. Those in the insulin-loaded groups were intraperitoneally injected with human insulin (2 U/kg body weight, Eli Lilly & Co., Kobe) 30 min prior to 18 F-FDG injection. We excluded one of the 96-weekold mice in the insulin-loaded group because the blood glucose level did not decrease after insulin loading. Each animal was initially anesthetized with 4% iso urane in air and maintained via spontaneous ventilation with 2% iso urane in air. 18 F-FDG (11.5 MBq/0.1 ml) was injected into the tail vein. Ninety minutes later, the animals were sacri ced and their organs were excised. The organs (muscle, heart, lungs, spleen, pancreas, white adipose tissue, testes, stomach, small intestine, large intestine, kidneys, liver, brown adipose tissue, and brain) and blood samples (blood and plasma) were weighed, and their radioactivity was determined with a gamma counter (WIZARD 2 2480; PerkinElmer, USA). After decay correction, the percentage of injected dose per gram of tissue was obtained and normalized to the animal weight [%ID/g tissue/kg body weight (%ID/g/kg)]. Blood samples for glucose concentration measurement were obtained from the control group and insulin-loaded groups.

Brain 18 F-FDG autoradiography study
Eight-week-old and 96-week-old male mice (n = 6, each group) were assigned to the control and insulinloaded groups. Those in the insulin-loaded groups received an intraperitoneal injection of human insulin (2 U/kg body weight, Eli Lilly & Co., Kobe) 30 min prior to 18 F-FDG injection. Each animal was initially anesthetized with 4% iso urane in air and maintained via spontaneous ventilation with 2% iso urane in air. 18 F-FDG (11.5 MBq/0.1 ml) was injected into the tail vein. Ninety minutes later, the animals were sacri ced, then brains were rapidly removed, placed in Brain Matrix (Stoelting Co, USA) and cut into coronal slices (2 mm/slice) to obtain 8-9 coronal slices that were exposed to a phosphor imaging plate (Fuji Imaging Plate BAS-SR 2025 for 18 F; Fuji Photo Film Co., Ltd., Tokyo, Japan) with a set of calibrated standards 51 . This autoradiographic exposure was performed overnight to detect the distribution of 18 F-FDG. Autoradiography images were analyzed using a computerized imaging analysis system (raytest, CR35, Version 2.1.0, Straubenhardt, Germany) with the image analysis software AIDA (Version 5.1 SP2, Straubenhardt, Germany). To determine brain radioactivity concentration, the cortex, striatum, thalamus and hippocampus were de ned using Aida Image Analyzer software. The roles of interests (ROIs), namely, the cortex, striatum, thalamus, and hippocampus in the left and right hemispheres in all mice were marked on the same anatomical plane with reference to the corresponding brain coronal slices (Fig.  8). The radioactivity in each ROI was determined per unit area, the percentage of injected dose per pixel of the cortex, striatum, thalamus, and hippocampus was obtained and normalized to the animal weight [%ID/pixel/kg body weight (%ID/p/kg)]. Finally, the average of the left and right values around each of the four regions was obtained. Blood samples for glucose concentration measurement were obtained from the control and insulin-loaded mice groups.

Statistical analyses
All data are expressed as mean ± standard deviation. Statistical analyses were performed using the unpaired Student's t-test to evaluate the signi cance of differences between the young and super-aged mice in body weight, blood glucose concentration, and 18 F-FDG distribution. Signi cance was assumed at P < 0.05.

Data availability
The data generated and/or analyzed in this study are available from the corresponding author on reasonable request. Control, control group; Insulin, insulin-loaded group *P < 0.05, ** P < 0.01, ***P < 0.001, ****P < 0.0001 vs control value Table 3. Body weight (g) and blood glucose concentration (mg/dl) in brain 18   Data are shown in parentheses (mean±SD).