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Background/aim The effects of dietary protein and carbohydrate content on the plasma amino acid profile of patients with diabetes are not fully understood. Therefore, we examined whether there are effects of diets with differing proportions of protein and carbohydrate on the plasma amino acid concentrations of control (CT) mice and mice with type 2 diabetes (db). Materials and methods We used db mice as an animal model of type 2 diabetes which are genetically deficient in leptin receptor. Diets with differing proportions of protein and carbohydrates (L diet: low protein/carbohydrate ratio, H diet: high protein/carbohydrate ratio) were supplied. db Mice were fed with a restriction on the basis of the consumption by CT-L mice, such that equivalent amounts of energy and fat were consumed. In CT mice fed the L or H diets, there was no significant difference in ad libitum food intake. Results There were significant interactions between diet and genotype with respect to water intake, urine volume, urinary glucose concentration, and plasma isoleucine, leucine, valine, branched-chain amino acids, and serine concentrations. db-H mice showed significantly higher water intake, urine volume, and urinary glucose than db-L mice. db Mice fed the L or H diets had similar plasma amino acid profiles, except for valine. In contrast, CT-H mice showed significantly higher valine and branched-chain amino acids and lower serine concentrations than CT-L mice. Thus, the CT-H mice were more similar to db mice fed either of the diets. Conclusion There were different effects of the dietary protein or carbohydrate content on the plasma amino acid profiles between nondiabetic and diabetic mice. In particular, the profiles in nondiabetic conditions were different between the low- and high-protein diet conditions.

Background/aim Diabetic nephropathy is aggravated by a higher intake of total protein. The effects of diets with different proportions of protein and carbohydrate on diabetic retinopathy in db mice, a type-2 diabetes animal model, were examined, as well as diabetic nephropathy. Materials and methods Control and db mice at 5 weeks of age were fed the diets (% energy of protein/carbohydrate/fat; L-diet: 12/71/17; H-diet: 24/59/17) under ad libitum conditions and pair-feeding conditions for 6 weeks, respectively. Results Mice fed the H-diet showed significantly greater retinal thickness by optical coherence tomography, and lower mRNA levels of angiotensinogen. Comparing combinations of diets and genotypes, db-H mice showed significantly higher mRNA levels of angiotensin-converting enzyme, advanced glycosylation end product-specific receptor, and cluster of differentiation molecule 11b (a microglial marker) than db-L mice. Conclusion Dietary protein and carbohydrate proportions influenced retinal manifestations, including retinal thickness and gene expression in control and diabetic mice.

The effects of dietary and lighting conditions on diurnal rhythm of locomotor activity (LA) and body temperature (BT) using four adult male microminipigs were investigated. Different feeding times, diet and lighting conditions were applied sequentially for 3 weeks in each phase as follows: Phase I: Morning mealtime, normal diet, 12-h lights on; phase II: mealtime changed to afternoon; phase III: diet changed to high-fat diet; phase IV: lighting changed to 20-h on; and phase V: phase I repeated. LA was measured by an actigraph which was worn on the body of each pig. A BT recording module (Thermochron Type-SL) was implanted in the neck subcutaneously. Phase II increased BT compared with phase I. Phase III increased LA and BT compared with phase II. Phase IV increased LA compared with phase III. LA in phase V was higher compared with phase I. These results can be extrapolated to other diurnal animals such as humans. This study provides an example of the effects of diet and lighting on biological activities in microminipigs under low-invasive procedures measuring LA and BT, leading to low variations in these measures.

Different involvement of leptin signaling in food intake (FI) and body temperature (BT) in pups and adults has been suggested. However, the leptin receptor (Lepr) long-form-deficient (db) mouse line has not been fully examined in pups. In the most availabledbmouse line, wild-type (WT) mice have a mutation in the dedicator of cytokinesis 7 gene, namedmisty, which was recently revealed to be involved in neuronal development. Therefore, we established a line ofdbmice without themistymutation using natural mating. Adult (8 weeks of age) homozygousdb/dbmice displayed significantly higher core body weight (BW) and FI and significantly lower core BT than WT mice. However, postnatal (2 weeks of age)db/dbmice displayed similar BW and milk intake and significantly lower core BT than WT mice. Correspondingly, adult and postnataldb/dbmice exhibited altered mRNA levels of hypothalamic orexigenic and anorexigenic peptide in adults but not in pups. Additionally,db/dbmice displayed significantly lower mRNA levels of brown adipose tissue uncoupling protein 1 at both ages. In conclusion, thedbmouse line without themistymutation clearly showed the different involvements of the Lepr long form in FI and BT in pups and adults.

We previously showed the deleterious effects of increased dietary protein on renal manifestations and glucose metabolism in leptin receptor-deficient (db) mice. Here, we further examined its effects on glucose metabolism, including urinary C-peptide. We also orally administered mixtures corresponding to low- or high-protein diets to diabetic mice. In diet experiments, under pair-feeding (equivalent energy and fat) conditions using a metabolic cage, mice were fed diets with different protein content (L diet: 12 % protein, 71 % carbohydrate, 17 % fat; H diet: 24 % protein, 59 % carbohydrate, 17 % fat) for 15 days. In oral administration experiments, the respective mixtures (L mixture: 12 % proline, 71 % maltose or starch, 17 % linoleic acid; H mixture: 24 % proline, 59 % maltose or starch, 17 % linoleic acid) were supplied to mice. Biochemical parameters related to glucose metabolism were measured. The db–H diet mice showed significantly higher water intake, urinary volume, and glucose levels than db–L diet mice but similar levels of excreted urinary C-peptide. In contrast, control-H diet mice showed significantly higher C-peptide excretion than control-L diet mice. Both types of mice fed H diet excreted high levels of urinary albumin. When maltose mixtures were administered, db–L mixture mice showed significantly higher blood glucose after 30 min than db–H mixture mice. However, db mice administered starch–H mixture showed significantly higher blood glucose 120–300 min post-administration than db–L mixture mice, although both groups exhibited similar insulin levels. High-protein, low-carbohydrate diets deteriorated diabetic conditions and were associated with insufficient insulin secretion in db mice. Our findings may have implications for dietary management of diabetic symptoms in human patients.

The food habit is involved in the onset and development of lifestyle-related diseases. In this review I would like to describe a historical case of vitamin B1 deficiency, as well as our case study of fatty acid metabolism abnormality due to carnitine deficiency. In history, the army and navy personnel in Japan at the end of the 19th century received food rations based on a high-carbohydrate diet including white rice, resulting in the onset of beriberi. An epidemiological study by Kenkan Takaki revealed the relationship between the onset of beriberi and rice intake. Then, Takaki was successful in preventing the onset of beriberi by changing the diet. However, the primary cause had yet to be elucidated. Finally, Christian Eijkman established an animal model of beriberi (chickens) showing peripheral neuropathy, and he identified the existence of an anti-beriberi substance, vitamin B1. This is an example of the successful control of a disease by integrating the results of epidemiological and experimental studies. In our study using a murine model of fatty acid metabolism abnormality caused by carnitine deficiency, cardiac abnormality and fatty liver developed depending on the amount of dietary fat. In addition, the mice showed disturbance of orexin neuron activity related to the sleep-arousal system, which is involved in fatigue symptoms under fasting condition, one of the states showing enhanced fatty acid metabolism. These findings suggest that fatty acid toxicity is enhanced when the mice are more dependent on fatty acid metabolism. Almost simultaneously, a human epidemiological study showed that narcolepsy, which is caused by orexin system abnormality, is associated with the polymorphism of the gene coding for carnitine palmitoyltransferase 1B, which is involved in carnitine metabolism. To understand the pathological mechanism of fatty acid toxicity, not only an experimental approach using animal models, but also an epidemiological approach is necessary. The results will be applied to preventing and treating lifestyle-related diseases associated with fatty acid metabolism abnormality.

Exercise is effective for preventing the onset and development of type 2 diabetes mellitus (T2DM) in human cases; however, the effect of exercise on the pathophysiology using animal models of T2DM has not been fully evaluated.We applied voluntary exercise under pair-fed (P) conditions in db mice, an animal model of T2DM. Exercising (Ex) and sedentary (Se) mice were placed in a cage, equipped with a free or locked running wheel, for 4 weeks, respectively. The amount of food consumed by ad libitum-fed wild-type mice under the Se condition (ad-WT) was supplied to all mice, except ad libitum db mice (ad-db). Blood parameters and expression of the genes involved in nutrient metabolism were analyzed.PEx-db (pair-fed and exercising) mice showed significantly lower HbA1c, body weight and liver weight than PSe-db and ad-db mice. Decreased hepatic triglycerides in PEx-db mice corresponded to a lower expression of lipogenic enzyme genes in the liver. Moreover, PEx-db mice showed significantly lower plasma branched-chain amino acids (BCAA), arginine, proline, and tyrosine, in addition to increased skeletal muscle (SM) weight, than PSe-db and ad-db mice, in spite of little influence on the expression of the BCAA transaminase gene, in SM and WAT.We found that exercise under a food restriction condition decreases several amino acids, including BCAA, and may improve insulin sensitivity more than mere food restriction. We propose that the decreased concentration of blood amino acids may be a valuable marker evaluating the effects of exercise on diabetic conditions.

Dietary protein content is related clinically to the development of diabetic nephropathy. Here, we investigated how dietary protein content (12-24 % energy) within the range used by humans affected renal manifestations including the expressions of genes involved in the renin-angiotensin (RA) system in control and diabetic mice. Moreover, we examined the effects of dietary protein content on HbA1c and urinary glucose.Control (CT) and leptin receptor-deficient obese (db) mice, 5 weeks old, were fed the diets below. Under ad libitum conditions, mice were fed 12, 18, and 24 % energy from protein (L-, M-, and H-diets) for 8 weeks. Under pair-feeding conditions, db mice were supplied H-diet (db-Hp) to the equivalent energy to that consumed by db-L mice. Renal manifestations and values related to glucose and insulin were examined biochemically and pathologically.Under ad libitum conditions, db mice consumed food and water dose dependently of the dietary protein content, although they were consumed similarly by CT mice. CT-L mice showed lower urinary albumin and kidney weight, in association with lower mRNA levels of angiotensinogen and renin, than CT-H mice. Under pair-feeding conditions, db-L mice showed a lower ratio of kidney/body weight, HbA1(C), and urinary glucose, and a higher β-cell distribution rate in the pancreas than db-Hp mice.Low-protein intake in the range used by humans may relieve renal manifestations through the suppressed expression of genes in the renal RA system of CT mice. On the other hand, in db mice, low-protein intake improved hyperglycemia and the renal manifestations of diabetes.

Acupuncture, an alternative medicine, has been widely applied for people with sleep disturbances; therefore, the effects should be evaluated objectively. Micro-minipigs (MMPigs), the smallest miniature pigs for animal experiments, were used. Acupuncture was performed at two different points: Dafengmen is located on the head and is an anatomically similar point to human-Baihui (GV20), an effective acupoint for sleep disturbances in humans; pig-Baihui is on the back. The procedure was performed as follows: shallow, within 5 mm depth for several seconds; deep, 10–20 mm depth for 20 min. The sleep conditions were evaluated by actigraph, and the amount of catecholamine in pooled urine after acupuncture treatment. MMPigs with deep acupuncture at Dafengmen showed significantly efficient values on actigraph and catecholamine analysis as compared with untreated MMPigs. The effective acupoint for sleep conditions in the porcine model is at an anatomically similar point to humans, rather than the point determined by traditional Chinese medicine.

To determine the impact of long-term voluntary exercise, representing habitual exercise for the prevention of lifestyle-related diseases, on glucose, lipid, and amino acid metabolism in mice.Twenty-four mice aged 6 weeks were divided into three groups. Two groups (16 mice) were housed individually in either cages equipped with a running wheel (8 mice, exercising, Ex-mice) or without (8 mice, sedentary, Se-mice) for 24 weeks. The remaining group (8 mice) was sacrificed at 6 weeks of age. Biomarkers related to glucose, lipid, and amino acid metabolism were examined.Ex-mice ran voluntarily, predominantly in the dark. The distance per day peaked at 4 weeks and then decreased until 12 weeks to around the level seen at the beginning of the experimental period, and was maintained at 4.9 ± 0.2 km/day from 12 to 24 weeks. Ex-mice showed a similar adrenal weight and vitamin C content to Se-mice but had a significantly lower body weight and higher food intake. Ex-mice also showed a higher skeletal muscle weight, a lower white adipose tissue and liver weight, associated with lower plasma leptin and insulin-like growth factor-1 levels, and a lower hepatic triglyceride content. Analysis of plasma amino acids showed that Ex-mice had significantly higher phenylalanine, tyrosine, and glutamine levels, resulting in a significantly lower Fischer's ratio.We present an animal model of long-term voluntary exercise under low stress. Findings related to the effects of long-term voluntary exercise on lipid, and amino acid metabolism in our mouse model indicate that such an exercise regimen may affect pathophysiological states related to appetite and behavior.